Chain formation mediated by Escherichia coli immunoglobulin-binding proteins EibD and EibG depends on expression levels and localization of proteins

Chain formation mediated by Escherichia coli immunoglobulin-binding proteins EibD and EibG depends on expression levels and localization of proteins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chain formation mediated by Escherichia coli immunoglobulin-binding proteins EibD and EibG depends on expression levels and localization of proteins Lena-Sophie Swiatek, Katharina Schaufler, Jack C. Leo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6781270/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Escherichia coli expresses immunoglobulin-binding proteins (Eib), a subgroup of trimeric autotransporter adhesins (TAA). Subtypes of Eib proteins mediate unique chain-like adherence patterns and autoaggregation. This study investigates the mechanisms underlying chain formation by EibG and EibD; a chain-forming phenotype of the latter has not been previously described. Using constitutive expression systems, we demonstrate that low-level expression of EibG and EibD lead primarily to chain formation, whereas higher expression levels predominantly result in clump formation. Notably, chain and clump formation are not mutually exclusive and can occur simultaneously. Selective deletion of the full head domain, but not the N-terminal domain alone, abolished chain formation, highlighting its critical role. Fluorescence microscopy of mixed cultures showed that chains form through homotypic protein-protein interactions. Investigation revealed EibD and EibG were predominantly localized at cell poles, corresponding to sites of intercellular contact. Functional investigations showed that chain-forming strains exhibited enhanced adhesion to plastic surfaces, a key step in biofilm formation, without affecting autoaggregation. These showed Eib-mediated chain formation depends on protein expression levels, domain architecture, and localization, contributing to bacterial adhesion and potentially pathogenicity. Understanding interactions provides insights into TAA-mediated chain formation and autoaggregation. Biological sciences/Biochemistry/Proteins Biological sciences/Biological techniques/Imaging/Fluorescence imaging Biological sciences/Biological techniques/Genetic engineering Biological sciences/Biological techniques/Microbiology techniques Biological sciences/Biological techniques/Microscopy Biological sciences/Biological techniques/Microscopy/Confocal microscopy Biological sciences/Biological techniques/Microscopy/Phase contrast microscopy Biological sciences/Biological techniques/Software Biological sciences/Biological techniques/Structure determination/Molecular modelling Biological sciences/Genetics/Mutation Biological sciences/Genetics/Prokaryote Biological sciences/Microbiology/Bacteria/Bacterial pathogenesis Biological sciences/Microbiology/Bacteria/Bacterial structural biology Biological sciences/Microbiology/Biofilms Biological sciences/Microbiology/Pathogens Biological sciences/Structural biology/Molecular modelling Biological sciences/Biochemistry Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Structural biology Adhesion Autoaggregation Cellular localization Chain formation E. coli immunoglobulin-binding protein Trimeric autotransporter adhesins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The species Escherichia coli , comprising both commensal and pathogenic representatives, is well studied. However, especially multi-drug resistant isolates are a major public health threat[ 1 ]. It is a versatile organism and pathogen reflected by a multitude of proteins presented on its cell surface[ 2 ]. This includes adhesins, pili, and fimbriae, which mediate attachment to host tissues and surfaces and play a critical role in colonization and infection[ 3 , 4 ]. Once adhered, E. coli can form biofilms that enhance resilience to environmental stressors, immune responses, and antibiotics[ 5 ]. By that, they contribute to the persistence and pathogenicity of E. coli in host organisms and various environments[ 3 , 5 ]. Different highly specialized protein secretion systems exist in Gram-negative bacteria that ensure protein transport across the inner and outer membranes[ 6 ]. Proteins belonging to the type 5 secretion system utilize the Sec translocation machinery for crossing the inner membrane[ 7 ]. These proteins are characterized by a β-barrel domain that inserts into the outer membrane[ 8 , 9 ]. No cytosolic energy sources for translocation across the outer membrane is needed resulting in the term autotransporter[ 10 , 11 ]. Trimeric autotransporter adhesins (TAAs, Type 5c secretion systems) are a group of obligate homotrimeric proteins that mediate their own transport across the outer membrane, a process that likely involves the βbarrel assembly machinery (BAM) but remains poorly understood[ 11 – 14 ]. These proteins are recognized as key virulence factors in Gram-negative bacteria, contributing to diverse functions, including immune evasion, serum resistance, host cell adhesion and invasion, and biofilm formation[ 15 ]. TAAs consist of a Nterminal signal peptide enabling Sec-dependent inner membrane translocation and their outer membrane translocation pore is formed by conserved 12-stranded β-barrel domains with each protomer contributing four of the stands[ 8 , 16 – 18 ]. The extracellular region or passenger contains one or more right- and/or left-handed coiled-coiled regions, as well as globular domains forming ‘heads’. Some TAAs, such as the prototypical Yersinia adhesin YadA, have a single head atop a coiled-coil stalk and thus resemble a lollipop[ 19 ]. However, other TAAs are more complex, with a number of heads interspersed with stretches of coiled coil[ 20 ]. In addition, smaller minidomians such as the saddle or an insertion of a 3-stranded β-meander into a coiled-coil segment (FGG motifs) may be present.[ 21 , 22 ]. One subgroup of TAAs comprises the immunoglobulin (Ig)-binding proteins (Eib) found in E. coli , including several distinct proteins[ 23 , 24 ]. These proteins mediate non-immune binding to IgG and IgA Fc regions[ 25 ]. They are prevalent in Shiga toxin-producing E. coli (STEC) but are also found in commensal strains, suggesting a potential role in intestinal adhesion[ 26 ]. Among these, EibG has been described as mediating a unique chain-like adherence pattern (CLAP) on both human and bovine cells[ 27 ]. This observation indicates that chain formation by EibG is not host-specific and likely represents a bacteria-to-bacteria adhesion mechanism that also occurs in liquid culture[ 26 , 27 ]. Eib proteins are known to induce autoaggregation, which manifests as clump formation under specific conditions[ 21 , 28 ]. Similarly to other multifunctional proteins like YadA, which binds to collagen and other extracellular matrix components and mediates serum resistance, EibG may have additional roles beyond adhesion. Despite its significance, little is known about the underlying mechanism by which EibG facilitates chain formation. Here, we investigated how EibG mediates chain formation. Our results demonstrate that EibG and also EibD can mediate chain formation under the certain conditions. The chain formation depends on homotypic interactions of Eib proteins enriched at the cell contact sites and also on the expression levels of the protein. Understanding this mechanism in detail could provide insights into the broader biological functions of TAAs and their role in bacterial. 2. Results Expression levels influence chain formation While auto-aggregation and clump formation is a known phenomenon mediated by Eib proteins[ 21 , 28 ], previous studies indicated a CLAP for cells expressing EibG while adhering to human cells[ 27 ]. However, we found that Eib proteins, including EibG, formed clumps when expressed at high levels using a strong inducible promoter ( Supplementary Fig. 1 ). To evaluate the influence of expression levels on the chain formation we cloned different eib variants into a plasmid with a medium-low strength promotor for constitutive expression and transformed E. coli TOP10 cells with respective constructs. Using this system, we were able to show that chain formation is observed when cells expressed EibG and EibD at low levels (Fig. 1 ). By contrast, EibA (with an EibD signal peptide) expression in this system did not lead to chain formation. For EibD and EibG, both clump and chain formation were often observed simultaneously and were not mutually exclusive. Image analysis demonstrated that chain formation is reflected in an increase of particle size but decrease in circularity compared to control cells containing the empty vector and EibA expressing cells, which form small clumps. Cells expressing YadA, the well-studied Yersinia ssp. autotransporter protein[ 23 ], only showed clump formation at the analyzed conditions. Deletion of neck-domain but not N-terminal domain depletes the chain forming phenotype A crystal structure encompassing the YadA-like left-handed parallel-beta roll domain (LPBR) and the LPBR and coiled coil stalk of EibD has been previously solved[ 21 , 29 ]. However, no structural information has been available for the N-terminal domain (NTD) of this protein. Therefore, we used AlphaFold2[ 30 , 31 ] to model the full trimers of EibD and EibG, as well as EibA and YadA (Fig. 2 ). The Eib proteins share highly similar structures at the C-terminus, which includes the β-barrel membrane anchor, a left-handed coiled-coil region and the saddle minidomain. In EibD and EibG, the N-terminal half of the stalk is a right-handed coiled coil, whereas in EibA, this is left-handed. EibD and EibG share a similar bipartite head region encompassing the NTD with a novel fold and the LBPR. EibA lacks a LPBR, having only the NTD. The NTD of these three proteins has a triangular core with two α-helices packing against an antiparallel beta-sheet ( Supplementary Fig. 2 ). In addition, a β-hairpin packs against the longer α-helix to make the 'tip' of the upside-down triangle. The NTDs of the three Eib proteins have some differences, particularly in the way they connect to the LPBR domain, or stalk in the case of EibA ( Supplementary Fig. 2 ). The NTDs have limited structural similarity to the plectin repeats of periplakin, a eukaryotic cytoskeletal linker protein[ 32 ]. EibG and EibD are relatively similar in the region of the LPBR domain, but EibA lacks this domain. We therefore evaluated chain formation on cells constructively expressing EibG with deletion of the LPBR. This revealed that the deletion of the EibG LPBR domain does not prevent chain formation (Fig. 1 ). Although EibG and EibD both have the potential of chain formation, chain formation for EibG was more pronounced with longer chains. We originally used an EibD variant with a PelB signal peptide, often used for recombinant protein production, as this worked well in induced overproduction conditions[ 21 , 33 ]. However, under lower expression conditions, we noticed that with the PelB signal, almost no EibD protein was seen in the outer membrane ( Supplementary Fig. 3 ). When we re-introduced the native signal peptide, EibD was expressed well. The reasons for this are unclear, but this demonstrates that the signal peptide plays an important role in trimeric autotransport. To identify domains that influence chain formation we introduced domain deletions in EibG and domain swaps between EibG and EibD proteins and validated protein production and trimerisation in the outer membranes ( Supplementary Fig. 3 ). This revealed that deletion or alteration of the NTD does not inhibit chain formation. However, when additionally deleting the LPBR and neck or the whole passenger, the bacteria formed clumps or remained as individual cells but no chains were observed. After quantifying the microscopy images, this was reflected in decreased size of the particles with slight increase in circularity. Similar observations were made when exchanging the domains between EibG and EibD. Clump formation resulted in a high variability in particle size and a relatively constant circularity (Fig. 1 ). Chain forming phenotype is induced by homotypic protein-protein interaction predominantly at the cell poles We were interested in whether interaction of the Eib proteins with themselves or with other surface structures leads to chain formation. Therefore, we set up mixed cultures with an E. coli reporter strain expressing mNeonGreen but no TAA and the Eib-expressing strains and performed microscopy (Fig. 3 a). In the mixed culture containing the empty vector control, individual cells were observed only. In contrast, chain formation was again detected in the EibG and EibD cultures. These chains consisted exclusively of cells lacking the mNeonGreen signal, while individual mNeonGreen-positive cells were found dispersed around them. In the next step, we evaluated whether heterotypic chains or clumps formed in a mixed culture of cells expressing mNeonGreen and EibG or mCherry and EibD, respectively. Upon fluorescence-microscopic evaluation, it became evident that chains are exclusively formed through homotypic interactions, as non-TAA expressing cells did not participate in chain formation (Fig. 3 b). EibD and EibG-expressing cells did not appear to interact, confirming that the interactions are homotypic and specific to each TAA. However, the additional expression of the mCherry reporter in EibD-expressing cells enhanced the clump forming phenotype. Sporadically, EibG chains were found within the EibD clumps, though we consider this to be an unspecific interaction. Moreover, we were interested in whether the chain-forming phenotype is induced by polar localization of the respective Eib proteins. For that, we used EibD and EibG variants with a SpyTag[ 34 ] located at the Nterminus upon cleavage of the signal peptide. Detection was ensured by incubation with a previously purified SpyCatcher-sfGFP protein which forms a covalent bound with the SpyTag[ 35 ]. Evaluation of the fluorescent signal from confocal fluorescence microscopy revealed that EibD and EibG proteins could be found around the whole cell. However, increased signals were detected at the contact points of the individual cells within a chain (Fig. 4 ). Quantification of these signals revealed an increased signal intensity for the cell-to-cell contacts compared to the whole chains, being significantly higher for the majority of chains. We also investigated the localization in clumps. The high cellular densities within clumps make precise detection and quantification difficult. However, Nno distinct localization was observed. Both Eib variants were detected around the cells in clumps with some individual cells showing enhanced signals, most likely due to variable accessibility of the fluorescent lable within the clumps ( Supplementary Fig. 4 ). In order to check for specificity of the signal, the Eib-SpyTag variants were incubated with a SpyCatcher-EQ-sfGFP variant, which prevents formation of a covalent bound with the SpyTag. SpyCatcher-EQ-sfGFP resulted in diffuse, low-intensity non-specific signals ( Supplementary Fig. 5 ). Based on these findings, we propose the following model for chain formation in Eib-expressing cells (Fig. 5 ): at low expression levels, proteins are preferentially exposed at the cell poles. The high density at the poles leads to homotypic interactions that mediate chain formation. Some