Beyond the stress response: absence of RelA protein hampers the cell wall structuring and cell size in Liquorilactobacillus vini

Beyond the stress response: absence of RelA protein hampers the cell wall structuring and cell size in Liquorilactobacillus vini | 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 Research Article Beyond the stress response: absence of RelA protein hampers the cell wall structuring and cell size in Liquorilactobacillus vini Dayane da Silva Santos, Allyson Andrade Mendonça, Manoel Zúñiga, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4252796/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The role of (p)ppGpp alarmone produced by RelA protein is well documented as a response to stress and nutritional deficiency in bacteria. However, little information is reported for Lactobacillus sensu lato, including the remaining species of old genus Lactobacillus and the derived species recently allocated in new genera. The present study aimed to characterize for the first time the effect of the inactivation of relA in a representative of the Liquorilactobacillus genus. Results obtained have revealed an unexpected role of RelA protein on the cell wall homeostasis and cell size. L.vini ΔrelA showed low growth and increased cell size, related to overexpression of genes responsible for DNA transcription and repair and down-expression of the fusA gene. The low growth also resulted in the loss of the flagellum in Δ relA , which may also be associated with the fragility of the cell wall, ease of lysis and resistance to beta-lactams of the Δ relA mutant. Cell wall homeostasis was deregulated mainly by the down-expression of the gene encoding alanine ligase and murB , which may have triggered the overexpression of the genes encoding PBPs proteins. Growth data suggest that the absence of the RelA protein promoted a disconnection between the cell cycle and cell division in L. vini , and a consequent reduction in the growth rate of the culture, cell wall fragility and beta-lactam resistance, expanding the RelA function in Liquorilactobacillus beyond the stringent response. Stringent response (p)ppGpp Lactic acid bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Genetic and metabolic studies have been conducted on Liquorilactobacillus vini , formerly known as Lactobacillus vini , since its identification as quantitatively relevant bacterial species in the composition of the microbial population with in bioethanol industrial processes in Sweden and Brazil (Passoth et al. 2007 ; Lucena et al. 2010 ; de Lucena et al. 2012 ; Mendonça et al. 2016 , 2020 ; da Silva et al. 2019 ). Since then, research has focused on understanding the biological mechanisms responsible for the adaptation of this bacterium to the industrial environment. This involves both the metabolic capacity to use available nutrients through central metabolism and the cellular response to the different challenges posed by agents and stressful conditions in that environment. In bacteria, both regulation of central metabolism, from carbon distribution to energy balance, and the stress response seem to converge on a control mechanism that has been generically called the stringent response (SR) (Bange et al. 2021 ). The main biochemical elements mediating this mechanism are a set of molecules called alarmone. It consists of tetra (ppGpp) and penta (pppGpp) phosphorylated forms of guanidine synthesized from GDP or GTP, respectively, by ATP-dependent alarmone synthetases (Atkinson et al. 2011 ; Gaca et al. 2015 ; Krishnan and Chatterji 2020 ). Although initially defined as a response to amino acid shortage, the term SR has been expanded to include any regulatory effect exerted by the robust accumulation of (p)ppGpp, regardless of the triggering mechanism (Gaca et al. 2013 ). Bacterial growth is regulated by (p)ppGpp, which controls various stages of the genetic information flux (replication, transcription, ribosome maturation, and translation) and central metabolism. In addition, it regulates various physiological processes such as pathogenesis, persistence, motility and competence (Mu et al. 2023 ). According to Büke et al. ( 2022 ), (p)ppGpp serves as a critical regulator that coordinates cell size and growth control, lipid synthesis and other anabolic processes to maintain the integrity of the cell envelope in response to nutrient abundance or starvation (Gaca et al. 2013 ; Gonzalez and Collier 2014 ; Imholz et al. 2020 ; Mu et al. 2023 ). Gram-positive bacteria, such as L. vini , have a thick and sophisticated cell wall composed of peptidoglycan, surface-anchored proteins, teichoic acids, lipoteichoic acids and lipoproteins, which protect the bacteria from environmental stress (Rajagopal and Walker 2015 ). The major component of the cell wall is the peptidoglycan sacculus, a large glycan polymer composed of linear strands of alternating N-acetylglucosamine (GlcNac) and N-acetylmuramic acid (MurNac) units cross-linked by peptides composed of four to five amino acids of D or L configurations (Pasquina-Lemonche et al. 2020 ). The cell wall is a dynamic structure whose composition changes in response to varying environmental conditions in order to ensure cell survival. For instance, the tolerance to acid stress by HCl in L. vini is accompanied by a reorganisation of the bacterial cell wall which involves the down-expression of biosynthetic genes and over-expression of degradation genes (Mendonça et al. 2019 ). It has been proposed that this mechanism increases the mechanical resistance of the cell at the same time that provides internal glucose for maintenance energy during cell growth arrest (Alcántara and Zúñiga 2012 ; Mendonça et al. 2019 ). Nevertheless, the involvement of the SR mechanism in this process remains to be elucidated. The control of bacterial metabolism via the SR mechanism depends on the production of the signalling molecule (p)ppGpp by a series of alarmone synthetases. Among them, the RelA protein is a bifunctional enzyme by alternating phosphotransferase and pyrophosphohydrolase activities for synthesis and degradation, respectively, during cell growth phase (Atkinson et al. 2011 ; Bange et al. 2021 ). Therefore, it is paramount to study the function of this gene and its protein on the metabolism and stress response of L. vini in order to understand its adaptive mechanisms to industrial processes, such as the bioethanol fermentation. Thus, this work presents for the first time the inactivation of relA in a representative of the Liquorilactobacillus genus and reveals the unexpected role of RelA protein on the cell wall homeostasis and cell size in this genus. The environmental and industrial implications of this finding are discussed. Materials and Methods Strains and genetic modification The strain JP 7.8.9 of L. vini was isolated from a bioethanol fermentation process (de Lucena et al. 2012 ). E. coli DH10B was used as an intermediate host for cloning purposes. Lactococcus lactis NZ9000 was used as a host for plasmid pNG8048e (Kuipers et al. 1998 ). These bacteria were cultivated and maintained in MRS (Difco) at 37°C, LB (Oxoid) at 37°C and M17 (Oxoid) media supplemented with 5 g/L glucose at 30°C, respectively. Antibiotics were supplemented when required as follows: ampicillin at 100 µg/mL for E. coli , chloramphenicol and erythromycin at 5 µg/mL for L. lactis and erythromycin at 5 µg/mL for L. vini . Genomic DNA from strain JP 7.8.9 was extracted with the AxyPrep™Bacterial Genomic DNA Purification Miniprep kit (Axigen) following the manufacturer’s guidelines. The inactivation cassette was cloned in the pRV300 vector (Leloup et al. 1997 ). The relA gene nucleotide sequence was identified from the L. vini genome by using RAST server program and used for primer design. A XhoI restriction site (5’-TTTTACTCGAGAGGGCGATGTCTTGGAGTTG-3’) and a SpeI restriction site were added to the 5’ end of the forward (5’-TTTTACTCGAGAGGGCGATGTCTTGGAGTTG-3’) and reverse (5’-TTTTACTAGTTTGATGATCCCCAAGGGTGC) primers, respectively. These primers were used to amplify an amplicon of 445 bp corresponding to the internal region of the relA gene (from nucleotide 1202 to nucleotide 1646 of the gene) by PCR from L. vini genomic DNA, purified by using the QIAquick PCR purification kit (Qiagen), and quantified using a Nanodrop® device. Plasmid pRV300 was extracted by the QIAGEN Plasmid Midi kit from E. coli DH10B following the manufacturer’s guidelines. Purified pRV300 and relA fragment were digested with XhoI and SpeI , purified, quantified and ligated following the conventional DNA cloning protocols. The integration vector was introduced into the cells of E. coli