spillover from the poles may lead to low levels of protein along the lateral side of the cell, but because of the presumed low affinity of the homotypic interactions, the low protein density here would not support cooperative binding and consequently cells only attach at the poles. At higher expression levels the proteins are more evenly distributed and cooperative interactions can take place at the lateral sides as well, which leads to clump formation. This is indeed what we observe in our current setup, where chain formation and clump formation coexist. We do see some fluorescent signal on the lateral sides of cells in chains, but an enrichment at cell-cell contact sites, which is consistent with this model. Future work will aim to uncover how Eibs are preferentially targeted to the cell poles. Chain formation does not impact auto-aggregation but adhesion TAAs are known to promote auto-aggregation among other virulence-associated phenotypes like biofilm formation[ 28 ]. We tested auto-aggregation of chain-forming bacteria using sedimentation assays (Fig. 6 ). All wild-type strains demonstrated auto-aggregative phenotypes in contrast to the empty-vector control. None of the domain deletions and domain exchange mutants showed altered auto-aggregation behavior, except for the deletion of the almost the entire passenger domain (EibG∆saddle). In contrast to auto-aggregation, an alteration in chain formation severely affected the adhesive phenotype on plastic surfaces, which is recognized as the crucial initial step of biofilm formation. For all strains that did not show chain formation, namely cells expressing, EibA EibG∆neck, and EibG∆saddle, the intensity of crystal violet staining was equal to the control or significantly reduced compared to EibG wild-type expressing strains. This was also true for cells with low-level YadA expression, although higher levels of YadA have been shown to promote biofilm formation[ 28 ]. For EibA, weak biofilm formation has been demonstrated before[ 28 ]. By contrast, strains with the chain-forming phenotype showed increased adhesion. For the NTD deletion mutant a slight decrease in adhesion was observed consistent with the reduction in chain sizes. When comparing the mutants with domain swaps between EibD and EibG, the highest levels of adhesion became evident for the chain-forming EibG variant with the EibD N-terminal domain (EibD-NTD-EibG). When additionally exchanging the head and neck domains or the whole passenger a decrease in staining intensity was observed for the clump-forming mutants compared to the wild-type and the chain-forming mutant (EibD-NTD-EibG). Interestingly, while there was a significant difference in adhesive phenotypes between the chain-forming and chain-deficient strains, no notable variation was observed among the long-term macrocolonies of the EibG wild type and its respective mutant strains. Notably, distinct biofilm phenotypes were observed among the different Eib wild type variants ( Supplementary Fig. 6 ). 3. Discussion This study highlights the critical influence of expression levels, and structural domains on the aggregation behavior of Eib proteins in E. coli . The findings offer new insights into the mechanisms driving chain formation and adhesive phenotypes, while also suggesting the predominant localization of EibD and EibG at cell-cell contacts, particularly at the cell poles, contribute to these processes. The results demonstrated that lower expression levels of EibG and EibD promote chain formation, a phenotype absent in EibA. Note that despite clear chain formation clumps were also still observed for both. Static growth, microaerophilic conditions, and alkaline pH induce EibG expression in STEC while agitation and decreased temperature is repressive[ 36 ]. Under laboratory conditions, Eib proteins were highly stable, while protein levels differed significantly among different strains[ 37 ]. Previous studies described a CLAP for EibG and EibF-expressing strains when adhering to human or bovine cells only while a eibABCDE -postive strain does not show a CLAP[ 27 ]. This could be explained by the presence of EibA or higher expression levels in general leading to clumping that masks any potential CLAP. Here we demonstrated that chain formation is not only induced by EibG and EibF but also EibD when expressed at moderate levels. The chain formation phenomenon does not require adhesion to cells but occurs in liquid cultures too. The absence of chain formation by EibA suggests that while these proteins share common features, functional differences arise due to sequence and structural variations. Differences became particularly evident in the LPBR. Although EibA lacks the LPBR region, deletion of this domain in EibG and EibD did not inhibit chain formation. Deletion of the LPBR neither affected adhesion nor autoaggregation. These findings do not align with earlier reports that the LPBR region of TAAs facilitates homotypic interactions critical for aggregation[ 29 ]. Similarly, deletion or exchange of the NTD did not prevent chain formation, resulting in similar adhesion capacities as seen in wild-type EibG expressing cells. By contrast, deletion of the head domain, consisting of the NTD, the LPBR and the neck domain, resulted in disrupted chain formation, and significantly impaired adhesion. Different eibG allelic variants exist that show phenotypes differing in length and sequence variation within the LBPR and neck domains[ 26 ]. Together, these observations suggest that the domains of the EibG head contribute to autoaggregation and chain formation, so that only when the entire head is deleted these processes are affected. We observed that chain formation, rather than clump formation, is a key determinant of adhesion in these systems, while macrocolony morphologies did not differ among the EibG wild type and its mutants. Adhesion is the initial step in host colonization, which in turn is a prerequisite for causing severe pathologies[ 38 ]. By shedding light on the molecular determinants of chain formation, a growing body of knowledge may inform the development of strategies to prevent adhesion of pathogenic bacteria as demonstrated for FimH[ 39 , 40 ]. In line with previous reports, no chain or biofilm formation was observed for EibA, which has instead been implicated in immunoglobulin binding and serum resistance[ 41 ]. In contrast, autoaggregation is not affected by alteration of the chain-forming phenotype and occurs independently of clump or chain formation. We demonstrated that chain formation exclusively occurs between cells expressing EibG or EibD and not among a mixed population of cells expressing no or different Eib variants. Moreover, we hypothesized that the polar localization is essential for the chain forming phenotype. Polar localization of autotransporters is a known process in E. coli that has been extensively studied, e.g. for the type 5a autotransporters IcsA and Ag43 but applies to other membrane proteins as well[ 42 – 45 ]. These studies suggested that outer membrane proteins are targeted to the pole in the cytoplasm before secretion[ 46 , 47 ] with multiple factors such as protein sequence determinants, lipopolysaccharide integrity, expression levels and the activity of key proteins, e.g. YidC, DnaK or FtsQ being involved[ 42 , 45 – 48 ]. However, many of these localization studies were performed in the absence of signal peptides, so the validity of this aggregation/disaggregation model is not fully confirmed. Omitting the signal peptide could easily lead to the formation of intracellular inclusion bodies, which may or may not be representative of the native process when a signal peptide is included. This study provides evidence that the signal peptide plays a role in determining the type of aggregation through presumably altered surface expression. When EibD was expressed with a PelB signal peptide protein levels detected in the outer membrane were severely impaired, as was the chain formation. This is consistent with the function of signal peptides enabling Sec-dependent translocation of TAA into the periplasm[ 12 ]. The results of this study indicate that low-level expression of Eib proteins leads to their initial predominant localization at the cell poles, which later serve as contact points for the initiation of chain formation. The mechanism behind this still requires further investigation. Furthermore, we identified that complex structural features of the Eib proteins are required as prerequisites for mediating the type of intercellular interaction that enables chain formation. The interplay between structural domains and expression levels in regulating bacterial adhesion and biofilm formation has important implications for understanding the pathogenic potential of STEC and related strains. 4. Methods 4.1. Bacterial strains and cultivation The bacterial strains used in the study are listed in Table 1 . E. coli TOP10 cells were used as host cells for constitute expression of all protein variants with the pASK-IBA2con backbone and a chloramphenicol resistance cassette ( Supplementary Fig. 1 ). Bacterial strains were cultivated in LB-Lennox medium or auto-induction medium (AIM) containing 25 µg/mL chloramphenicol, 15 µg/mL tetracycline or 100 µg/mL ampicillin at 37°C. For cloning and protein expression, strains were cultivated shaking at 150 rpm. Whenever chain formation was induced, shaking was reduced to 30–50 rpm. Table 1 Bacterial strains . Strain names, the host with the obtained plasmids, as well as a short description and reference are listed. Deleted amino acids are shown in square brackets. Control strain was obtained from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA. *The pBAD-EibD/EibG were constructed based on pBAD/HIsA from Invitrogen Strain name Host Plasmid Description Reference Ctrl E. coli TOP10 pASK-IBA2con Control strain Invitrogen EibA E. coli TOP10 pASK-IBA2con_ eibA Constitutive expression of EibA This study EibD E. coli TOP10 pASK-IBA2con_ eibD Constitutive expression of EibD This study EibG E. coli TOP10 pASK-IBA2con_ eibG Constitutive expression of EibG This study EibG∆NTD E. coli TOP10 pASK-IBA2con_ eibG ∆n.term Constitutive expression of EibG with deleted N-terminal domain (NTD) [∆Q28-L173] This study EibG∆neck E. coli TOP10 pASK-IBA2con_ eibG ∆neck Constitutive expression of EibG with deletion up to and including the neck domain [∆Q28-V318] This study EibG∆saddle E. coli TOP10 pASK-IBA2con_ eibG ∆saddle Constitutive expression of EibG with deletion up to and including the saddle domain [∆Q28-A375] This study EibG∆LPBR E. coli TOP10 pASK-IBA2con_ eibG ∆LPBR Constitutive expression of EibG with deleted LPBR domain [∆V174-Y287] This study EibD-NTD-EibG E. coli TOP10 pASK-IBA2con_ eibD - n.term_ eibG Constitutive expression of EibG variant encoding the EibD N-terminal domain This study EibD-neck-EibG E. coli TOP10 pASK-IBA2con_ eibD - neck_ eibG Constitutive expression of EibG variant encoding the EibD neck domain This study EibD-pass-EibG E. coli TOP10 pASK-IBA2con_ eibD - pass_ eibG Constitutive expression of EibG variant encoding the EibD passenger domain This study YadA E. coli TOP10 pASK-IBA2con_ yadA Constitutive expression of YadA This study Ctrl-GFP E. coli TOP10 pACYC184-mNeonGreen Assessing mixed chain formation This study EibG-GFP E. coli TOP10 pACYC184-mNeonGreen Assessing mixed chain formation This study EibD-mCherry E. coli TOP10 pACYC184-mCherry Assessing mixed chain formation This study EibD-induced BL21∆ABCF pBAD Induction for determining surface expression This study* EibG-induced BL21∆ABCF pBAD Induction for determining surface expression This study* EibG-SpyTag E. coli TOP10 pASK-IBA2con_ eibG -SpyTag EibG variant with a 5’ Spy-Tag This study EibD-SpyTag E. coli TOP10 pASK-IBA2con_ eibD -SpyTag EibD variant with a 5’ Spy-Tag This study 4.2. Cloning and mutagenesis For inducible production of EibG and EibD, the coding sequences for the proteins were amplified from pGEMEBG[ 27 ] and pETDuetS-EibD[ 33 ], respectively. These were cloned into the arabinose-inducible vector pBAD/HisA (Invitrogen). To produce a plasmid giving low-level constitutive expression, we replaced the tet promoter of pASK-IBA2C (IBA Lifesciences GmbH, Goettingen, Germany) with the constitutive promoter J23105 ( https://parts.igem.org/Part:BBa_J23105 ) by site-directed mutagenesis[ 49 ]. eibD and eibG were subcloned into this vector, as were eibA (with an EibD signal peptide, from pETDues-EibA[ 33 ]) and YadA (from Yersinia enterocolitica O:3, amplified from genomic DNA of strain 6471/76[ 50 ]). For fluorescent labelling, we used either mNeonGreen or mCherry, the coding sequences for which were inserted into the plasmid pACYC184[ 51 ]. The SpyTag[ 35 ] sequence was introduced using mutagenesis primers to insert the tag into the pASK-IBA2C-eibG and pASK-IBA2C-eibD vectors, respectively, at the 5′ end of the genes to produce N-terminally tagged proteins. All cloning procedures were performed with Gibson assembly[ 52 ] using the 2 x HiFi Master Mix (New England Biolabs, Ipswich, UK) and VeriFi™ Mix Red (PCR Biosystems Ltd., London, UK) for amplification of fragments or Q5 polymerase (New England Biolabs, Ipswich, UK) for site-direct mutagenesis. All primers are listed ( Supplementary table 1 ). Domain deletions were performed based on a previously published protocol[ 49 ]. The annotation of domains was performed based on AlphaFold[ 53 ] ( Supplementary Fig. 1 ). Chemically competent E. coli TOP10 cells[ 54 ] were transformed with the respective cloning products. Clones were validated by colony PCR using PCRBIO Taq Mix Red (PCR Biosystems Ltd) and Sanger sequencing (Source Bioscience Genomics, Cambridge, UK). 4.3. Bright field and epifluorescence microscopy Chain formation was evaluated using bright field microscopy. Overnight cultures were diluted (1:50) in 10 mL of fresh medium and incubated at 37°C with shaking at 30 rpm for at least 5 h. After incubation, 1 mL of the culture was gently transferred to a 2 mL microcentrifuge tube using a 1000 µL pipette tip with the tip cut to minimize shear stress on the bacteria. The sample was allowed to sediment for 30 minutes at room temperature. During the sedimentation period, microscopy slides were prepared using 1 mL of 1% agarose in PBS on each slide, allowing it to solidify. Following sedimentation, the bacteria were resuspended by gently pipetting up and down three times with the cut tip. A small drop of the resuspended bacteria was transferred to a prepared microscopy slide. Microscopy was performed using a Nikon Eclipse epifluorescence microscope (Nikon, Amstelveen, The Netherlands) at 1000x magnification with immersion oil. For potential formation of mixed chains was assessed by using TOP10 cells either expressing EibG and mNeonGreen or EibD and mCherry, or TAA-expressing cells (non-fluorescent) with TOP10 cells expressing mNeonGreen. mNeonGreen and mCherry were introduce on a pACYC184-based plasmid, either with chloramphenicol resistance or tetracycline resistance. The samples were prepared with minor modifications. Individual overnight cultures of the described strains (with chloramphenicol or chloramphenicol and tetracycline for mNeonGreen/EibG and mCherry/EibD strains) were equally diluted in fresh medium (OD 600 0.05) and incubated at 37°C with shaking at 30 rpm for at least 5 h. Bright field and epifluorescence mode were used simultaneously to identify (i) Eib and NeonGreen (Eib-negative) expressing cells respectively or (ii) EibG and mNeonGreen or EibD and mCherry expressing cells. Overlay of images was ensured using microscopic software (NIS-Elements, Nikon v.5.11.01). 4.4. Quantification of clump and chain formation For quantification of clump and chain formation compared to individual cells we used the Fiji software[ 55 ]. For that, microscopic images were exported as TIF files and imported into Fiji. Ten semi-randomly taken microscopic images were used from four biological replicates each. If multiple color channels exist, they were reduced to one and background removal was performed. The threshold was adjusted and the particles were analyzed in default mode to identify the cells. Shape descriptions were measured as the read out analyzing the individual particles (Size: 0-Infinity; Circularity: 0.00–1.00). 4.5. Expression and Purification of SpyCatcher-sfGFP and SpyCatcher-EQ-sfGFP SpyCatcher proteins were produced as described before with minor modifications[ 35 ]. In brief, E. coli BL21Gold(DE3) cells harboring either pET22-SpyCatcher-sfGFP or pET22-SpyCatcherEQ-sfGFP plasmids were inoculated separately into 5 mL AIM supplemented with 100 µg/mL ampicillin in the morning and grown at 37°C for several hours. The cultures were then transferred into 1 L of AIM containing 100 µg/mL ampicillin and antifoam for incubation overnight at 37°C in a LEX10 bioreactor (Epiphyte3, Toronto, Ontario, Canada). The following day, cells were harvested by centrifugation (15 min at 5000 x g ) and resuspended in 30 mL PBS, then stored at − 20°C until purification. Frozen cell suspensions were thawed and treated with 0.1 mg/mL of lysozyme, a pinch of DNase I, 1 mM MgCl₂, and 1 mM MnCl₂. Bacterial cells were lysed using a probe sonicator for six 30-second cycles. The lysate was clarified by centrifugation at 30,000 x g for 60 minutes. The supernatant was applied to a Ni-NTA affinity column connected to an FPLC unit. Proteins were eluted using an imidazole gradient (10 mM – 500 mM). Fractions were collected, and 40 µL aliquots from each were prepared for SDS-PAGE as described above. 4.6. Evaluation of protein localization using SpyCatcher and confocal microscopy The EibG-SpyTag and EibG-SpyTag expressing-strains were grown overnight, diluted in the morning and incubated at 37°C with shaking at 50 rpm. After 4 h of growth, SpyCatcher-sfGFP or SpyCatcher-sfGFP-EQ was added to the culture to a final concentration of 0.02 mg/mL. The culture was gently swirled to ensure proper mixing and then incubated for an additional 1.5 h at 37°C with shaking reduced to 30 rpm. After the incubation period, the bacterial clumps were allowed to settle for a few minutes without shaking. A 400 µL sample from the bottom of the tube was transferred to a 2 mL tube and carefully resuspended, using a pipette with the tip cut off. To wash the cells, 400 µL PBS was added and the mixture was centrifuged at 1000 x g for 3 min. The supernatant was discarded, and the bacterial pellet was gently resuspended in 800 µL PBS with minimal pipetting, followed by gentle swirling and flicking of the tube to remove the bacteria from the bottom. The washing step was repeated. Afterwards, the bacterial pellet was gently resuspended in 200 µL of PBS and a 6 µL aliquot of the resuspended sample was transferred to an agarose pad using a pipette with a cut-end tip for imaging using Nikon AXR with NSPARC. To analyze the fluorescence intensity at the bacterial cell-to-cell contact points, images were processed using Fiji software[ 55 ]. A single bacterial chain was first selected using the selection tool. This selection was duplicated to preserve the original image and facilitate further analysis. A maximum intensity projection of the Z-stack was created. The fluorescence image was subsequently split into individual color channels. The area corresponding to cell-to-cell contact was identified within the bright field image using the freehand selection tool. The selection was transferred to the fluorescence channel. The selected region of interest (ROI) was added to the ROI manager and mean fluorescence intensity within this ROI was measured. This was repeated for all cell-to-cell contacts within the chain and then for the whole chain as well. The data were then exported. To evaluate differences in fluorescence intensity, statistical analysis was performed using an R-script to generate plots and conduct a paired t-test. This test compared the fluorescence signal across the entire bacterial chain with the mean fluorescence intensity at the cell-to-cell contact points to determine if significant differences existed. 4.7. Sedimentation Overnight cultures (10 mL) were inoculated from single cultures and grown for 16 h overnight at 37°C shaking at 30 rpm. The following day, the cultures were carefully inverted to ensure uniform mixing and transferred into long glass tubes. The OD 600 was measured every 15–30 min by taking 200 µL from the top pf the cultures using the plate reader (SPECTROstar Nano; BMG Labtech, Ortenberg, Germany). The cultures were incubated at 37°C statically in-between. Reduction in OD 600 relative to the initial OD was measured as degree of sedimentation. 4.8. Crystal violet assay For analyzing biofilm formation, the OD 600 of overnight cultures was adjusted to 0.2 and 100 µL of OD-adjusted culture was inoculated into four wells of a Nunc™ MicroWell™ 96-Well-mikrotiter plate (ThermoFisher Scientific, Waltham, MA, USA). As negative control, the respective medium alone was included. Plates were sealed with PARAFILM®, and incubated statically at 37°C for 48 h. Afterwards, the culture was carefully aspirated and the adhered biofilm was washed three times with 200 µL of PBS to remove non-adherent cells. The plates were then incubated at 60°C for at least 60 minutes to fix the biofilm. The adhered biofilm was stained by adding 150 µL of 1% (w/v) crystal violet (Sigma Aldrich; Merck, Darmstadt, Germany) for 20 min. The plates were rinsed three times with distilled water and left inverted to dry. To solubilize the stained biofilm, 150 µL of 33% (v/v) glacial acetic acid (Scientific Laboratory Supplies, Nottingham, UK) was added per well. After complete solubilization, the solution was transferred to a new plate. The OD at 540 nm (OD 540 ) was measured (SPECTROstar Nano; BMG Labtech). 4.9. Software and statistics For all cloning approaches, we used SnapGene Viewer (GSL Biotech LLC v7.1.2.0) in combination with ApE-A plasmid Editor (v3.1.6) and Oligo Calculator (v3.27)[ 56 ]. Evaluation of microscopy was done with Nikon NIS (v5.11) together with Fiji (ImageJ Version 2.35). NIS Element (v6.10.01). Data evaluation and visualization was performed using GraphPad Prism (v10.2.1) and RStudio (v2023.06.2 + 561). Paired t-test or One-way ANOVA with Dunnett’s post-hoc test was used for multiple comparisons when applicable. We used large language models, such as ChatGPT (GPT-4), to assist in revising portions of the manuscript. All AI-generated content was thoroughly reviewed, edited, and approved by the authors to ensure its accuracy and integrity. Abbreviations TAAs Trimeric autotransporter adhesins CLAP Cchain-like adherence pattern GFP Green fluorescent protein Eib Immunoglobulin-binding proteins STEC Shiga toxin-producing E. coli OD 600 optical density at 600 nm OD 540 optical density at 540 nm SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis LPBR left-handed parallel beta-roll Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Data availability statement Raw data microscopic images and metadata are stored at FigShare (DOI: 10.6084/m9.figshare.29166725). Gene and plasmid sequences are publically available and can be found as: eibA (AF151091.1), eibD (AAF63040.1), eibG (ADJ17717.1), yadA (WP_032488477.1) pASK-IBA2con (2-1321-000; IBA Lifesciences GmbH, Goettingen, Germany). The tet promoter of pASK-IBA2C was replaced with the constitutive promoter J23105 (https://parts.igem.org/Part:BBa_J23105) by site-directed mutagenesis. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the German Federal Ministry of Education and Research (BMBF) within the “ DISPATch _MRGN-Disarming pathogens as a different strategy to fight antimicrobial-resistant Gram-negatives” project (grant 01KI2410), and internal funding from Nottingham Trent University. International collaboration was supported by the Erasmus+ program. Authors' contributions Conceptualization, J.L.; methodology, J.L., and L.S.; software, J.L., and L.S.; validation, J.L., K.S., and L.S.; formal analysis, J.L. and L.S.; investigation, J.L., and L.S.; resources, J.L., and K.S.; data curation, J.L., and L.S..; writing-original draft preparation, L.S.; writing-review and editing, J.L., K.S., and L.S.; visualization, J.L. and L.S.; supervision, J.L., and L.S.; project administration, J.L.; funding acquisition, J.L., K.S., and L.S.. All authors have read and agreed to the published version of the manuscript. Acknowledgments We thank Jonas Øgaard for his assistance with the image analysis. We also thank Dr. Graham Hickman associated with School of Science & Technology Imaging Suite for providing access to and for his support with the confocal microscopy facility. Our gratitude also goes to the members of the Antibiotic Resistance, Omics, and Microbiota Group for their support and valuable discussions. We would also like to thank Dr. Sunao Iyoda (National Institute for Infectious Diseases, Japan) for providing the eibG plasmid. Authors Information Lena-Sophie Swiatek [email protected] Katharina Schaufler [email protected] Jack C. Leo [email protected] References Geneva: World Health Organization. WHO Bacterial Priority Pathogens List, 2024: Bacterial Pathogens of Public Health Importance to Guide Research, Development and Strategies to Prevent and Control Antimicrobial Resistance.. (2024). Ageorges, V. et al. Molecular determinants of surface colonisation in diarrhoeagenic Escherichia coli (DEC): From bacterial adhesion to biofilm formation. FEMS Microbiol. Rev. 44 , 314–350 (2020). Kaper, J. B., Nataro, J. P. & Mobley, H. L. T. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2 , 123–140 (2004). Le Bouguénec, C. Adhesins and invasins of pathogenic Escherichia coli. Int. J. Med. Microbiol. 295 , 471–478 (2005). Sharma, G. et al. Escherichia coli biofilm: development and therapeutic strategies. J. Appl. Microbiol. 121 , 309–319 (2016). Christie, P. J. The Rich Tapestry of Bacterial Protein Translocation Systems Peter. Protein J. 38 , 389–408 (2019). Henderson, I. R., Navarro-Garcia, F., Desvaux, M. & Fernandez, R. C. Ala’Aldeen, D. Type V Protein Secretion Pathway: the Autotransporter Story. Microbiol. Mol. Biol. Rev. 68 , 692–744 (2004). Linke, D., Riess, T., Autenrieth, I. B., Lupas, A. & Kempf, V. A. J. Trimeric autotransporter adhesins: variable structure, common function. Trends Microbiol. 14 , 264–270 (2006). Clarke, K. R. et al. Phylogenetic Classification and Functional Review of Autotransporters. Front. Immunol. 13 , 1–20 (2022). Klauser, T., Pohlner, J. & Meyer, T. F. The secretion pathway of IgA protease-type proteins in gram‐negative bacteria. BioEssays 15 , 799–805 (1993). Leo, J. C., Grin, I. & Linke, D. Type V secretion: Mechanism(S) of autotransport through the bacterial outer membrane. Philos. Trans. R Soc. B Biol. Sci. 367 , 1088–1101 (2012). Kiessling, A. R., Malik, A. & Goldman, A. Recent advances in the understanding of trimeric autotransporter adhesins. Med. Microbiol. Immunol. 209 , 233–242 (2020). Sikdar, R. & Bernsteina, H. D. Sequential translocation of polypeptides across the bacterial outer membrane through the trimeric autotransporter pathway. MBio 10, (2019). Chauhan, N. et al. Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA). Mol. Microbiol. 111 , 844–862 (2019). Meuskens, I., Saragliadis, A., Leo, J. C. & Linke, D. Type V secretion systems: An overview of passenger domain functions. Front. Microbiol. 10 , 1–19 (2019). Szczesny, P. & Lupas, A. Domain annotation of trimeric autotransporter adhesins - daTAA. Bioinformatics 24 , 1251–1256 (2008). Meng, G., Surana, N. K., Geme, S., Waksman, G. & J. W. & Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter. EMBO J. 25 , 2297–2304 (2006). Shahid, S. A. et al. Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals. Nat. Methods . 9 , 1212–1217 (2012). Hoiczyk, E., Roggenkamp, A., Reichenbecher, M., Lupas, A. & Heesemann, J. Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. EMBO J. 19 , 5989–5999 (2000). Hartmann, M. D. et al. Complete fiber structures of complex trimeric autotransporter adhesins conserved in enterobacteria. Proc. Natl. Acad. Sci. U. S. A. 109, 20907–20912 (2012). Leo, J. C. et al. The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins. Structure 19 , 1021–1030 (2011). Bassler, J., Alvarez, H., Hartmann, B., Lupas & M. D. & A. N. A domain dictionary of trimeric autotransporter adhesins. Int. J. Med. Microbiol. 305 , 265–275 (2015). Sandt, C. H. & Hill, C. W. Nonimmune binding of human immunoglobulin A (IgA) and IgG Fc by distinct sequence segments of the EibF cell surface protein of Escherichia coli. Infect. Immun. 69 , 7293–7303 (2001). Sandt, C. H. & Hill, C. W. Four different genes responsible for nonimmune immunoglobulin-binding activities within a single strain of Escherichia coli. Infect. Immun. 68 , 2205–2214 (2000). Leo, J. C. & Goldman, A. The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc. Mol. Immunol. 46 , 1860–1866 (2009). Merkel, V. et al. Distribution and phylogeny of immunoglobulin-binding protein G in shiga toxin-producing Escherichia coli and its association with adherence phenotypes. Infect. Immun. 78 , 3625–3636 (2010). Lu, Y. et al. A new immunoglobulin-binding protein, EibG, is responsible for the chain-like adhesion phenotype of locus of enterocyte effacement-negative, shiga toxin-producing Escherichia coli. Infect. Immun. 74 , 5747–5755 (2006). Khalil, H. S., Øgaard, J. & Leo, J. C. Coaggregation properties of trimeric autotransporter adhesins. Microbiologyopen 9 , 1–22 (2020). Nummelin, H. et al. The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel β-roll. EMBO J. 23 , 701–711 (2004). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583–589 (2021). Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods . 19 , 679–682 (2022). Boczonadi, V. & Määttä, A. Functional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins. Methods in Enzymology vol. 569 (Elsevier Inc., 2016). Mikula, K. M. et al. The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation. J. Bacteriol. 194 , 827–838 (2012). Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. U. S. A. 109, (2012). Duodu, R., Linke, D. & Leo, J. C. Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System. Methods Mol. Biol. 2778 , 53–63 (2024). Kuczius, T. et al. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC). PLoS One . 10 , 1–16 (2015). Rubin, D., Zhang, W., Karch, H. & Kuczius, T. Distinct expression of immunoglobulin-binding proteins in shiga toxin-producing Escherichia coli implicates high protein stability and a characteristic phenotype. Toxins (Basel) 9 , (2017). Aprikian, P. et al. The bacterial fimbrial tip acts as a mechanical force sensor. PLoS Biol 9 , (2011). Mydock-Mcgrane, L. K., Cusumano, Z. T. & Janetka, J. W. Mannose-derived FimH antagonists: A promising anti-virulence therapeutic strategy for urinary tract infections and Crohns disease. Expert Opin. Ther. Pat. 26 , 175–197 (2016). Mydock-McGranea, L. K., Hannana, T. J. & Janetka, J. W. Rational Design Strategies for FimH Antagonists: New Drugs on the Horizon for Urinary Tract Infection and Crohn’s Disease. Expert Opin. Drug Discov . 