DH10B by electroporation. Cells were subsequently spread on LB medium plates containing ampicillin at 100 µg/mL, IPTG at 0.1 mM and X-gal at 80 µg/mL according to standard protocol. Transformant colonies were picked and cultivated in LB plus ampicillin. Plasmids were extracted as indicated above and the interruption of the relA gene was checked by PCR. The resulting plasmid was named pRVrelA. A protocol for L. vini transformation was standardized as follows: JP7.8.9 cells were cultivated in MRS broth for 24 h at 37 o C and used to inoculate MRS broth containing glycine (from 10 to 100 g/L) to an initial optical density (OD) of 0.1 at 600 nm (OD 600 ). The cultures were incubated at 37 o C until reaching 0.6 OD 600 and the cell were collected by centrifugation at 5000 g at 4 o C, and resuspended to the same culture volume in transformation buffer (0.3 M sucrose, 5 mM sodium phosphate pH 7.4 and 1mM MgCl 2 in Milli-Q water). This washing procedure was repeated twice and the cells were finally re-suspended in transformation buffer to 1/100 of the initial volume, dispensed in 50 µL aliquots and stored at -80 o C until use. The transformation efficiency of the electrocompetent L. vini cells was tested with the plasmid pNG8048e at concentrations ranging from 100 ng to 500 ng. The electroporation conditions were set to: 100 to 400 Ω parallel resistance, 25 µF capacitance and 1,000 to 2,000 V pulse voltage. Following the electroporation, the cells were mixed in the cuvette with the recovery medium consisting of MRS supplemented with CaCl 2 (2 mM), MgCl 2 (20 mM) and sucrose (300 mM) and transferred to microtubes. The cell suspensions were incubated for 3 h at 37 o C and then plated on MRS plate containing erythromycin (5 µg/mL). The plates were incubated for five days at 37 o C in anaerobic jars and the number of transformant colonies was recorded to stablish the best electroporation setup condition for subsequent transformation with pRVrelA. Cell growth and cell size analysis Cells grown in MRS broth for 24 hours were used to inoculate aliquots of 150 µL of MRS dispensed in a microtiter plate to an initial OD 600 of 0.05. The plates were incubated in a Sinergy HT multireader device (Biotek, Switzerland) at 37°C and the variation of culture OD 600 was recorded automatically every 30 min for 48 hours. Samples were collected from the exponential growth phase and the cell size measurements were made at a 2000× magnification in a Nikon Eclipse Ni-U microscope with bright field optics, and photomicrographs were taken using DIC optics with a Nikon DS-Fi2 camera. 20 cells for strains JP 789 and its isogenic Δ relA mutant were measured and mean differences were assessed by using the Student's t-test. Normality of data distribution was checked with the Shapiro Wilk test. Cell lysis experiments Cells from exponential growth phase in MRS medium (1.0 OD 600 ) were collected by centrifugation and resuspended to one-tenth of the original volume in STE buffer (300 mM sucrose, 100 mM Tris-HCl, and 50mM EDTA-NaOH, pH 8). Aliquots of 150 µl of the buffered cell suspensions were transferred to a microtiter plate and incubated at 37°C. OD variations were recorded every 30 min for 10 h in a Sinergy HT multireader device to check for cell integrity. Alternatively, the cells were suspended in STE buffer containing lysozyme at 10 mg/ml to test the resistance to this lytic enzyme. The experiments were performed in biological duplicate with technical triplicates. Therefore, the numbers represent the mean value of six measurements (± SD) for each sample. RNA isolation, cDNA synthesis and gene expression For RNA isolation, cells were suspended in 1 mL MRS to 3.5 OD 600 . After three hours at 37°C, cells were collected by centrifugation and subjected to cell lysis by lysozyme (Mendonça et al. 2019 ). Afterwards, total RNA was extracted with TRIzol® LS (Invitrogen Corp.) according to the manufacturer’s instructions (Oberg et al. 2011 ). The upper phase was collected in new tubes and the RNA was purified with the illustra RNAspin Mini RNA Isolation kit (GE Healthcare) following the manufacturer’s instructions. RNA integrity was evaluated directly on 1 % agarose gel prepared with TAE buffer (40 mM of tris, 10 mM of EDTA, 20 mM of acetic acid) and stained with 0.5 mg ml − 1 of ethidium bromide, and its concentration was determined in a Nanodrop spectrophotometer (Thermo Fisher, USA). RNA samples were stored at − 80°C until use. The cDNA was synthetized with the ImProm-II Reverse Transcription System (Promega, USA), following the manufacturer’s instructions. Each reaction was performed with 0.5 µg of RNA in a final volume of 20 µl. RT negative controls were also prepared with the same amount of RNA diluted in water (same concentration as cDNA sample) in order to estimate contamination with genomic DNA. The samples were stored at − 20°C until use. The cDNA was used for qPCR in biological duplicates and technical triplicates. Expression of genes involved in transcription regulation ( rpoD, rpoE, sigV and rpoB ), DNA maintenance ( recA , pcrA and mutL ) and translation elongation ( fusA ), in teichoic acid biosynthesis ( dltD ), peptidoglycan precursor biosynthesis ( glmS , glmU and murB ), cell wall assembly ( pbp1ABα and pbp1ABb ), cell wall degradation ( nagA ), alanine metabolism ( ddl, alaR and alaT ) and pbpX. The Cq values of each replicate from reference genes and tested genes were used for normalization and relative expression quantification according to MIQE guidelines (Vandesompele et al. 2002 ; Huggett et al. 2005 ). Normalization data are available in the supplementary material. The candidate genes for data normalization were the same as those used by Mendonça et al. ( 2019 ) and, among them, fusA and rpoB genes were used in this study. Flagellum presence and motility assay To confirm the presence of the L. vini flagellum, fluorescence microscopy was used. Widefield microscopy was performed using an arc for excitation and a charge-coupled device camera for detection (set of blue filters and 100x objective). To prepare the slides, cells were grown in MRS broth (24 hours at 37°C), washed with saline solution (0.9% sodium chloride), 20 µl aliquots were deposited onto glass slides and incubated at room temperature for 30 minutes. A total of 2 µL of DAPI solution (4.6diamidino2-phenylindole) or Calcofluor White and 10% (w/v) potassium hydroxide were deposited on the slides so that the staining covered the entire site of initial bacterial deposition. The slides were incubated in darkness at room temperature for 20 minutes. The images were captured using a Hamamatsu CCD camera coupled to an Olympus BX51 epifluorescence microscope or Leica DM5500B fluorescence microscope. For the motility assay, the parental JP 789 strain and its mutant strain Δ relA were inoculated in MRS agar (1.5%) Petri dishes. Aliquots of 2 µl of the bacterial cultures were added to the centre of the plate. Subsequently, semi-solid MRSagar (0.5%) medium was added and the plates were incubated at 37°C for 48 hours. The assay was performed in duplicate and the halos formed were measured with a vernier caliper. Antibiotic resistance Minimal inhibitory concentrations (MICs) of ampicillin, amoxicillin, imipenem, penicillin G and cephalothin were determined for L. vini JP7.8.9 and Δ relA strains. Bacterial cells were cultivated on MRS broth for 24 h and subsequently diluted to OD 600 0.002 with MRS broth. The MIC was determined by the micro-dilution method starting with 2 µg/mL of antibiotic and followed by serial dilutions until 0.031 µg/mL. MIC value was defined at the first concentration that completely inhibited bacterial growth. The strain of Enterococcus. faecalis ATCC 29212 was used as a sensitive reference (CLSI 2010 ). The plates were incubated for 48 h at 37°C. All the tests were performed in duplicate. Results Liquorilactobacillus vini transformation For the construction of the isogenic Δ relA mutant strain, the first step was to establishes a transformation procedure for L. vini JP 7.8.9. The highest number of transformants with the reference plasmid pNG8048e was 38 transformants/µg of plasmid DNA input. This was achieved through the condition and parameters set as: glycine at 20 g/L in MRS, for the preparation of electrocompetent cells, and the electroporation setup of 200 Ω of resistance, 25 µF capacitance and 1.500 V pulse voltage. Decreasing of glycine concentrations, voltage pulse above 1.750 V or resistance below 100 Ω abolished the transformability completely. The confirmation of L. vini transformant with the reference plasmid were performed by plasmid purification followed by