12 , 711–731 (2017). Wells, T. J., Totsika, M. & Schembri, M. A. Autotransporters of Escherichia coli: A sequence-based characterization. Microbiology 156 , 2459–2469 (2010). Gray, A. N., Li, Z., Henderson-Frost, J. & Goldberg, M. B. Biogenesis of YidC cytoplasmic membrane substrates is required for positioning of autotransporter IcsA at future poles. J. Bacteriol. 196 , 624–632 (2014). Doyle, M. T., Grabowicz, M. & Morona, R. A small conserved motif supports polarity augmentation of Shigella flexneri IcsA. Microbiol. (United Kingdom) . 161 , 2087–2097 (2015). Brandon, L. D. et al. IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed. Mol. Microbiol. 50 , 45–60 (2003). Vejborg, R. M. & Klemm, P. Cellular chain formation in Escherichia coli biofilms. Microbiology 155 , 1407–1417 (2009). Jain, S. et al. Polar localization of the autotransporter family of large bacterial virulence proteins. J. Bacteriol. 188 , 4841–4850 (2006). Fixen, K. R. et al. Genetic reporter system for positioning of proteins at the bacterial pole. MBio 3, (2012). Janakiraman, A., Fixen, K. R., Gray, A. N., Niki, H. & Goldberg, M. B. A genome-scale proteomic screen identifies a role for DnaK in chaperoning of polar autotransporters in Shigella. J. Bacteriol. 191 , 6300–6311 (2009). Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8 , 91 (2008). Skurnik, M. Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis. J. Appl. Bacteriol. 56 , 355–363 (1984). Chang, A. C. Y. & Cohen, S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134 , 1141–1156 (1978). Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods . 6 , 343–345 (2009). Varadi, M. et al. AlphaF old Prot ein Structure D atabase in 2024. Nucleic Acids Res. 52 , D368–D375 (2024). Chung, C. T., Niemela, S. L. & Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U. S. A. 86, 2172–2175 (1989). Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods . 9 , 676–682 (2012). Kibbe, W. A. & OligoCalc An online oligonucleotide properties calculator. Nucleic Acids Res. 35 , 43–46 (2007). Additional Declarations No competing interests reported. Supplementary Files SupplementSwiateketalSciRep.docx Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Aug, 2025 Reviews received at journal 15 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 23 Jun, 2025 Reviewers agreed at journal 23 Jun, 2025 Reviewers invited by journal 21 Jun, 2025 Editor assigned by journal 21 Jun, 2025 Editor invited by journal 10 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 First submitted to journal 30 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6781270","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475334971,"identity":"f98ccee6-b414-4f2a-9ca5-472ab619ade7","order_by":0,"name":"Lena-Sophie Swiatek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABaElEQVRIie2SP2vCQBTA33GQLC9mDVT0ExROAmkLtv0qJ4FM6uIitNCAkC71A5QKfoVO0vHkIF1Cu2booHR1sHRRSLWXVqPYP3Oh+cHl7t693z3eEYCcnD9IYb1g9HNGNSi8+FAEC4hQO66lQcwU7auiASXXvlpYAEL8pmyFKDW2FdhR9O5wCkm1eaBTQebBSdHsd6R9fFfF/ZuOkNMEmgW9K2DSzhR8cC0SeK2jjsapEbhohZrrNiIPnaeQC4HQUjmc9KJMseps782XtVuJjJIBRQjRlo1AohPXmRDWshaoHHVbppQn9gySVDGnZD64wHJovsqjYLlSGKyUxaYKOhZoH1UAjIGqFSJ1SSBWCl8r/qaXunOY9sKkxqSxuMdK6NmVrmrKiT0uIqEUjNiwF64VU4/sOH0x9ijH41l0dlqS8tlST1dyYnc4aidQ619eVUaT892/gKcfsRvd8M0R/zk7Jycn59/zDn50hliM3SYZAAAAAElFTkSuQmCC","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research HZI","correspondingAuthor":true,"prefix":"","firstName":"Lena-Sophie","middleName":"","lastName":"Swiatek","suffix":""},{"id":475334972,"identity":"5dd6c66a-a9b3-4d93-91e3-8acb177bf833","order_by":1,"name":"Katharina Schaufler","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research HZI","correspondingAuthor":false,"prefix":"","firstName":"Katharina","middleName":"","lastName":"Schaufler","suffix":""},{"id":475334973,"identity":"bdc92a31-6735-4b02-90bf-e33782e0a7d9","order_by":2,"name":"Jack C. Leo","email":"","orcid":"","institution":"Nottingham Trent University","correspondingAuthor":false,"prefix":"","firstName":"Jack","middleName":"C.","lastName":"Leo","suffix":""}],"badges":[],"createdAt":"2025-05-30 05:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6781270/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6781270/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-22967-3","type":"published","date":"2025-10-09T15:58:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85688572,"identity":"114884be-d14f-4e23-8237-135ef712e873","added_by":"auto","created_at":"2025-06-30 16:26:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":901643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of chain vs. clump formation\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Bright field microcopy was performed after 16 h incubation (37 °C, 30 rpm) of the indicated cultures to differentiate between individual cells, clumps and chains. One mL of cells were taken from the bottom of the tube using a cut 1 mL tip, transferred to a 2 mL tube and then onto a prepare objective slide after gentle resuspension (3x). The scale bar in the bottom right corner indicates 10 µm. pIBA2Ccon is the empty vector. For an explanation of the various deletion constructs, refer to Supplementary Figure 2b. (\u003cstrong\u003eb\u003c/strong\u003e+\u003cstrong\u003ec\u003c/strong\u003e) Ten semi-randomly selected pictures were evaluated to obtain their size and circularity using Fiji (ImageJ) and the average was calculated. This was performed for four biological replicates each and the mean value was plotted. One-way ANOVA with Dunnett’s post-hoc test was used for multiple comparisons to the control group (\u003cem\u003ep\u003c/em\u003e-value: * 0.01 – 0.05; ** 0.001 – 0.01).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/045d4e0b53d9356ea57ae961.png"},{"id":85688976,"identity":"4e8034a1-ea2a-4e9a-84ae-3ae7c92b290e","added_by":"auto","created_at":"2025-06-30 16:34:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":508677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlphaFold2 models of TAAs used in this study\u003c/strong\u003e. The TAAs all display a lollipop-like architecture, with a C-terminal beta-barrel, a stalk and a head region. Structural features are indicated. NTD = N-terminal domain, LPBR = left-handed parallel beta-roll domain. The three chains of the trimers are colored differently. The figure was produced using PyMOL (Schroedinger).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/b7a1d1d6452ae163b3d965e4.png"},{"id":85688574,"identity":"922b6e14-bc3b-4ca7-b99b-9196d365e9bb","added_by":"auto","created_at":"2025-06-30 16:26:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":528838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessing mixed chain formation.\u003c/strong\u003e Mixed cultures were set up containing (\u003cstrong\u003eA\u003c/strong\u003e) mNeonGreen expressing TOP cells and either, pIBA2 (ctrl), EibD, EibG or EibD with a PelB signal peptide (EibD-PelB) expressing cells or (\u003cstrong\u003eB\u003c/strong\u003e) Cells expressing both mNeonGreen and EibG or mCherry and EibD. Combination of bright field and fluorescence microscopy was used to analyze mixed chain formation. The scale bar in the bottom right corner indicates 10 µm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/9d0955cf295da0c3cf1b3504.png"},{"id":85688573,"identity":"74969ec5-3deb-4745-af3d-256f6d0b27a2","added_by":"auto","created_at":"2025-06-30 16:26:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":578989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular localization of Eib proteins.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e+\u003cstrong\u003eB\u003c/strong\u003e) EibD and (\u003cstrong\u003eC\u003c/strong\u003e+\u003cstrong\u003eD\u003c/strong\u003e) EibG proteins, each with an N-terminal SpyTag, were expressed in E. coli TOP10 cells and labeled with previously purified SpyCatcher protein. (\u003cstrong\u003eA\u003c/strong\u003e+\u003cstrong\u003eC\u003c/strong\u003e) Localization of the proteins was assessed by quantifying the fluorescence signal at cell-to-cell contacts compared to the signal along the entire bacterial chain. Significant differences were evaluated by performing a paired t-test (\u003cem\u003ep-\u003c/em\u003evalues are indicated). Representative microscopic images of quantified chains are shown for (\u003cstrong\u003eB\u003c/strong\u003e) EibD- and (\u003cstrong\u003eD\u003c/strong\u003e) EibG-expressing cells, with cell-to-cell contacts exhibiting pronounced fluorescence indicated by arrows. The scale bar in the bottom right corner indicates 5 µm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/fa28f3c0ff25f3b4a56fdde1.png"},{"id":85688577,"identity":"2e7da0c7-6ca3-4d06-8d3f-9335c79ce3d4","added_by":"auto","created_at":"2025-06-30 16:26:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":242614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel for Eib-mediated chain formation\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) At medium expression levels, most protein is located at the bacterial polls, with some spillover onto the lateral sides. However, the low density of adhesins and presumably low individual affinities between Eib molecules means that cooperative binding happens mainly at polar contacts, where the density of adhesins is high enough to allow stable interactions. (\u003cstrong\u003eb\u003c/strong\u003e) At high expression levels, Eibs cover the entire cell at high densities, leading to clump formation as stable interactions can happen at all sides of the cell.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/b682c70c95311fe6d18f9e7a.png"},{"id":85688576,"identity":"927d0d0b-9652-4ed7-bf7f-63edfd654cc0","added_by":"auto","created_at":"2025-06-30 16:26:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":712411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of chain formation on auto-aggregation and biofilm formation\u003c/strong\u003e. For analysis of auto-aggregation strains were cultivated overnight (10 mL, 37°C, 30 rpm), cultures were mixed by inverting the samples and then transferred to long glass tubes. The OD\u003csub\u003e600\u003c/sub\u003e was measured every 15 to 30 min by taking 200 µL from top of the culture in three biological replicates and the mean value and standard deviations are plotted. (\u003cstrong\u003ea\u003c/strong\u003e) EibD, EibG, \u003cdel\u003e\u0026nbsp;\u003c/del\u003eand EibA expressing wild type cells (\u003cstrong\u003eb\u003c/strong\u003e) deletion mutants including deletion of LPBR (\u003cstrong\u003ec\u003c/strong\u003e) exchange mutants. (\u003cstrong\u003ed\u003c/strong\u003e+\u003cstrong\u003ee\u003c/strong\u003e) Crystal violet assay for the different Eib expressing cells: The cells were seeded into a 96-well microtiter plate and then incubated for 48 h at 37 °C before crystal violet staining. Upon solubilization of the strain the OD was measured at 540 nm. The mean value and standard deviation is shown of three biological replicates. One-way ANOVA with Dunnett’s post-hoc test was used for multiple comparisons to the control group (\u003cstrong\u003ed\u003c/strong\u003e) or EibG (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/225e9ec250434cda294b8bb8.png"},{"id":93421154,"identity":"30ab591e-8ff1-46af-a9aa-843329f27aea","added_by":"auto","created_at":"2025-10-13 16:10:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4811767,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/49b464c6-1e45-4137-a020-49efc56aba4f.pdf"},{"id":85688578,"identity":"de567f2e-94c9-465f-9ea4-6a230bad6880","added_by":"auto","created_at":"2025-06-30 16:26:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2544711,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementSwiateketalSciRep.docx","url":"https://assets-eu.researchsquare.com/files/rs-6781270/v1/6ed1a53ca77b81de9fa4a900.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chain formation mediated by Escherichia coli immunoglobulin-binding proteins EibD and EibG depends on expression levels and localization of proteins","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe species \u003cem\u003eEscherichia coli\u003c/em\u003e, comprising both commensal and pathogenic representatives, is well studied. However, especially multi-drug resistant isolates are a major public health threat[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is a versatile organism and pathogen reflected by a multitude of proteins presented on its cell surface[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This includes adhesins, pili, and fimbriae, which mediate attachment to host tissues and surfaces and play a critical role in colonization and infection[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Once adhered, \u003cem\u003eE. coli\u003c/em\u003e can form biofilms that enhance resilience to environmental stressors, immune responses, and antibiotics[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. By that, they contribute to the persistence and pathogenicity of \u003cem\u003eE. coli\u003c/em\u003e in host organisms and various environments[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent highly specialized protein secretion systems exist in Gram-negative bacteria that ensure protein transport across the inner and outer membranes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Proteins belonging to the type 5 secretion system utilize the Sec translocation machinery for crossing the inner membrane[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These proteins are characterized by a β-barrel domain that inserts into the outer membrane[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. No cytosolic energy sources for translocation across the outer membrane is needed resulting in the term autotransporter[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Trimeric autotransporter adhesins (TAAs, Type 5c secretion systems) are a group of obligate homotrimeric proteins that mediate their own transport across the outer membrane, a process that likely involves the βbarrel assembly machinery (BAM) but remains poorly understood[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These proteins are recognized as key virulence factors in Gram-negative bacteria, contributing to diverse functions, including immune evasion, serum resistance, host cell adhesion and invasion, and biofilm formation[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. TAAs consist of a Nterminal signal peptide enabling Sec-dependent inner membrane translocation and their outer membrane translocation pore is formed by conserved 12-stranded β-barrel domains with each protomer contributing four of the stands[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The extracellular region or passenger contains one or more right- and/or left-handed coiled-coiled regions, as well as globular domains forming \u0026lsquo;heads\u0026rsquo;. Some TAAs, such as the prototypical \u003cem\u003eYersinia\u003c/em\u003e adhesin YadA, have a single head atop a coiled-coil stalk and thus resemble a lollipop[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, other TAAs are more complex, with a number of heads interspersed with stretches of coiled coil[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, smaller minidomians such as the saddle or an insertion of a 3-stranded β-meander into a coiled-coil segment (FGG motifs) may be present.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne subgroup of TAAs comprises the immunoglobulin (Ig)-binding proteins (Eib) found in \u003cem\u003eE. coli\u003c/em\u003e, including several distinct proteins[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These proteins mediate non-immune binding to IgG and IgA Fc regions[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. They are prevalent in Shiga toxin-producing \u003cem\u003eE. coli\u003c/em\u003e (STEC) but are also found in commensal strains, suggesting a potential role in intestinal adhesion[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Among these, EibG has been described as mediating a unique chain-like adherence pattern (CLAP) on both human and bovine cells[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This observation indicates that chain formation by EibG is not host-specific and likely represents a bacteria-to-bacteria adhesion mechanism that also occurs in liquid culture[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEib proteins are known to induce autoaggregation, which manifests as clump formation under specific conditions[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly to other multifunctional proteins like YadA, which binds to collagen and other extracellular matrix components and mediates serum resistance, EibG may have additional roles beyond adhesion. Despite its significance, little is known about the underlying mechanism by which EibG facilitates chain formation. Here, we investigated how EibG mediates chain formation. Our results demonstrate that EibG and also EibD can mediate chain formation under the certain conditions. The chain formation depends on homotypic interactions of Eib proteins enriched at the cell contact sites and also on the expression levels of the protein. Understanding this mechanism in detail could provide insights into the broader biological functions of TAAs and their role in bacterial.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e \u003cb\u003eExpression levels influence chain formation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhile auto-aggregation and clump formation is a known phenomenon mediated by Eib proteins[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], previous studies indicated a CLAP for cells expressing EibG while adhering to human cells[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, we found that Eib proteins, including EibG, formed clumps when expressed at high levels using a strong inducible promoter (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). To evaluate the influence of expression levels on the chain formation we cloned different \u003cem\u003eeib\u003c/em\u003e variants into a plasmid with a medium-low strength promotor for constitutive expression and transformed \u003cem\u003eE. coli\u003c/em\u003e TOP10 cells with respective constructs. Using this system, we were able to show that chain formation is observed when cells expressed EibG and EibD at low levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By contrast, EibA (with an EibD signal peptide) expression in this system did not lead to chain formation. For EibD and EibG, both clump and chain formation were often observed simultaneously and were not mutually exclusive. Image analysis demonstrated that chain formation is reflected in an increase of particle size but decrease in circularity compared to control cells containing the empty vector and EibA expressing cells, which form small clumps. Cells expressing YadA, the well-studied \u003cem\u003eYersinia\u003c/em\u003e ssp. autotransporter protein[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], only showed clump formation at the analyzed conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of neck-domain but not N-terminal domain depletes the chain forming phenotype\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA crystal structure encompassing the YadA-like left-handed parallel-beta roll domain (LPBR) and the LPBR and coiled coil stalk of EibD has been previously solved[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, no structural information has been available for the N-terminal domain (NTD) of this protein. Therefore, we used AlphaFold2[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] to model the full trimers of EibD and EibG, as well as EibA and YadA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The Eib proteins share highly similar structures at the C-terminus, which includes the β-barrel membrane anchor, a left-handed coiled-coil region and the saddle minidomain. In EibD and EibG, the N-terminal half of the stalk is a right-handed coiled coil, whereas in EibA, this is left-handed. EibD and EibG share a similar bipartite head region encompassing the NTD with a novel fold and the LBPR. EibA lacks a LPBR, having only the NTD. The NTD of these three proteins has a triangular core with two α-helices packing against an antiparallel beta-sheet (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). In addition, a β-hairpin packs against the longer α-helix to make the 'tip' of the upside-down triangle. The NTDs of the three Eib proteins have some differences, particularly in the way they connect to the LPBR domain, or stalk in the case of EibA (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). The NTDs have limited structural similarity to the plectin repeats of periplakin, a eukaryotic cytoskeletal linker protein[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEibG and EibD are relatively similar in the region of the LPBR domain, but EibA lacks this domain. We therefore evaluated chain formation on cells constructively expressing EibG with deletion of the LPBR. This revealed that the deletion of the EibG LPBR domain does not prevent chain formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although EibG and EibD both have the potential of chain formation, chain formation for EibG was more pronounced with longer chains. We originally used an EibD variant with a PelB signal peptide, often used for recombinant protein production, as this worked well in induced overproduction conditions[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, under lower expression conditions, we noticed that with the PelB signal, almost no EibD protein was seen in the outer membrane (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). When we re-introduced the native signal peptide, EibD was expressed well. The reasons for this are unclear, but this demonstrates that the signal peptide plays an important role in trimeric autotransport.\u003c/p\u003e \u003cp\u003eTo identify domains that influence chain formation we introduced domain deletions in EibG and domain swaps between EibG and EibD proteins and validated protein production and trimerisation in the outer membranes (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). This revealed that deletion or alteration of the NTD does not inhibit chain formation. However, when additionally deleting the LPBR and neck or the whole passenger, the bacteria formed clumps or remained as individual cells but no chains were observed. After quantifying the microscopy images, this was reflected in decreased size of the particles with slight increase in circularity. Similar observations were made when exchanging the domains between EibG and EibD. Clump formation resulted in a high variability in particle size and a relatively constant circularity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChain forming phenotype is induced by homotypic protein-protein interaction predominantly at the cell poles\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe were interested in whether interaction of the Eib proteins with themselves or with other surface structures leads to chain formation. Therefore, we set up mixed cultures with an \u003cem\u003eE. coli\u003c/em\u003e reporter strain expressing mNeonGreen but no TAA and the Eib-expressing strains and performed microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the mixed culture containing the empty vector control, individual cells were observed only. In contrast, chain formation was again detected in the EibG and EibD cultures. These chains consisted exclusively of cells lacking the mNeonGreen signal, while individual mNeonGreen-positive cells were found dispersed around them.\u003c/p\u003e \u003cp\u003eIn the next step, we evaluated whether heterotypic chains or clumps formed in a mixed culture of cells expressing mNeonGreen and EibG or mCherry and EibD, respectively. Upon fluorescence-microscopic evaluation, it became evident that chains are exclusively formed through homotypic interactions, as non-TAA expressing cells did not participate in chain formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). EibD and EibG-expressing cells did not appear to interact, confirming that the interactions are homotypic and specific to each TAA. However, the additional expression of the mCherry reporter in EibD-expressing cells enhanced the clump forming phenotype. Sporadically, EibG chains were found within the EibD clumps, though we consider this to be an unspecific interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, we were interested in whether the chain-forming phenotype is induced by polar localization of the respective Eib proteins. For that, we used EibD and EibG variants with a SpyTag[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] located at the Nterminus upon cleavage of the signal peptide. Detection was ensured by incubation with a previously purified SpyCatcher-sfGFP protein which forms a covalent bound with the SpyTag[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Evaluation of the fluorescent signal from confocal fluorescence microscopy revealed that EibD and EibG proteins could be found around the whole cell. However, increased signals were detected at the contact points of the individual cells within a chain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Quantification of these signals revealed an increased signal intensity for the cell-to-cell contacts compared to the whole chains, being significantly higher for the majority of chains.\u003c/p\u003e \u003cp\u003eWe also investigated the localization in clumps. The high cellular densities within clumps make precise detection and quantification difficult. However, Nno distinct localization was observed. Both Eib variants were detected around the cells in clumps with some individual cells showing enhanced signals, most likely due to variable accessibility of the fluorescent lable within the clumps (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn order to check for specificity of the signal, the Eib-SpyTag variants were incubated with a SpyCatcher-EQ-sfGFP variant, which prevents formation of a covalent bound with the SpyTag. SpyCatcher-EQ-sfGFP resulted in diffuse, low-intensity non-specific signals (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these findings, we propose the following model for chain formation in Eib-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e): at low expression levels, proteins are preferentially exposed at the cell poles. The high density at the poles leads to homotypic interactions that mediate chain formation. Some spillover from the poles may lead to low levels of protein along the lateral side of the cell, but because of the presumed low affinity of the homotypic interactions, the low protein density here would not support cooperative binding and consequently cells only attach at the poles. At higher expression levels the proteins are more evenly distributed and cooperative interactions can take place at the lateral sides as well, which leads to clump formation. This is indeed what we observe in our current setup, where chain formation and clump formation coexist. We do see some fluorescent signal on the lateral sides of cells in chains, but an enrichment at cell-cell contact sites, which is consistent with this model. Future work will aim to uncover how Eibs are preferentially targeted to the cell poles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChain formation does not impact auto-aggregation but adhesion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTAAs are known to promote auto-aggregation among other virulence-associated phenotypes like biofilm formation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We tested auto-aggregation of chain-forming bacteria using sedimentation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). All wild-type strains demonstrated auto-aggregative phenotypes in contrast to the empty-vector control. None of the domain deletions and domain exchange mutants showed altered auto-aggregation behavior, except for the deletion of the almost the entire passenger domain (EibG∆saddle).\u003c/p\u003e \u003cp\u003eIn contrast to auto-aggregation, an alteration in chain formation severely affected the adhesive phenotype on plastic surfaces, which is recognized as the crucial initial step of biofilm formation. For all strains that did not show chain formation, namely cells expressing, EibA EibG∆neck, and EibG∆saddle, the intensity of crystal violet staining was equal to the control or significantly reduced compared to EibG wild-type expressing strains. This was also true for cells with low-level YadA expression, although higher levels of YadA have been shown to promote biofilm formation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For EibA, weak biofilm formation has been demonstrated before[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. By contrast, strains with the chain-forming phenotype showed increased adhesion. For the NTD deletion mutant a slight decrease in adhesion was observed consistent with the reduction in chain sizes. When comparing the mutants with domain swaps between EibD and EibG, the highest levels of adhesion became evident for the chain-forming EibG variant with the EibD N-terminal domain (EibD-NTD-EibG). When additionally exchanging the head and neck domains or the whole passenger a decrease in staining intensity was observed for the clump-forming mutants compared to the wild-type and the chain-forming mutant (EibD-NTD-EibG).\u003c/p\u003e \u003cp\u003eInterestingly, while there was a significant difference in adhesive phenotypes between the chain-forming and chain-deficient strains, no notable variation was observed among the long-term macrocolonies of the EibG wild type and its respective mutant strains. Notably, distinct biofilm phenotypes were observed among the different Eib wild type variants (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThis study highlights the critical influence of expression levels, and structural domains on the aggregation behavior of Eib proteins in \u003cem\u003eE. coli\u003c/em\u003e. The findings offer new insights into the mechanisms driving chain formation and adhesive phenotypes, while also suggesting the predominant localization of EibD and EibG at cell-cell contacts, particularly at the cell poles, contribute to these processes.