digestion with Bam HI. This enzyme was chosen due the doble restriction site in the reference plasmid which delivers a restriction fragments size profile specific. The agarose gel electrophoresis showed the presence of the pNG8048e plasmid in all transformant cells tested (Fig. 1 ). Thus, we established for the first time an efficient protocol for transformation of L. vini. This protocol was employed to inactivates the relA gene in the wild-type strain with the pRV300 vector bearing 445 pb of internal region of the target gene. The recombination between the internal region in the vector and the homologous region in the target gene inserted the whole plasmid construction and disrupts the coding sequence of the gene. This allows the integrated vector to be replicated and segregated through the cell division and turn these cells resistant to erythromycin due the erm b gene in the vector. As the pRV-300 cannot replicates in L. vini because the lack of the repA gene, the drug resistance could only arise in the tranformants through genomic integration. The efficiency of transformation with this integrative plasmid was 10 transformants/µg. To confirm the site of integration, were performed a PCR reaction with the same primers employed in the relA target region amplification and primers targeting the gene dltB . In the wild-type strains the PCRs for both targeted gene were positive but to the relA mutants only was possible to amplify the fragment of the dltB indicating the gene structure alteration. The relA mutant cells are more elongated and display slow growth Growth and division are central to cell size and in the present work we verified the importance of (p)ppGpp to these parameters in L.vini . Growth curve profile in MRS (Fig. 2 A) showed that Δ relA grew slower (growth rate of 0.07 h − 1 ; 9 h 50 min of doubling time) than the parental strain JP 789 (growth rate of 0.28 h − 1 ; 2 h 27 min of doubling time). Furthermore, microscopic analyses revealed that the mutant cells are more elongated than the parental cells (Fig. 2 B), with mean values of 2.8 and 1.8 µm in length, respectively. Statistical analysis revealed the significance of cell size differences p < 0.01 (Fig. 2 C). Following the microscopic inspection of the bacterial morphology, we noticed that the flagellum that was present in the parental strain JP7.8.9 was not observed in the Δ relA mutant strain (Fig. 3 A,D). In addition, the motility assay also pointed out that the Δ relA strain lost the agility of movement in the semi-solid culture medium, due to the impact that the absence of the relA gene caused in the biosynthesis of the flagella and/or anchoring of the flagella (Fig. 3 E). As previously stated, alarmones are a pleiotropic bacterial regulator that reprograms growth under stressful conditions. The low growth rate and morphological alterations displayed by the Δ relA mutant (Fig. 2 ) suggested that the homeostasis of the bacterial metabolism was possibly also affected by the absence of RelA protein, even in the absence of stressing conditions. In view of this, we tested the expression of genes that encode transcription factors known to influence cell growth. The relative expression analysis revealed that the lack of the RelA protein led to the overexpression the genes rpoD (encoding the sigma factor 70), sigV (encoding the sigma factor 5) and rpoB (encoding the β subunit of the bacterial RNA polymerase) relative to its parental strain (Fig. 4). These genes are related to cell maintenance, extracytoplasmic stress and gene transcription, respectively. Conversely, there was a down-expression of rpoE gene that encodes the sigma factor 24 (Fig. 4), which was shown to respond to heat shock. Unexpectedly, it was observed the overexpression of mutL gene, whose protein works to the mismatch repair mechanism, and the down-expression of fusA gene that encodes the elongation factor involved in protein translation (Fig. 4). The altered levels of all these housekeeping genes may have triggered a metabolic disorganization that might account for the phenotypic traits of the Δ relA mutant. Absence of relA gene affected cell wall homeostasis The observed increased cell size and the lack of flagellum in Δ relA strain led us to determine the expression of genes involved in cell wall biogenesis (Fig. 5 ). It was found that the genes involved in alanine metabolism ( alaT and ddl ) were down-expressed, with emphasis on ddl , which was 5 times less expressed in Δ relA compared to the parental strain. The alaR gene showed equal expression in the parent and the mutant. Genes gmlS and gmlU were over expressed in Δ relA . However, murB and dltD genes were up-regulated, as were the pbp1AB α and pbp1AB β involved in the structuring of the cell wall, On the other hand, nagA and pbpX genes were down-regulated. We next tested the response of the Δ relA mutant to stressing conditions, since RelA protein would be involved in the SR regulatory mechanism that also involves stress response. During the preliminary assays to set up the conditions for stress tolerance tests, we noticed a constant drop in the population of viable cells of the Δ relA strain when suspended in buffer even before starting exposure to stressors. This observation, together with the fact that genes involved in cell wall maintenance were affected by the absence of RelA, led us to analyse the stability of the bacterial cell wall by quantifying the rate of spontaneous cell lysis of the strains. Cells grown in MRS were collected in the exponential phase of growth, resuspended in STE buffer and the suspension was immediately evaluated for variation in cell density. The data clearly showed that mutant strain cell suspensions presented a significant drop in OD value while the parental strain maintained a practically constant OD throughout 10 h of incubation (Fig. 6 ), indicating the indeed the absence of RelA protein fragilizes the bacterial cell wall. Δ relA strain is more resistant to β-lactam Results in Fig. 5 showed that the absence of RelA protein increases the expression of two PBP proteins involved with transglycosylation and transpeptidation reactions for the construction of the bacterial cell wall. These proteins are the target of β-lactam antibiotics. Thus, MIC values to β-lactam antibiotics for both parental and mutant cells were determined. The results showed very consistent increments in MICs for five different β-Lactams in the mutant cells compared to the parental JP 789, varying from 2x for penicillin to 8x for cephalothin (Table 1 ). Therefore, the overexpression of PBP gene coincided with the increment of cell resistance to β-Lactams antibiotics. Table 1 Minimal Inhibitory Concentration (MIC) of antibiotics affecting the cell wall synthesis of Liquorilactobacillus vini JP7.8.9 and its isogenic mutant Δ relA mutant. The results represent the values of six experiments for each antibiotic in microtitration plates. MIC (µg/ml) Antibiotic Subclass JP7.8.9 Δ relA Penicillin G Penicillin 0.125 0.250 Ampicillin Penicillin 0.250 1.0 Amoxicillin Penicillin 0.250 1.0 Cephalothin Cephalosporin 0.250 > 2 Imipenem Carbapenem 1.0 > 2 Discussion The SR mediated by the alarmone (p)ppGpp is a survival system under adverse conditions and has been characterized in model organisms such as Escherichia coli and Bacillus subtilis . However, the function of these alarmones goes far beyond the SR, they may also control the cell cycle (Büke et al. 2022 ), growth (Mu et al. 2023 ) and antibiotic resistance (Salzer and Wolz 2023 ). In lactic acid bacteria such as Enterococcus faecalis , the alarmones are involved in the classic SR (Abranches et al. 2009 ) and their absence results in changes in the fermentative profile, production of high levels of H 2 O 2 and causes unbalancing of the cell metabolism (Gaca et al. 2013 ). In L.vini strain defective for the relA gene, it was observed phenotypes under non-limiting conditions that go beyond a SR. The growth rate was slowed down cell size was increased (Fig. 2 ). Büke et al (Büke et al. 2022 ) defined in their work with E. coli that decreasing ppGpp production resulted in progressively slower growth and larger cells and considered that changes in ppGpp concentration dynamically affected cell size and its control. In the present work, we showed that the absence of the RelA protein in L.vini also modulated the expression of genes related to cell growth (Fig. 5 ). fusA gene was down-regulated in the relA mutant. This gene encodes the elongation factor G (EF-G) in bacteria which is an essential component of the protein translation machinery (Norén et al. 2007 ) and directly impacts growth. In contrast, the gene encoding the sigma 70 factor responsible for the transcription of maintenance genes was overexpressed in L.vini relA mutant. The Δ relA strain also showed, even in the absence of DNA damage agents, the overexpression of mutL (Fig. 5 ). Its product MutL is a key component of the DNA mismatch repair system (Putnam 2021 ). mutL overexpression may be indicative of problems in DNA replication in Δ relA cells, which might partly explain the low growth of the Δ relA strain (Fig. 2 ). Bacterial flagella are synthesized by systems strictly controlled by nutritional and environmental conditions (Lemke et al. 2009 ). L. vini was described as a mobile bacterium (Rodas et al. 2006 ). In agreement with this, strain JP 7.8.9 also possesses a flagellum (Fig. 3 A,B) that allows the cells to spread on the surface of semi-solid media (Fig. 3 E). However, this flagellum was lost or reduced beyond our detection limit when relA gene was interrupted (Fig. 3 C,D), limiting the mobility capacity of the cells (Fig. 3 E). Studies on E. coli have shown that motility is closely linked to the cell growth rate (Sim et al. 2017 ). In addition, it was shown that ppGpp can inhibit the expression of genes in the flagellar building cascade during the stationary phase and after starvation, thus coordinating the assembly of the flagella (Lemke et al. 2009 ). In Vibrio alginolyticus , the lack of spoT , which also synthesizes (p)ppGpp, resulted in the loss of the flagellum (Yin et al. 2022 ), and a previous study revealed that the swimming ability of V. alginolyticus Δ relA Δ spoT double mutant decreased dramatically (Yin et al. 2021 ). Since L. vini apparently does not encode a spoT homolog (de Lucena et al. 2012 ), the single Δ relA mutant should behave like Δ relA Δ spoT double mutants in other bacterial species. However, the effect of relA gene interruption in Bacillus subitilis was just the opposite, with the mutant cells showing more flagella and turning more mobile, despite the low growth rate (Ababneh and Herman 2015 ). Furthermore, a classic work published in 1969 by Vaituzis and Doetsch pointed out the relationship between the cell wall, cytoplasmic membrane, and bacterial motility, and explained the importance of the cell wall in anchoring bacterial flagella (Vaituzis and Doetsch 1969 ). Hence, the loss of the flagella might also be linked to the apparent weakening of the cell wall also observed in the Δ relA strain (Fig. 6 ). In a previous report, we showed that treatment of L. vini cells with organic acids, as well as with hydrochloric acid, induced a restructuring of the bacterial cell wall as a consequence of the differential regulation of key genes (Mendonça et al. 2019 ). Some of these genes were tested in this work for their basal expression in a Δ relA strain relative to the parental strain, given the suspicion that the absence of the RelA protein weakens the bacterial cell wall. The results obtained showed that murB , alaT and ddl genes were under-expressed in the mutant strain (Fig. 5 ), especially ddl . The murB gene encodes a UDP-N-acetylmuramate dehydrogenase involved in the biosynthesis of N-acetylmuramic acid (Matsuo et al. 2003 ); alaT encodes the alanine aminotransferase, which catalyses reversible transamination between alanine and α-ketoglutarate, forming pyruvate and glutamate (Welch et al. 1975 ); ddl encodes a D-alanyl-D-alanine ligase involved in the peptidoglycan crossbridge peptide synthesis. Ddl is an essential protein for the proper growth and development of bacterial cell walls (Pederick et al. 2023 ). These three enzymes are directly involved in the biogenesis of peptidoglycan. Thus, the observed lysis profile of ΔrelA could be explained by the weakening of the cell wall, although the induction of endogenous lysines or activation of prophages in the mutant strain cannot be discharged. However, the results in expression of several genes involved in cell wall synthesis and maintenance and increased resistance to b-lactam antibiotics were indicative of alterations in the bacterial cell wall. In this hypothesis, failure in the correct construction of this cell component by the down-expression of relevant genes would lead to the weakening of the cell wall (Fig. 6 ). Besides, it was observed the overexpression of gmlS (encoding a glucosamine synthase), gmlU (glucosamine-1-phosphate N-acetyltransferase) and dltD (in this work representing the operon dlt involved in the biosynthesis of teichoic acid), indicating the synthesis of N-acetylglucosamine. However, the down expression of murB gene might interfere in the synthesis of UDP-MurNAC as essential component of PG formation. Moreover, overexpression of pbp1ABα and pbp1ABb , especially the second gene, was also observed (Fig. 4). These genes encode a transglycosylation and a transpeptidation enzymes, respectively, involved in the cross-linking of the peptides to the glucan chain in the cell wall structure. Together, the overexpression of these genes might represent a counter-action mechanism triggered in the mutant cells to maintain the integrity of the peptidoglycan in face of the shortage of N-acetylmuramic acid and D-alanyl-D-alanyl dipeptide used to build-up a stiff cell wall. A study with Streptococcus pneumonie , lactic acid bacteria, showed that the enzymes responsible for cell wall biosynthesis are connected to the alarmone through their ability to improve errors in protein translation (Aggarwal et al. 2021 ). In L.vini Δ relA mutant, the protein elongation factor was negatively regulated (Fig. 4), but the correlation between these two factors still seems uncertain. Δ relA mutant showed resistance to the tested beta-lactams (Table 1 ) and simultaneously, greater expression of the genes encoding PBPs proteins due to defects in the cell wall (Fig. 5 ). There are many mechanisms for bacteria to resist the action of β-lactams, a class of antibiotics that primarily acts on PBPs (Penicillin-Binding Proteins) (Sun et al. 2014 ). Interestingly, the inactivation of RelA together with the overexpression of the pbp genes provided a moderate increase in resistance to β-lactam antibiotics, in opposition to what has been seen in other bacteria (Das and Bhadra 2020 ; Salzer and Wolz 2023 ). Thus, the increased number of antibiotic-targeted PBPs substrates would account for this antibiotic resistance. Conclusion The role of (p)ppGpp alarmones in controlling cell cycle, cell growth and stress response in connection with the nutritional and physic-chemical conditions of the environment is well documented for Gram negative and positive bacteria. However, little information is reported for Lactobacillus sensu lato, including the remaining species of old genus Lactobacillus and the derived species recently allocated in new genera, as it is the case of L. vini . In the present work, we revealed for the first time the relevance of this sensor for the maintenance of the cell wall structure and for the synchrony between cell cycle and cell division in L. vini . However, the question that remains open is whether cell wall structural alterations are affecting cell division or whether metabolic deficiency due to the absence of RelA would affect both cell wall structure and cell division regulation. Experiments are underway to determine the influence of the relA gene, and consequently the production of alarmone, on the carbohydrate and energy metabolism of L. vini , which will contribute to the understanding of this sensor in the control of the cell cycle. Declarations Supplementary Information. The online version contains supplementary material available at https://doi.org/……... Author contributions DSS and AAM carried out the experiment, analysed the data, prepared the draft version of the manuscript and prepared figures and tables. CE carried out the experiment and analysed the data. MZC analysed the data and revised the final version of the manuscript. MAMJ analysed the data, prepared the draft version and revised the final version of the manuscript. All authors reviewed and approved the manuscript for submission. Funding DSS acknowledge the Fundação de Amparo a Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for the PHD scholarship support (grant IBPG-0670-2.02/22). This research was funded with resouces from Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant 441640/2023-0). Conflict of interest The authors declare no competing interests. Ethical approval This research article does not include any human or animal studies performed by any author. 