\u003c/p\u003e \u003cp\u003eThe results demonstrated that lower expression levels of EibG and EibD promote chain formation, a phenotype absent in EibA. Note that despite clear chain formation clumps were also still observed for both. Static growth, microaerophilic conditions, and alkaline pH induce EibG expression in STEC while agitation and decreased temperature is repressive[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Under laboratory conditions, Eib proteins were highly stable, while protein levels differed significantly among different strains[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous studies described a CLAP for EibG and EibF-expressing strains when adhering to human or bovine cells only while a \u003cem\u003eeibABCDE\u003c/em\u003e-postive strain does not show a CLAP[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This could be explained by the presence of EibA or higher expression levels in general leading to clumping that masks any potential CLAP. Here we demonstrated that chain formation is not only induced by EibG and EibF but also EibD when expressed at moderate levels. The chain formation phenomenon does not require adhesion to cells but occurs in liquid cultures too. The absence of chain formation by EibA suggests that while these proteins share common features, functional differences arise due to sequence and structural variations.\u003c/p\u003e \u003cp\u003eDifferences became particularly evident in the LPBR. Although EibA lacks the LPBR region, deletion of this domain in EibG and EibD did not inhibit chain formation. Deletion of the LPBR neither affected adhesion nor autoaggregation. These findings do not align with earlier reports that the LPBR region of TAAs facilitates homotypic interactions critical for aggregation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, deletion or exchange of the NTD did not prevent chain formation, resulting in similar adhesion capacities as seen in wild-type EibG expressing cells. By contrast, deletion of the head domain, consisting of the NTD, the LPBR and the neck domain, resulted in disrupted chain formation, and significantly impaired adhesion. Different \u003cem\u003eeibG\u003c/em\u003e allelic variants exist that show phenotypes differing in length and sequence variation within the LBPR and neck domains[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Together, these observations suggest that the domains of the EibG head contribute to autoaggregation and chain formation, so that only when the entire head is deleted these processes are affected.\u003c/p\u003e \u003cp\u003eWe observed that chain formation, rather than clump formation, is a key determinant of adhesion in these systems, while macrocolony morphologies did not differ among the EibG wild type and its mutants. Adhesion is the initial step in host colonization, which in turn is a prerequisite for causing severe pathologies[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. By shedding light on the molecular determinants of chain formation, a growing body of knowledge may inform the development of strategies to prevent adhesion of pathogenic bacteria as demonstrated for FimH[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In line with previous reports, no chain or biofilm formation was observed for EibA, which has instead been implicated in immunoglobulin binding and serum resistance[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In contrast, autoaggregation is not affected by alteration of the chain-forming phenotype and occurs independently of clump or chain formation.\u003c/p\u003e \u003cp\u003eWe demonstrated that chain formation exclusively occurs between cells expressing EibG or EibD and not among a mixed population of cells expressing no or different Eib variants. Moreover, we hypothesized that the polar localization is essential for the chain forming phenotype. Polar localization of autotransporters is a known process in \u003cem\u003eE. coli\u003c/em\u003e that has been extensively studied, e.g. for the type 5a autotransporters IcsA and Ag43 but applies to other membrane proteins as well[\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These studies suggested that outer membrane proteins are targeted to the pole in the cytoplasm before secretion[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] with multiple factors such as protein sequence determinants, lipopolysaccharide integrity, expression levels and the activity of key proteins, e.g. YidC, DnaK or FtsQ being involved[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, many of these localization studies were performed in the absence of signal peptides, so the validity of this aggregation/disaggregation model is not fully confirmed. Omitting the signal peptide could easily lead to the formation of intracellular inclusion bodies, which may or may not be representative of the native process when a signal peptide is included.\u003c/p\u003e \u003cp\u003eThis study provides evidence that the signal peptide plays a role in determining the type of aggregation through presumably altered surface expression. When EibD was expressed with a PelB signal peptide protein levels detected in the outer membrane were severely impaired, as was the chain formation. This is consistent with the function of signal peptides enabling Sec-dependent translocation of TAA into the periplasm[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of this study indicate that low-level expression of Eib proteins leads to their initial predominant localization at the cell poles, which later serve as contact points for the initiation of chain formation. The mechanism behind this still requires further investigation. Furthermore, we identified that complex structural features of the Eib proteins are required as prerequisites for mediating the type of intercellular interaction that enables chain formation. The interplay between structural domains and expression levels in regulating bacterial adhesion and biofilm formation has important implications for understanding the pathogenic potential of STEC and related strains.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Bacterial strains and cultivation\u003c/h2\u003e \u003cp\u003eThe bacterial strains used in the study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eE. coli\u003c/em\u003e TOP10 cells were used as host cells for constitute expression of all protein variants with the pASK-IBA2con backbone and a chloramphenicol resistance cassette (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Bacterial strains were cultivated in LB-Lennox medium or auto-induction medium (AIM) containing 25 \u0026micro;g/mL chloramphenicol, 15 \u0026micro;g/mL tetracycline or 100 \u0026micro;g/mL ampicillin at 37\u0026deg;C. For cloning and protein expression, strains were cultivated shaking at 150 rpm. Whenever chain formation was induced, shaking was reduced to 30\u0026ndash;50 rpm.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eBacterial strains\u003c/b\u003e. Strain names, the host with the obtained plasmids, as well as a short description and reference are listed. Deleted amino acids are shown in square brackets. Control strain was obtained from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA. *The pBAD-EibD/EibG were constructed based on pBAD/HIsA from Invitrogen\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCtrl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl strain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG∆NTD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibG\u003c/em\u003e∆n.term\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG with deleted N-terminal domain (NTD) [∆Q28-L173]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG∆neck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibG\u003c/em\u003e∆neck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG with deletion up to and including the neck domain [∆Q28-V318]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG∆saddle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibG\u003c/em\u003e∆saddle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG with deletion up to and including the saddle domain [∆Q28-A375]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG∆LPBR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibG\u003c/em\u003e∆LPBR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG with deleted LPBR domain [∆V174-Y287]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD-NTD-EibG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibD\u003c/em\u003e- n.term_\u003cem\u003eeibG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG variant encoding the EibD N-terminal domain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD-neck-EibG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibD\u003c/em\u003e- neck_\u003cem\u003eeibG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG variant encoding the EibD neck domain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD-pass-EibG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibD\u003c/em\u003e- pass_\u003cem\u003eeibG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of EibG variant encoding the EibD passenger domain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYadA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eyadA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConstitutive expression of YadA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCtrl-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epACYC184-mNeonGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAssessing mixed chain formation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epACYC184-mNeonGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAssessing mixed chain formation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD-mCherry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epACYC184-mCherry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAssessing mixed chain formation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD-induced\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBL21∆ABCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epBAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInduction for determining surface expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG-induced\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBL21∆ABCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epBAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInduction for determining surface expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibG-SpyTag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibG\u003c/em\u003e-SpyTag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEibG variant with a 5\u0026rsquo; Spy-Tag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEibD-SpyTag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epASK-IBA2con_\u003cem\u003eeibD\u003c/em\u003e-SpyTag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEibD variant with a 5\u0026rsquo; Spy-Tag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Cloning and mutagenesis\u003c/h2\u003e \u003cp\u003eFor inducible production of EibG and EibD, the coding sequences for the proteins were amplified from pGEMEBG[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and pETDuetS-EibD[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], respectively. These were cloned into the arabinose-inducible vector pBAD/HisA (Invitrogen). To produce a plasmid giving low-level constitutive expression, we replaced the \u003cem\u003etet\u003c/em\u003e promoter of pASK-IBA2C (IBA Lifesciences GmbH, Goettingen, Germany) with the constitutive promoter J23105 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://parts.igem.org/Part:BBa_J23105\u003c/span\u003e\u003cspan address=\"https://parts.igem.org/Part:BBa_J23105\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by site-directed mutagenesis[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. \u003cem\u003eeibD\u003c/em\u003e and \u003cem\u003eeibG\u003c/em\u003e were subcloned into this vector, as were \u003cem\u003eeibA\u003c/em\u003e (with an EibD signal peptide, from pETDues-EibA[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]) and YadA (from \u003cem\u003eYersinia enterocolitica\u003c/em\u003e O:3, amplified from genomic DNA of strain 6471/76[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]). For fluorescent labelling, we used either mNeonGreen or mCherry, the coding sequences for which were inserted into the plasmid pACYC184[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The SpyTag[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] sequence was introduced using mutagenesis primers to insert the tag into the pASK-IBA2C-eibG and pASK-IBA2C-eibD vectors, respectively, at the 5\u0026prime; end of the genes to produce N-terminally tagged proteins. All cloning procedures were performed with Gibson assembly[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] using the 2 x HiFi Master Mix (New England Biolabs, Ipswich, UK) and VeriFi\u0026trade; Mix Red (PCR Biosystems Ltd., London, UK) for amplification of fragments or Q5 polymerase (New England Biolabs, Ipswich, UK) for site-direct mutagenesis. All primers are listed (\u003cb\u003eSupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/b\u003e). Domain deletions were performed based on a previously published protocol[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The annotation of domains was performed based on AlphaFold[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Chemically competent \u003cem\u003eE. coli\u003c/em\u003e TOP10 cells[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] were transformed with the respective cloning products. Clones were validated by colony PCR using PCRBIO Taq Mix Red (PCR Biosystems Ltd) and Sanger sequencing (Source Bioscience Genomics, Cambridge, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Bright field and epifluorescence microscopy\u003c/h2\u003e \u003cp\u003eChain formation was evaluated using bright field microscopy. Overnight cultures were diluted (1:50) in 10 mL of fresh medium and incubated at 37\u0026deg;C with shaking at 30 rpm for at least 5 h. After incubation, 1 mL of the culture was gently transferred to a 2 mL microcentrifuge tube using a 1000 \u0026micro;L pipette tip with the tip cut to minimize shear stress on the bacteria. The sample was allowed to sediment for 30 minutes at room temperature. During the sedimentation period, microscopy slides were prepared using 1 mL of 1% agarose in PBS on each slide, allowing it to solidify. Following sedimentation, the bacteria were resuspended by gently pipetting up and down three times with the cut tip. A small drop of the resuspended bacteria was transferred to a prepared microscopy slide. Microscopy was performed using a Nikon Eclipse epifluorescence microscope (Nikon, Amstelveen, The Netherlands) at 1000x magnification with immersion oil. For potential formation of mixed chains was assessed by using TOP10 cells either expressing EibG and mNeonGreen or EibD and mCherry, or TAA-expressing cells (non-fluorescent) with TOP10 cells expressing mNeonGreen. mNeonGreen and mCherry were introduce on a pACYC184-based plasmid, either with chloramphenicol resistance or tetracycline resistance. The samples were prepared with minor modifications. Individual overnight cultures of the described strains (with chloramphenicol or chloramphenicol and tetracycline for mNeonGreen/EibG and mCherry/EibD strains) were equally diluted in fresh medium (OD\u003csub\u003e600\u003c/sub\u003e 0.05) and incubated at 37\u0026deg;C with shaking at 30 rpm for at least 5 h. Bright field and epifluorescence mode were used simultaneously to identify (i) Eib and NeonGreen (Eib-negative) expressing cells respectively or (ii) EibG and mNeonGreen or EibD and mCherry expressing cells. Overlay of images was ensured using microscopic software (NIS-Elements, Nikon v.5.11.