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Microbiology (United Kingdom) 165:26–36. https://doi.org/10.1099/mic.0.000738 Mendonça AA, De Lucena BTL, de Morais MAMMCMA, de Morais MAMMCMA (2016) First identification of Tn916-like element in industrial strains of Lactobacillus vini that spread the tet-M resistance gene. FEMS Microbiol Lett 363:fnv240. https://doi.org/10.1093/femsle/fnv240 Mu H, Han F, Wang Q, et al (2023) Recent functional insights into the magic role of (p)ppGpp in growth control. Comput Struct Biotechnol J 21:168–175. https://doi.org/10.1016/j.csbj.2022.11.063 Norén T, Åkerlund T, Wullt M, et al (2007) Mutations in fusA Associated with Posttherapy Fusidic Acid Resistance in Clostridium difficile . Antimicrob Agents Chemother 51:1840–1843. https://doi.org/10.1128/AAC.01283-06 Oberg AL, French AJ, Sarver AL, et al (2011) miRNA Expression in Colon Polyps Provides Evidence for a Multihit Model of Colon Cancer. PLoS One 6:e20465. https://doi.org/10.1371/journal.pone.0020465 Pasquina-Lemonche L, Burns J, Turner RD, et al (2020) The architecture of the Gram-positive bacterial cell wall. Nature 582:294–297. https://doi.org/10.1038/s41586-020-2236-6 Passoth V, Blomqvist J, Schnürer J (2007) Dekkera bruxellensis and Lactobacillus vini form a stable ethanol-producing consortium in a commercial alcohol production process. Appl Environ Microbiol 73:4354–6. https://doi.org/10.1128/AEM.00437-07 Pederick JL, Woolman JC, Bruning JB (2023) Comparative functional and structural analysis of Pseudomonas aeruginosa d ‐alanine– d ‐alanine ligase isoforms as prospective antibiotic targets. FEBS J 290:5536–5553. https://doi.org/10.1111/febs.16932 Putnam CD (2021) Strand discrimination in DNA mismatch repair. DNA Repair (Amst) 105:103161. https://doi.org/10.1016/j.dnarep.2021.103161 Rajagopal M, Walker S (2015) Envelope Structures of Gram-Positive Bacteria. pp 1–44 Rodas AM, Chenoll E, Macián MC, et al (2006) Lactobacillus vini sp. nov., a wine lactic acid bacterium homofermentative for pentoses. Int J Syst Evol Microbiol 56:513–517. https://doi.org/10.1099/ijs.0.63877-0 Salzer A, Wolz C (2023) Role of (p)ppGpp in antibiotic resistance, tolerance, persistence and survival in Firmicutes. microLife 4:. https://doi.org/10.1093/femsml/uqad009 Sim M, Koirala S, Picton D, et al (2017) Growth rate control of flagellar assembly in Escherichia coli strain RP437. Sci Rep 7:41189. https://doi.org/10.1038/srep41189 Sun S, Selmer M, Andersson DI (2014) Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica. PLoS One 9:e97202. https://doi.org/10.1371/journal.pone.0097202 Vaituzis Z, Doetsch RN (1969) Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility. J Bacteriol 100:512–521. https://doi.org/10.1128/jb.100.1.512-521.1969 Vandesompele J, De Preter K, Pattyn F, et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034.1. https://doi.org/10.1186/gb-2002-3-7-research0034 Welch SG, Mills PR, Gaensslen RE (1975) Phenotypic distributions of red cell glutamate-pyruvate transaminase (E.C.2.6.1.2) isoenzymes in British and New York populations. Humangenetik 27:59–62. https://doi.org/10.1007/BF00283506 Yin W-L, Xie Z-Y, Zeng Y-H, et al (2022) Two (p)ppGpp Synthetase Genes, relA and spoT, Are Involved in Regulating Cell Motility, Exopolysaccharides Production, and Biofilm Formation of Vibrio alginolyticus. Front Microbiol 13:. https://doi.org/10.3389/fmicb.2022.858559 Yin W-L, Zhang N, Xu H, et al (2021) Stress adaptation and virulence in Vibrio alginolyticus is mediated by two (p)ppGpp synthetase genes, relA and spoT. Microbiol Res 253:126883. https://doi.org/10.1016/j.micres.2021.126883 Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.jpeg suplementarymaterial.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-4252796","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290688655,"identity":"f6e0ea3e-df3d-4743-aff3-db6d1358eb7e","order_by":0,"name":"Dayane da Silva Santos","email":"","orcid":"","institution":"Federal University of Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Dayane","middleName":"da Silva","lastName":"Santos","suffix":""},{"id":290688656,"identity":"a97beca5-ef0d-4496-b589-5e23e81fa70b","order_by":1,"name":"Allyson Andrade Mendonça","email":"","orcid":"","institution":"Federal University of Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Allyson","middleName":"Andrade","lastName":"Mendonça","suffix":""},{"id":290688657,"identity":"4ee4130d-f9b2-4179-9cb0-4bc6147c0e16","order_by":2,"name":"Manoel Zúñiga","email":"","orcid":"","institution":"Institute of Agrochemistry and Food Technology (IATA-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"Manoel","middleName":"","lastName":"Zúñiga","suffix":""},{"id":290688658,"identity":"a4ab7ac7-dcbd-4276-a2bc-f31481dfec9b","order_by":3,"name":"Marcos Antonio de Morais Jr","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3QMQuCQBTA8XcItrxqNYr6BMGFYEvfpOUi0Okg6As41SI1+y2EFkdFcDJamyoXZ9sMGjopqEWjreH+w9304+49AJns3wsAoQ9KAEDsH4gOKvuBlM3sb2S43qd5fodpuxulYdE7WV7cDC/EP1USI7H0jrsC7m5MGiEuuRe35pQky2oSmKrStIF7CdAIkHHv7BgaWbFqcshUBe8laeRhgcyiMY6LWnIUr6BaEqQBImOCGFBPMkXMonHXwYWYhY1cMYs2S+o+ZhKxsQnfYmN3vTls0BIby69+NXmlPS/ilCcCfAXviheRyWQy2UcPQ9BVLA8VJzoAAAAASUVORK5CYII=","orcid":"","institution":"Federal University of Pernambuco","correspondingAuthor":true,"prefix":"","firstName":"Marcos","middleName":"Antonio","lastName":"de Morais","suffix":"Jr"},{"id":290688659,"identity":"3bc01c0d-11df-43ed-a48f-0d1ae9d13a8b","order_by":4,"name":"Carolina Elsztein","email":"","orcid":"","institution":"Federal University of Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Elsztein","suffix":""}],"badges":[],"createdAt":"2024-04-11 13:37:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4252796/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4252796/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54785030,"identity":"ef9fbc47-379c-4b0f-ad2f-ebadb35a3d06","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22769,"visible":true,"origin":"","legend":"\u003cp\u003ePlasmid extraction from \u003cem\u003eLiquorilactobacillus vini \u003c/em\u003etransformant cells (T1 to T3) harbouring the plasmid pNG8048e. After purification, the plasmid solution was submitted to digestion with \u003cem\u003eBamHI\u003c/em\u003e restriction enzyme and separated in 1% agarose gel electrophorese. The purified and \u003cem\u003eBamHI-\u003c/em\u003edigested pNG8048e (pNG) was used as positive control. Plasmid extraction from untransformed JP 7.8.9 strain (WT) showing its native plasmids was used as negative control.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/171fbf81a26b0fef94ac84da.jpg"},{"id":54785031,"identity":"9288e043-ac64-465b-9754-54c4f30636fa","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37452,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003e JP7.8.9 (circles) and its isogenic D\u003cem\u003erelA\u003c/em\u003e mutant in MRS (Panel a). Cells were collected and their lengths measured (panel b). The differences in length between the two strains were statistically tested (panel c).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/b8f42e7d723cfed2537bbb3a.jpg"},{"id":54785034,"identity":"22f954e2-8b98-4d58-b58b-1c6350889584","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46365,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy of \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003eJP7.8.9 (panels a and b) and its isogenic D\u003cem\u003erelA\u003c/em\u003e mutant (panels c and d) and motility assay of these strains in semi-solid MRS-Agar (panel e). The arrows indicate the presence of flagellum.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/49feef95adfd708916713e6f.jpg"},{"id":54785036,"identity":"6b0119e3-1876-4c48-aa22-9f4dd08cdf25","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18905,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003e genes involved in transcription regulation (black columns), DNA maintenance (gray columns) and translation elongation (white columns) in the isogenic D\u003cem\u003erelA\u003c/em\u003e mutant relative to its parental strain JP7.8.9 in exponential growth phase in MRS medium.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/289b8ac19736d4153fb6720a.jpg"},{"id":54785032,"identity":"49079eca-504b-4b8d-ad24-bc5653d41273","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25375,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of key genes involved in cell wall biogenesis in \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003e D\u003cem\u003erelA \u003c/em\u003emutant relative to its parental strain JP7.8.9 cultivated to exponential growth phase in MRS. The results represent the mean value of three biological replicates (± standard deviation).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/9f8567e732a332ed3dc997d1.jpg"},{"id":54785206,"identity":"f1f00ce3-b0a8-4e66-8e8e-8c0cff87e78c","added_by":"auto","created_at":"2024-04-16 18:06:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":37490,"visible":true,"origin":"","legend":"\u003cp\u003eLysis curve of bacterial cells suspended in STE buffer. Cells of \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003e were cultivated in MRS to exponential growth phase and suspended in STE buffer in absence and absence of lysozyme.\u0026nbsp; JP7.8.9 no lysozyme (white circles) and its isogenic mutant D\u003cem\u003erelA\u003c/em\u003e no lysozyme (white triangle) and lysozyme presence JP7.8.9 (black circles) and its isogenic mutant D\u003cem\u003erelA\u003c/em\u003e\u0026nbsp; (black triangle). Variations of OD\u003csub\u003e600\u003c/sub\u003e of the suspensions were monitored for 10 h. The results represent the mean value of three biological replicates.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/7fbda832a92dc85e795b3577.jpg"},{"id":56537654,"identity":"95474d7b-475e-47dc-ace4-8f11b9909d2c","added_by":"auto","created_at":"2024-05-15 13:29:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":795999,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/0493c4ce-8731-4d5f-8acb-2bc673c0e348.pdf"},{"id":54785033,"identity":"453456da-89af-4f66-9786-c3a42fd0c5c7","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":52847,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/4eee1d056f981f4f4584f4bc.jpeg"},{"id":54785035,"identity":"ec20cd4f-f067-499e-8c05-6263b6e36033","added_by":"auto","created_at":"2024-04-16 17:58:01","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5998,"visible":true,"origin":"","legend":"","description":"","filename":"suplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4252796/v1/031c4b3a5bbb9711943f7848.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Beyond the stress response: absence of RelA protein hampers the cell wall structuring and cell size in Liquorilactobacillus vini","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGenetic and metabolic studies have been conducted on \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003e, formerly known as \u003cem\u003eLactobacillus vini\u003c/em\u003e, since its identification as quantitatively relevant bacterial species in the composition of the microbial population with in bioethanol industrial processes in Sweden and Brazil (Passoth et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lucena et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; de Lucena et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mendon\u0026ccedil;a et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since then, research has focused on understanding the biological mechanisms responsible for the adaptation of this bacterium to the industrial environment. This involves both the metabolic capacity to use available nutrients through central metabolism and the cellular response to the different challenges posed by agents and stressful conditions in that environment.\u003c/p\u003e \u003cp\u003eIn bacteria, both regulation of central metabolism, from carbon distribution to energy balance, and the stress response seem to converge on a control mechanism that has been generically called the stringent response (SR) (Bange et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The main biochemical elements mediating this mechanism are a set of molecules called alarmone. It consists of tetra (ppGpp) and penta (pppGpp) phosphorylated forms of guanidine synthesized from GDP or GTP, respectively, by ATP-dependent alarmone synthetases (Atkinson et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Gaca et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Krishnan and Chatterji \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although initially defined as a response to amino acid shortage, the term SR has been expanded to include any regulatory effect exerted by the robust accumulation of (p)ppGpp, regardless of the triggering mechanism (Gaca et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Bacterial growth is regulated by (p)ppGpp, which controls various stages of the genetic information flux (replication, transcription, ribosome maturation, and translation) and central metabolism. In addition, it regulates various physiological processes such as pathogenesis, persistence, motility and competence (Mu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to B\u0026uuml;ke et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), (p)ppGpp serves as a critical regulator that coordinates cell size and growth control, lipid synthesis and other anabolic processes to maintain the integrity of the cell envelope in response to nutrient abundance or starvation (Gaca et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gonzalez and Collier \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Imholz et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGram-positive bacteria, such as \u003cem\u003eL. vini\u003c/em\u003e, have a thick and sophisticated cell wall composed of peptidoglycan, surface-anchored proteins, teichoic acids, lipoteichoic acids and lipoproteins, which protect the bacteria from environmental stress (Rajagopal and Walker \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The major component of the cell wall is the peptidoglycan sacculus, a large glycan polymer composed of linear strands of alternating N-acetylglucosamine (GlcNac) and N-acetylmuramic acid (MurNac) units cross-linked by peptides composed of four to five amino acids of D or L configurations (Pasquina-Lemonche et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The cell wall is a dynamic structure whose composition changes in response to varying environmental conditions in order to ensure cell survival. For instance, the tolerance to acid stress by HCl in \u003cem\u003eL. vini\u003c/em\u003e is accompanied by a reorganisation of the bacterial cell wall which involves the down-expression of biosynthetic genes and over-expression of degradation genes (Mendon\u0026ccedil;a et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been proposed that this mechanism increases the mechanical resistance of the cell at the same time that provides internal glucose for maintenance energy during cell growth arrest (Alc\u0026aacute;ntara and Z\u0026uacute;\u0026ntilde;iga \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mendon\u0026ccedil;a et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Nevertheless, the involvement of the SR mechanism in this process remains to be elucidated.\u003c/p\u003e \u003cp\u003eThe control of bacterial metabolism via the SR mechanism depends on the production of the signalling molecule (p)ppGpp by a series of alarmone synthetases. Among them, the RelA protein is a bifunctional enzyme by alternating phosphotransferase and pyrophosphohydrolase activities for synthesis and degradation, respectively, during cell growth phase (Atkinson et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bange et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, it is paramount to study the function of this gene and its protein on the metabolism and stress response of \u003cem\u003eL. vini\u003c/em\u003e in order to understand its adaptive mechanisms to industrial processes, such as the bioethanol fermentation. Thus, this work presents for the first time the inactivation of \u003cem\u003erelA\u003c/em\u003e in a representative of the \u003cem\u003eLiquorilactobacillus\u003c/em\u003e genus and reveals the unexpected role of RelA protein on the cell wall homeostasis and cell size in this genus. The environmental and industrial implications of this finding are discussed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and genetic modification\u003c/h2\u003e \u003cp\u003eThe strain JP 7.8.9 of \u003cem\u003eL. vini\u003c/em\u003e was isolated from a bioethanol fermentation process (de Lucena et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eE. coli\u003c/em\u003e DH10B was used as an intermediate host for cloning purposes. \u003cem\u003eLactococcus lactis\u003c/em\u003e NZ9000 was used as a host for plasmid pNG8048e (Kuipers et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). These bacteria were cultivated and maintained in MRS (Difco) at 37\u0026deg;C, LB (Oxoid) at 37\u0026deg;C and M17 (Oxoid) media supplemented with 5 g/L glucose at 30\u0026deg;C, respectively. Antibiotics were supplemented when required as follows: ampicillin at 100 \u0026micro;g/mL for \u003cem\u003eE. coli\u003c/em\u003e, chloramphenicol and erythromycin at 5 \u0026micro;g/mL for \u003cem\u003eL. lactis\u003c/em\u003e and erythromycin