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Quantification of clump and chain formation\u003c/h2\u003e \u003cp\u003eFor quantification of clump and chain formation compared to individual cells we used the Fiji software[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. For that, microscopic images were exported as TIF files and imported into Fiji. Ten semi-randomly taken microscopic images were used from four biological replicates each. If multiple color channels exist, they were reduced to one and background removal was performed. The threshold was adjusted and the particles were analyzed in default mode to identify the cells. Shape descriptions were measured as the read out analyzing the individual particles (Size: 0-Infinity; Circularity: 0.00\u0026ndash;1.00).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Expression and Purification of SpyCatcher-sfGFP and SpyCatcher-EQ-sfGFP\u003c/h2\u003e \u003cp\u003eSpyCatcher proteins were produced as described before with minor modifications[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In brief, \u003cem\u003eE. coli\u003c/em\u003e BL21Gold(DE3) cells harboring either pET22-SpyCatcher-sfGFP or pET22-SpyCatcherEQ-sfGFP plasmids were inoculated separately into 5 mL AIM supplemented with 100 \u0026micro;g/mL ampicillin in the morning and grown at 37\u0026deg;C for several hours. The cultures were then transferred into 1 L of AIM containing 100 \u0026micro;g/mL ampicillin and antifoam for incubation overnight at 37\u0026deg;C in a LEX10 bioreactor (Epiphyte3, Toronto, Ontario, Canada). The following day, cells were harvested by centrifugation (15 min at 5000 x \u003cem\u003eg\u003c/em\u003e) and resuspended in 30 mL PBS, then stored at \u0026minus;\u0026thinsp;20\u0026deg;C until purification. Frozen cell suspensions were thawed and treated with 0.1 mg/mL of lysozyme, a pinch of DNase I, 1 mM MgCl₂, and 1 mM MnCl₂. Bacterial cells were lysed using a probe sonicator for six 30-second cycles. The lysate was clarified by centrifugation at 30,000 x \u003cem\u003eg\u003c/em\u003e for 60 minutes. The supernatant was applied to a Ni-NTA affinity column connected to an FPLC unit. Proteins were eluted using an imidazole gradient (10 mM \u0026ndash; 500 mM). Fractions were collected, and 40 \u0026micro;L aliquots from each were prepared for SDS-PAGE as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Evaluation of protein localization using SpyCatcher and confocal microscopy\u003c/h2\u003e \u003cp\u003eThe EibG-SpyTag and EibG-SpyTag expressing-strains were grown overnight, diluted in the morning and incubated at 37\u0026deg;C with shaking at 50 rpm. After 4 h of growth, SpyCatcher-sfGFP or SpyCatcher-sfGFP-EQ was added to the culture to a final concentration of 0.02 mg/mL. The culture was gently swirled to ensure proper mixing and then incubated for an additional 1.5 h at 37\u0026deg;C with shaking reduced to 30 rpm. After the incubation period, the bacterial clumps were allowed to settle for a few minutes without shaking. A 400 \u0026micro;L sample from the bottom of the tube was transferred to a 2 mL tube and carefully resuspended, using a pipette with the tip cut off. To wash the cells, 400 \u0026micro;L PBS was added and the mixture was centrifuged at 1000 x \u003cem\u003eg\u003c/em\u003e for 3 min. The supernatant was discarded, and the bacterial pellet was gently resuspended in 800 \u0026micro;L PBS with minimal pipetting, followed by gentle swirling and flicking of the tube to remove the bacteria from the bottom. The washing step was repeated. Afterwards, the bacterial pellet was gently resuspended in 200 \u0026micro;L of PBS and a 6 \u0026micro;L aliquot of the resuspended sample was transferred to an agarose pad using a pipette with a cut-end tip for imaging using Nikon AXR with NSPARC. To analyze the fluorescence intensity at the bacterial cell-to-cell contact points, images were processed using Fiji software[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. A single bacterial chain was first selected using the selection tool. This selection was duplicated to preserve the original image and facilitate further analysis. A maximum intensity projection of the Z-stack was created. The fluorescence image was subsequently split into individual color channels. The area corresponding to cell-to-cell contact was identified within the bright field image using the freehand selection tool. The selection was transferred to the fluorescence channel. The selected region of interest (ROI) was added to the ROI manager and mean fluorescence intensity within this ROI was measured. This was repeated for all cell-to-cell contacts within the chain and then for the whole chain as well. The data were then exported. To evaluate differences in fluorescence intensity, statistical analysis was performed using an R-script to generate plots and conduct a paired t-test. This test compared the fluorescence signal across the entire bacterial chain with the mean fluorescence intensity at the cell-to-cell contact points to determine if significant differences existed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Sedimentation\u003c/h2\u003e \u003cp\u003eOvernight cultures (10 mL) were inoculated from single cultures and grown for 16 h overnight at 37\u0026deg;C shaking at 30 rpm. The following day, the cultures were carefully inverted to ensure uniform mixing and transferred into long glass tubes. The OD\u003csub\u003e600\u003c/sub\u003e was measured every 15\u0026ndash;30 min by taking 200 \u0026micro;L from the top pf the cultures using the plate reader (SPECTROstar Nano; BMG Labtech, Ortenberg, Germany). The cultures were incubated at 37\u0026deg;C statically in-between. Reduction in OD\u003csub\u003e600\u003c/sub\u003e relative to the initial OD was measured as degree of sedimentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Crystal violet assay\u003c/h2\u003e \u003cp\u003eFor analyzing biofilm formation, the OD\u003csub\u003e600\u003c/sub\u003e of overnight cultures was adjusted to 0.2 and 100 \u0026micro;L of OD-adjusted culture was inoculated into four wells of a Nunc\u0026trade; MicroWell\u0026trade; 96-Well-mikrotiter plate (ThermoFisher Scientific, Waltham, MA, USA). As negative control, the respective medium alone was included. Plates were sealed with PARAFILM\u0026reg;, and incubated statically at 37\u0026deg;C for 48 h. Afterwards, the culture was carefully aspirated and the adhered biofilm was washed three times with 200 \u0026micro;L of PBS to remove non-adherent cells. The plates were then incubated at 60\u0026deg;C for at least 60 minutes to fix the biofilm. The adhered biofilm was stained by adding 150 \u0026micro;L of 1% (w/v) crystal violet (Sigma Aldrich; Merck, Darmstadt, Germany) for 20 min. The plates were rinsed three times with distilled water and left inverted to dry. To solubilize the stained biofilm, 150 \u0026micro;L of 33% (v/v) glacial acetic acid (Scientific Laboratory Supplies, Nottingham, UK) was added per well. After complete solubilization, the solution was transferred to a new plate. The OD at 540 nm (OD\u003csub\u003e540\u003c/sub\u003e) was measured (SPECTROstar Nano; BMG Labtech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Software and statistics\u003c/h2\u003e \u003cp\u003eFor all cloning approaches, we used SnapGene Viewer (GSL Biotech LLC v7.1.2.0) in combination with ApE-A plasmid Editor (v3.1.6) and Oligo Calculator (v3.27)[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Evaluation of microscopy was done with Nikon NIS (v5.11) together with Fiji (ImageJ Version 2.35). NIS Element (v6.10.01). Data evaluation and visualization was performed using GraphPad Prism (v10.2.1) and RStudio (v2023.06.2\u0026thinsp;+\u0026thinsp;561). Paired t-test or One-way ANOVA with Dunnett\u0026rsquo;s post-hoc test was used for multiple comparisons when applicable. We used large language models, such as ChatGPT (GPT-4), to assist in revising portions of the manuscript. All AI-generated content was thoroughly reviewed, edited, and approved by the authors to ensure its accuracy and integrity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTAAs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Trimeric autotransporter adhesins\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCLAP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cchain-like adherence pattern\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGFP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Green fluorescent protein\u003c/p\u003e\n\u003cp\u003eEib\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunoglobulin-binding proteins\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSTEC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Shiga toxin-producing \u003cem\u003eE.\u0026nbsp;coli\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOD\u003csub\u003e600\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;optical density at 600 nm\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOD\u003csub\u003e540\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;optical density at 540 nm\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSDS-PAGE\u0026nbsp; \u0026nbsp; \u0026nbsp;sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLPBR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;left-handed parallel beta-roll\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eData availability statement\u003c/h3\u003e\n\u003cp\u003eRaw data microscopic images and metadata are stored at FigShare\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(DOI: 10.6084/m9.figshare.29166725). Gene and plasmid sequences are publically available and can be found as: \u003cem\u003eeibA\u003c/em\u003e (AF151091.1), \u003cem\u003eeibD\u003c/em\u003e (AAF63040.1), \u003cem\u003eeibG\u003c/em\u003e (ADJ17717.1), \u003cem\u003eyadA\u003c/em\u003e (WP_032488477.1) pASK-IBA2con (2-1321-000; IBA Lifesciences GmbH, Goettingen, Germany). The \u003cem\u003etet\u003c/em\u003e promoter of pASK-IBA2C was replaced with the constitutive promoter J23105 (https://parts.igem.org/Part:BBa_J23105) by site-directed mutagenesis.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the German Federal Ministry of Education and Research (BMBF) within the “\u003cem\u003eDISPATch\u003c/em\u003e_MRGN-Disarming pathogens as a different strategy to fight antimicrobial-resistant Gram-negatives” project (grant 01KI2410), and internal funding from Nottingham Trent University. International collaboration was supported by the Erasmus+ program.\u003c/p\u003e\n\u003ch3\u003eAuthors' contributions\u003c/h3\u003e\n\u003cp\u003eConceptualization, J.L.; methodology, J.L., and L.S.; software, J.L., and L.S.; validation, J.L., K.S., and L.S.; formal analysis, J.L. and L.S.; investigation, J.L., and L.S.; resources, J.L., and K.S.; data curation, J.L., and L.S..; writing-original draft preparation, L.S.; writing-review and editing, J.L., K.S., and L.S.; visualization, J.L. and L.S.; supervision, J.L., and L.S.; project administration, J.L.; funding acquisition, J.L., K.S., and L.S..\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgments\u003c/h3\u003e\n\u003cp\u003eWe thank Jonas Øgaard for his assistance with the image analysis. We also thank Dr. Graham Hickman associated with School of Science \u0026amp; Technology Imaging Suite for providing access to and for his support with the confocal microscopy facility. Our gratitude also goes to the members of the Antibiotic Resistance, Omics, and Microbiota Group for their support and valuable discussions. We would also like to thank Dr. Sunao Iyoda (National Institute for Infectious Diseases, Japan) for providing the \u003cem\u003eeibG\u003c/em\u003e plasmid.\u003c/p\u003e\n\u003ch3\u003eAuthors Information\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eLena-Sophie Swiatek\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003eKatharina Schaufler\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003eJack C. Leo \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGeneva: World Health Organization. WHO Bacterial Priority Pathogens List, 2024: Bacterial Pathogens of Public Health Importance to Guide Research, Development and Strategies to Prevent and Control Antimicrobial Resistance.. (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgeorges, V. et al. Molecular determinants of surface colonisation in diarrhoeagenic Escherichia coli (DEC): From bacterial adhesion to biofilm formation. \u003cem\u003eFEMS Microbiol. Rev.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, 314\u0026ndash;350 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaper, J. B., Nataro, J. P. \u0026amp; Mobley, H. L. T. Pathogenic Escherichia coli. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 123\u0026ndash;140 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Bougu\u0026eacute;nec, C. Adhesins and invasins of pathogenic Escherichia coli. \u003cem\u003eInt. J. Med. Microbiol.\u003c/em\u003e \u003cb\u003e295\u003c/b\u003e, 471\u0026ndash;478 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma, G. et al. Escherichia coli biofilm: development and therapeutic strategies. \u003cem\u003eJ. Appl. Microbiol.\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e, 309\u0026ndash;319 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristie, P. J. The Rich Tapestry of Bacterial Protein Translocation Systems Peter. \u003cem\u003eProtein J.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 389\u0026ndash;408 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenderson, I. R., Navarro-Garcia, F., Desvaux, M. \u0026amp; Fernandez, R. C. Ala\u0026rsquo;Aldeen, D. Type V Protein Secretion Pathway: the Autotransporter Story. \u003cem\u003eMicrobiol. Mol. Biol. Rev.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, 692\u0026ndash;744 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLinke, D., Riess, T., Autenrieth, I. B., Lupas, A. \u0026amp; Kempf, V. A. J. Trimeric autotransporter adhesins: variable structure, common function. \u003cem\u003eTrends Microbiol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 264\u0026ndash;270 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClarke, K. R. et al. Phylogenetic Classification and Functional Review of Autotransporters. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1\u0026ndash;20 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlauser, T., Pohlner, J. \u0026amp; Meyer, T. F. The secretion pathway of IgA protease-type proteins in gram‐negative bacteria. \u003cem\u003eBioEssays\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 799\u0026ndash;805 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeo, J. C., Grin, I. \u0026amp; Linke, D. Type V secretion: Mechanism(S) of autotransport through the bacterial outer membrane. \u003cem\u003ePhilos. Trans. R Soc. B Biol. Sci.\u003c/em\u003e \u003cb\u003e367\u003c/b\u003e, 1088\u0026ndash;1101 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiessling, A. R., Malik, A. \u0026amp; Goldman, A. Recent advances in the understanding of trimeric autotransporter adhesins. \u003cem\u003eMed. Microbiol. Immunol.\u003c/em\u003e \u003cb\u003e209\u003c/b\u003e, 233\u0026ndash;242 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSikdar, R. \u0026amp; Bernsteina, H. D. Sequential translocation of polypeptides across the bacterial outer membrane through the trimeric autotransporter pathway. \u003cem\u003eMBio\u003c/em\u003e 10, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauhan, N. et al. Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA). \u003cem\u003eMol. Microbiol.\u003c/em\u003e \u003cb\u003e111\u003c/b\u003e, 844\u0026ndash;862 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeuskens, I., Saragliadis, A., Leo, J. C. \u0026amp; Linke, D. Type V secretion systems: An overview of passenger domain functions. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1\u0026ndash;19 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzczesny, P. \u0026amp; Lupas, A. Domain annotation of trimeric autotransporter adhesins - daTAA. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 1251\u0026ndash;1256 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng, G., Surana, N. K., Geme, S., Waksman, G. \u0026amp; J. W. \u0026amp; Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter. \u003cem\u003eEMBO J.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 2297\u0026ndash;2304 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahid, S. A. et al. Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals. \u003cem\u003eNat. Methods\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 1212\u0026ndash;1217 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoiczyk, E., Roggenkamp, A., Reichenbecher, M., Lupas, A. \u0026amp; Heesemann, J. Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. \u003cem\u003eEMBO J.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 5989\u0026ndash;5999 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartmann, M. D. et al. Complete fiber structures of complex trimeric autotransporter adhesins conserved in enterobacteria. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 109, 20907\u0026ndash;20912 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeo, J. C. et al. The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins. \u003cem\u003eStructure\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 1021\u0026ndash;1030 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBassler, J., Alvarez, H., Hartmann, B., Lupas \u0026amp; M. D. \u0026amp; A. N. A domain dictionary of trimeric autotransporter adhesins. \u003cem\u003eInt. J. Med. Microbiol.\u003c/em\u003e \u003cb\u003e305\u003c/b\u003e, 265\u0026ndash;275 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandt, C. H. \u0026amp; Hill, C. W. Nonimmune binding of human immunoglobulin A (IgA) and IgG Fc by distinct sequence segments of the EibF cell surface protein of Escherichia coli. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 7293\u0026ndash;7303 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandt, C. H. \u0026amp; Hill, C. W. Four different genes responsible for nonimmune immunoglobulin-binding activities within a single strain of Escherichia coli. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, 2205\u0026ndash;2214 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeo, J. C. \u0026amp; Goldman, A. The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc. \u003cem\u003eMol. Immunol.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 1860\u0026ndash;1866 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerkel, V. et al. Distribution and phylogeny of immunoglobulin-binding protein G in shiga toxin-producing Escherichia coli and its association with adherence phenotypes. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e78\u003c/b\u003e, 3625\u0026ndash;3636 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, Y. et al. A new immunoglobulin-binding protein, EibG, is responsible for the chain-like adhesion phenotype of locus of enterocyte effacement-negative, shiga toxin-producing Escherichia coli. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 5747\u0026ndash;5755 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil, H. S., \u0026Oslash;gaard, J. \u0026amp; Leo, J. C. Coaggregation properties of trimeric autotransporter adhesins. \u003cem\u003eMicrobiologyopen\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 1\u0026ndash;22 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNummelin, H. et al. The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel β-roll. \u003cem\u003eEMBO J.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 701\u0026ndash;711 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJumper, J. et al. Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e596\u003c/b\u003e, 583\u0026ndash;589 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMirdita, M. et al. ColabFold: making protein folding accessible to all. \u003cem\u003eNat. Methods\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e, 679\u0026ndash;682 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoczonadi, V. \u0026amp; M\u0026auml;\u0026auml;tt\u0026auml;, A. \u003cem\u003eFunctional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins. Methods in Enzymology\u003c/em\u003evol. 569 (Elsevier Inc., 2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikula, K. M. et al. The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e194\u003c/b\u003e, 827\u0026ndash;838 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 109, (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuodu, R., Linke, D. \u0026amp; Leo, J. C. Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e \u003cb\u003e2778\u003c/b\u003e, 53\u0026ndash;63 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuczius, T. et al. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC). \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 1\u0026ndash;16 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRubin, D., Zhang, W., Karch, H. \u0026amp; Kuczius, T. Distinct expression of immunoglobulin-binding proteins in shiga toxin-producing Escherichia coli implicates high protein stability and a characteristic phenotype. \u003cem\u003eToxins (Basel)\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAprikian, P. et al. The bacterial fimbrial tip acts as a mechanical force sensor. \u003cem\u003ePLoS Biol\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMydock-Mcgrane, L. K., Cusumano, Z. T. \u0026amp; Janetka, J. W. Mannose-derived FimH antagonists: A promising anti-virulence therapeutic strategy for urinary tract infections and Crohns disease. \u003cem\u003eExpert Opin. Ther. Pat.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 175\u0026ndash;197 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMydock-McGranea, L. K., Hannana, T. J. \u0026amp; Janetka, J. W. Rational Design Strategies for FimH Antagonists: New Drugs on the Horizon for Urinary Tract Infection and Crohn\u0026rsquo;s Disease. \u003cem\u003eExpert Opin. Drug Discov\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 711\u0026ndash;731 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWells, T. J., Totsika, M. \u0026amp; Schembri, M. A. Autotransporters of Escherichia coli: A sequence-based characterization. \u003cem\u003eMicrobiology\u003c/em\u003e \u003cb\u003e156\u003c/b\u003e, 2459\u0026ndash;2469 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGray, A. N., Li, Z., Henderson-Frost, J. \u0026amp; Goldberg, M. B. Biogenesis of YidC cytoplasmic membrane substrates is required for positioning of autotransporter IcsA at future poles. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e196\u003c/b\u003e, 624\u0026ndash;632 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoyle, M. T., Grabowicz, M. \u0026amp; Morona, R. A small conserved motif supports polarity augmentation of Shigella flexneri IcsA. \u003cem\u003eMicrobiol. (United Kingdom)\u003c/em\u003e. \u003cb\u003e161\u003c/b\u003e, 2087\u0026ndash;2097 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrandon, L. D. et al. IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed. \u003cem\u003eMol. Microbiol.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 45\u0026ndash;60 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVejborg, R. M. \u0026amp; Klemm, P. Cellular chain formation in Escherichia coli biofilms. \u003cem\u003eMicrobiology\u003c/em\u003e \u003cb\u003e155\u003c/b\u003e, 1407\u0026ndash;1417 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, S. et al. Polar localization of the autotransporter family of large bacterial virulence proteins. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e188\u003c/b\u003e, 4841\u0026ndash;4850 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFixen, K. R. et al. Genetic reporter system for positioning of proteins at the bacterial pole. \u003cem\u003eMBio\u003c/em\u003e 3, (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanakiraman, A., Fixen, K. R., Gray, A. N., Niki, H. \u0026amp; Goldberg, M. B. A genome-scale proteomic screen identifies a role for DnaK in chaperoning of polar autotransporters in Shigella. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e191\u003c/b\u003e, 6300\u0026ndash;6311 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, H. \u0026amp; Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. \u003cem\u003eBMC Biotechnol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 91 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkurnik, M. Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis. \u003cem\u003eJ. Appl. Bacteriol.\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e, 355\u0026ndash;363 (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang, A. C. Y. \u0026amp; Cohen, S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cb\u003e134\u003c/b\u003e, 1141\u0026ndash;1156 (1978).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. \u003cem\u003eNat. Methods\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, 343\u0026ndash;345 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaradi, M. et al. AlphaF old Prot ein Structure D atabase in 2024. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, D368\u0026ndash;D375 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung, C. T., Niemela, S. L. \u0026amp; Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 86, 2172\u0026ndash;2175 (1989).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindelin, J. et al. Fiji: An open-source platform for biological-image analysis. \u003cem\u003eNat. Methods\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 676\u0026ndash;682 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKibbe, W. A. \u0026amp; OligoCalc An online oligonucleotide properties calculator. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 43\u0026ndash;46 (2007).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adhesion, Autoaggregation, Cellular localization, Chain formation, E. coli immunoglobulin-binding protein, Trimeric autotransporter adhesins","lastPublishedDoi":"10.21203/rs.3.rs-6781270/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6781270/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e expresses immunoglobulin-binding proteins (Eib), a subgroup of trimeric autotransporter adhesins (TAA). Subtypes of Eib proteins mediate unique chain-like adherence patterns and autoaggregation. This study investigates the mechanisms underlying chain formation by EibG and EibD; a chain-forming phenotype of the latter has not been previously described. Using constitutive expression systems, we demonstrate that low-level expression of EibG and EibD lead primarily to chain formation, whereas higher expression levels predominantly result in clump formation. Notably, chain and clump formation are not mutually exclusive and can occur simultaneously. Selective deletion of the full head domain, but not the N-terminal domain alone, abolished chain formation, highlighting its critical role. Fluorescence microscopy of mixed cultures showed that chains form through homotypic protein-protein interactions. Investigation revealed EibD and EibG were predominantly localized at cell poles, corresponding to sites of intercellular contact. Functional investigations showed that chain-forming strains exhibited enhanced adhesion to plastic surfaces, a key step in biofilm formation, without affecting autoaggregation. These showed Eib-mediated chain formation depends on protein expression levels, domain architecture, and localization, contributing to bacterial adhesion and potentially pathogenicity. Understanding interactions provides insights into TAA-mediated chain formation and autoaggregation.\u003c/p\u003e","manuscriptTitle":"Chain formation mediated by Escherichia coli immunoglobulin-binding proteins EibD and EibG depends on expression levels and localization of proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 16:25:58","doi":"10.21203/rs.3.rs-6781270/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-08T10:10:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T09:54:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T07:25:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52583946853906807993292231973969971590","date":"2025-06-23T17:22:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82784907164629607352726724029022498478","date":"2025-06-23T06:32:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-21T12:27:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-21T12:25:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-11T03:15:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T09:37:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-30T05:35:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36fd8753-d853-442a-8d46-ea7d00ca9a3a","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50473972,"name":"Biological sciences/Biochemistry/Proteins"},{"id":50473973,"name":"Biological sciences/Biological techniques/Imaging/Fluorescence imaging"},{"id":50473974,"name":"Biological sciences/Biological techniques/Genetic engineering"},{"id":50473975,"name":"Biological sciences/Biological techniques/Microbiology techniques"},{"id":50473976,"name":"Biological sciences/Biological techniques/Microscopy"},{"id":50473977,"name":"Biological sciences/Biological techniques/Microscopy/Confocal microscopy"},{"id":50473978,"name":"Biological sciences/Biological techniques/Microscopy/Phase contrast microscopy"},{"id":50473979,"name":"Biological sciences/Biological techniques/Software"},{"id":50473980,"name":"Biological sciences/Biological techniques/Structure determination/Molecular modelling"},{"id":50473981,"name":"Biological sciences/Genetics/Mutation"},{"id":50473982,"name":"Biological sciences/Genetics/Prokaryote"},{"id":50473983,"name":"Biological sciences/Microbiology/Bacteria/Bacterial pathogenesis"},{"id":50473984,"name":"Biological sciences/Microbiology/Bacteria/Bacterial structural biology"},{"id":50473985,"name":"Biological sciences/Microbiology/Biofilms"},{"id":50473986,"name":"Biological sciences/Microbiology/Pathogens"},{"id":50473987,"name":"Biological sciences/Structural biology/Molecular modelling"},{"id":50473988,"name":"Biological sciences/Biochemistry"},{"id":50473989,"name":"Biological sciences/Microbiology"},{"id":50473990,"name":"Biological sciences/Molecular biology"},{"id":50473991,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2025-10-13T16:09:36+00:00","versionOfRecord":{"articleIdentity":"rs-6781270","link":"https://doi.org/10.1038/s41598-025-22967-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-09 15:58:23","publishedOnDateReadable":"October 9th, 2025"},"versionCreatedAt":"2025-06-30 16:25:58","video":"","vorDoi":"10.1038/s41598-025-22967-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22967-3","workflowStages":[]},"version":"v1","identity":"rs-6781270","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6781270","identity":"rs-6781270","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}