at 5 \u0026micro;g/mL for \u003cem\u003eL. vini\u003c/em\u003e. Genomic DNA from strain JP 7.8.9 was extracted with the AxyPrep\u0026trade;Bacterial Genomic DNA Purification Miniprep kit (Axigen) following the manufacturer\u0026rsquo;s guidelines. The inactivation cassette was cloned in the pRV300 vector (Leloup et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003erelA\u003c/em\u003e gene nucleotide sequence was identified from the \u003cem\u003eL. vini\u003c/em\u003e genome by using RAST server program and used for primer design. A \u003cem\u003eXhoI\u003c/em\u003e restriction site (5\u0026rsquo;-TTTTACTCGAGAGGGCGATGTCTTGGAGTTG-3\u0026rsquo;) and a \u003cem\u003eSpeI\u003c/em\u003e restriction site were added to the 5\u0026rsquo; end of the forward (5\u0026rsquo;-TTTTACTCGAGAGGGCGATGTCTTGGAGTTG-3\u0026rsquo;) and reverse (5\u0026rsquo;-TTTTACTAGTTTGATGATCCCCAAGGGTGC) primers, respectively. These primers were used to amplify an amplicon of 445 bp corresponding to the internal region of the \u003cem\u003erelA\u003c/em\u003e gene (from nucleotide 1202 to nucleotide 1646 of the gene) by PCR from \u003cem\u003eL. vini\u003c/em\u003e genomic DNA, purified by using the QIAquick PCR purification kit (Qiagen), and quantified using a Nanodrop\u0026reg; device. Plasmid pRV300 was extracted by the QIAGEN Plasmid Midi kit from \u003cem\u003eE. coli\u003c/em\u003e DH10B following the manufacturer\u0026rsquo;s guidelines. Purified pRV300 and \u003cem\u003erelA\u003c/em\u003e fragment were digested with \u003cem\u003eXhoI\u003c/em\u003e and \u003cem\u003eSpeI\u003c/em\u003e, purified, quantified and ligated following the conventional DNA cloning protocols. The integration vector was introduced into the cells of \u003cem\u003eE. coli\u003c/em\u003e DH10B by electroporation. Cells were subsequently spread on LB medium plates containing ampicillin at 100 \u0026micro;g/mL, IPTG at 0.1 mM and X-gal at 80 \u0026micro;g/mL according to standard protocol. Transformant colonies were picked and cultivated in LB plus ampicillin. Plasmids were extracted as indicated above and the interruption of the \u003cem\u003erelA\u003c/em\u003e gene was checked by PCR. The resulting plasmid was named pRVrelA.\u003c/p\u003e \u003cp\u003eA protocol for \u003cem\u003eL. vini\u003c/em\u003e transformation was standardized as follows: JP7.8.9 cells were cultivated in MRS broth for 24 h at 37 \u003csup\u003eo\u003c/sup\u003eC and used to inoculate MRS broth containing glycine (from 10 to 100 g/L) to an initial optical density (OD) of 0.1 at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e). The cultures were incubated at 37 \u003csup\u003eo\u003c/sup\u003eC until reaching 0.6 OD\u003csub\u003e600\u003c/sub\u003e and the cell were collected by centrifugation at 5000 g at 4 \u003csup\u003eo\u003c/sup\u003eC, and resuspended to the same culture volume in transformation buffer (0.3 M sucrose, 5 mM sodium phosphate pH 7.4 and 1mM MgCl\u003csub\u003e2\u003c/sub\u003e in Milli-Q water). This washing procedure was repeated twice and the cells were finally re-suspended in transformation buffer to 1/100 of the initial volume, dispensed in 50 \u0026micro;L aliquots and stored at -80 \u003csup\u003eo\u003c/sup\u003eC until use. The transformation efficiency of the electrocompetent \u003cem\u003eL. vini\u003c/em\u003e cells was tested with the plasmid pNG8048e at concentrations ranging from 100 ng to 500 ng. The electroporation conditions were set to: 100 to 400 Ω parallel resistance, 25 \u0026micro;F capacitance and 1,000 to 2,000 V pulse voltage. Following the electroporation, the cells were mixed in the cuvette with the recovery medium consisting of MRS supplemented with CaCl\u003csub\u003e2\u003c/sub\u003e (2 mM), MgCl\u003csub\u003e2\u003c/sub\u003e (20 mM) and sucrose (300 mM) and transferred to microtubes. The cell suspensions were incubated for 3 h at 37 \u003csup\u003eo\u003c/sup\u003eC and then plated on MRS plate containing erythromycin (5 \u0026micro;g/mL). The plates were incubated for five days at 37 \u003csup\u003eo\u003c/sup\u003eC in anaerobic jars and the number of transformant colonies was recorded to stablish the best electroporation setup condition for subsequent transformation with pRVrelA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell growth and cell size analysis\u003c/h2\u003e \u003cp\u003eCells grown in MRS broth for 24 hours were used to inoculate aliquots of 150 \u0026micro;L of MRS dispensed in a microtiter plate to an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.05. The plates were incubated in a Sinergy HT multireader device (Biotek, Switzerland) at 37\u0026deg;C and the variation of culture OD\u003csub\u003e600\u003c/sub\u003e was recorded automatically every 30 min for 48 hours.\u003c/p\u003e \u003cp\u003eSamples were collected from the exponential growth phase and the cell size measurements were made at a 2000\u0026times; magnification in a Nikon Eclipse Ni-U microscope with bright field optics, and photomicrographs were taken using DIC optics with a Nikon DS-Fi2 camera. 20 cells for strains JP 789 and its isogenic Δ\u003cem\u003erelA\u003c/em\u003e mutant were measured and mean differences were assessed by using the Student's t-test. Normality of data distribution was checked with the Shapiro Wilk test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell lysis experiments\u003c/h2\u003e \u003cp\u003eCells from exponential growth phase in MRS medium (1.0 OD\u003csub\u003e600\u003c/sub\u003e) were collected by centrifugation and resuspended to one-tenth of the original volume in STE buffer (300 mM sucrose, 100 mM Tris-HCl, and 50mM EDTA-NaOH, pH 8). Aliquots of 150 \u0026micro;l of the buffered cell suspensions were transferred to a microtiter plate and incubated at 37\u0026deg;C. OD variations were recorded every 30 min for 10 h in a Sinergy HT multireader device to check for cell integrity. Alternatively, the cells were suspended in STE buffer containing lysozyme at 10 mg/ml to test the resistance to this lytic enzyme. The experiments were performed in biological duplicate with technical triplicates. Therefore, the numbers represent the mean value of six measurements (\u0026plusmn;\u0026thinsp;SD) for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, cDNA synthesis and gene expression\u003c/h2\u003e \u003cp\u003eFor RNA isolation, cells were suspended in 1 mL MRS to 3.5 OD\u003csub\u003e600\u003c/sub\u003e. After three hours at 37\u0026deg;C, cells were collected by centrifugation and subjected to cell lysis by lysozyme (Mendon\u0026ccedil;a et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Afterwards, total RNA was extracted with TRIzol\u0026reg; LS (Invitrogen Corp.) according to the manufacturer\u0026rsquo;s instructions (Oberg et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The upper phase was collected in new tubes and the RNA was purified with the illustra RNAspin Mini RNA Isolation kit (GE Healthcare) following the manufacturer\u0026rsquo;s instructions. RNA integrity was evaluated directly on 1 % agarose gel prepared with TAE buffer (40 mM of tris, 10 mM of EDTA, 20 mM of acetic acid) and stained with 0.5 mg ml\u0026thinsp;\u0026minus;\u0026thinsp;1 of ethidium bromide, and its concentration was determined in a Nanodrop spectrophotometer (Thermo Fisher, USA). RNA samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eThe cDNA was synthetized with the ImProm-II Reverse Transcription System (Promega, USA), following the manufacturer\u0026rsquo;s instructions. Each reaction was performed with 0.5 \u0026micro;g of RNA in a final volume of 20 \u0026micro;l. RT negative controls were also prepared with the same amount of RNA diluted in water (same concentration as cDNA sample) in order to estimate contamination with genomic DNA. The samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. The cDNA was used for qPCR in biological duplicates and technical triplicates. Expression of genes involved in transcription regulation (\u003cem\u003erpoD, rpoE, sigV\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e), DNA maintenance (\u003cem\u003erecA\u003c/em\u003e, \u003cem\u003epcrA\u003c/em\u003e and \u003cem\u003emutL\u003c/em\u003e) and translation elongation (\u003cem\u003efusA\u003c/em\u003e), in teichoic acid biosynthesis (\u003cem\u003edltD\u003c/em\u003e), peptidoglycan precursor biosynthesis (\u003cem\u003eglmS\u003c/em\u003e, \u003cem\u003eglmU\u003c/em\u003e and \u003cem\u003emurB\u003c/em\u003e), cell wall assembly (\u003cem\u003epbp1ABα\u003c/em\u003e and \u003cem\u003epbp1ABb\u003c/em\u003e), cell wall degradation (\u003cem\u003enagA\u003c/em\u003e), alanine metabolism (\u003cem\u003eddl, alaR\u003c/em\u003e and \u003cem\u003ealaT\u003c/em\u003e) and \u003cem\u003epbpX.\u003c/em\u003e The Cq values of each replicate from reference genes and tested genes were used for normalization and relative expression quantification according to MIQE guidelines (Vandesompele et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Huggett et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Normalization data are available in the supplementary material. The candidate genes for data normalization were the same as those used by Mendon\u0026ccedil;a et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and, among them, \u003cem\u003efusA\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e genes were used in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFlagellum presence and motility assay\u003c/h2\u003e \u003cp\u003eTo confirm the presence of the \u003cem\u003eL. vini\u003c/em\u003e flagellum, fluorescence microscopy was used. Widefield microscopy was performed using an arc for excitation and a charge-coupled device camera for detection (set of blue filters and 100x objective). To prepare the slides, cells were grown in MRS broth (24 hours at 37\u0026deg;C), washed with saline solution (0.9% sodium chloride), 20 \u0026micro;l aliquots were deposited onto glass slides and incubated at room temperature for 30 minutes. A total of 2 \u0026micro;L of DAPI solution (4.6diamidino2-phenylindole) or Calcofluor White and 10% (w/v) potassium hydroxide were deposited on the slides so that the staining covered the entire site of initial bacterial deposition. The slides were incubated in darkness at room temperature for 20 minutes. The images were captured using a Hamamatsu CCD camera coupled to an Olympus BX51 epifluorescence microscope or Leica DM5500B fluorescence microscope.\u003c/p\u003e \u003cp\u003eFor the motility assay, the parental JP 789 strain and its mutant strain Δ\u003cem\u003erelA\u003c/em\u003e were inoculated in MRS agar (1.5%) Petri dishes. Aliquots of 2 \u0026micro;l of the bacterial cultures were added to the centre of the plate. Subsequently, semi-solid MRSagar (0.5%) medium was added and the plates were incubated at 37\u0026deg;C for 48 hours. The assay was performed in duplicate and the halos formed were measured with a vernier caliper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic resistance\u003c/h2\u003e \u003cp\u003eMinimal inhibitory concentrations (MICs) of ampicillin, amoxicillin, imipenem, penicillin G and cephalothin were determined for \u003cem\u003eL. vini\u003c/em\u003e JP7.8.9 and Δ\u003cem\u003erelA\u003c/em\u003e strains. Bacterial cells were cultivated on MRS broth for 24 h and subsequently diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.002 with MRS broth. The MIC was determined by the micro-dilution method starting with 2 \u0026micro;g/mL of antibiotic and followed by serial dilutions until 0.031 \u0026micro;g/mL. MIC value was defined at the first concentration that completely inhibited bacterial growth. The strain of \u003cem\u003eEnterococcus. faecalis\u003c/em\u003e ATCC 29212 was used as a sensitive reference (CLSI \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The plates were incubated for 48 h at 37\u0026deg;C. All the tests were performed in duplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLiquorilactobacillus vini\u003c/strong\u003e \u003cstrong\u003etransformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the construction of the isogenic \u0026Delta;\u003cem\u003erelA\u003c/em\u003e mutant strain, the first step was to establishes a transformation procedure for \u003cem\u003eL. vini\u003c/em\u003e JP 7.8.9. The highest number of transformants with the reference plasmid pNG8048e was 38 transformants/\u0026micro;g of plasmid DNA input. This was achieved through the condition and parameters set as: glycine at 20 g/L in MRS, for the preparation of electrocompetent cells, and the electroporation setup of 200 Ω of resistance, 25 \u0026micro;F capacitance and 1.500 V pulse voltage. Decreasing of glycine concentrations, voltage pulse above 1.750 V or resistance below 100 Ω abolished the transformability completely. The confirmation of \u003cem\u003eL. vini\u003c/em\u003e transformant with the reference plasmid were performed by plasmid purification followed by digestion with \u003cem\u003eBam\u003c/em\u003eHI. This enzyme was chosen due the doble restriction site in the reference plasmid which delivers a restriction fragments size profile specific. The agarose gel electrophoresis showed the presence of the pNG8048e plasmid in all transformant cells tested (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, we established for the first time an efficient protocol for transformation of \u003cem\u003eL. vini.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis protocol was employed to inactivates the \u003cem\u003erelA\u003c/em\u003e gene in the wild-type strain with the pRV300 vector bearing 445 pb of internal region of the target gene. The recombination between the internal region in the vector and the homologous region in the target gene inserted the whole plasmid construction and disrupts the coding sequence of the gene. This allows the integrated vector to be replicated and segregated through the cell division and turn these cells resistant to erythromycin due the \u003cem\u003eerm\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e gene in the vector. As the pRV-300 cannot replicates in \u003cem\u003eL. vini\u003c/em\u003e because the lack of the \u003cem\u003erepA\u003c/em\u003e gene, the drug resistance could only arise in the tranformants through genomic integration. The efficiency of transformation with this integrative plasmid was 10 transformants/\u0026micro;g. To confirm the site of integration, were performed a PCR reaction with the same primers employed in the \u003cem\u003erelA\u003c/em\u003e target region amplification and primers targeting the gene \u003cem\u003edltB\u003c/em\u003e. In the wild-type strains the PCRs for both targeted gene were positive but to the \u003cem\u003erelA\u003c/em\u003e mutants only was possible to amplify the fragment of the \u003cem\u003edltB\u003c/em\u003e indicating the gene structure alteration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e \u003cstrong\u003erelA\u003c/strong\u003e \u003cstrong\u003emutant cells are more elongated and display slow growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth and division are central to cell size and in the present work we verified the importance of (p)ppGpp to these parameters in \u003cem\u003eL.vini\u003c/em\u003e. Growth curve profile in MRS (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) showed that \u0026Delta;\u003cem\u003erelA\u003c/em\u003e grew slower (growth rate of 0.07 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 9 h 50 min of doubling time) than the parental strain JP 789 (growth rate of 0.28 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 2 h 27 min of doubling time). Furthermore, microscopic analyses revealed that the mutant cells are more elongated than the parental cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), with mean values of 2.8 and 1.8 \u0026micro;m in length, respectively. Statistical analysis revealed the significance of cell size differences p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eFollowing the microscopic inspection of the bacterial morphology, we noticed that the flagellum that was present in the parental strain JP7.8.9 was not observed in the \u0026Delta;\u003cem\u003erelA\u003c/em\u003e mutant strain (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA,D). In addition, the motility assay also pointed out that the \u0026Delta;\u003cem\u003erelA\u003c/em\u003e strain lost the agility of movement in the semi-solid culture medium, due to the impact that the absence of the \u003cem\u003erelA\u003c/em\u003e gene caused in the biosynthesis of the flagella and/or anchoring of the flagella (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003eAs previously stated, alarmones are a pleiotropic bacterial regulator that reprograms growth under stressful conditions. The low growth rate and morphological alterations displayed by the \u0026Delta;\u003cem\u003erelA\u003c/em\u003e mutant (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) suggested that the homeostasis of the bacterial metabolism was possibly also affected by the absence of RelA protein, even in the absence of stressing conditions. In view of this, we tested the expression of genes that encode transcription factors known to influence cell growth. The relative expression analysis revealed that the lack of the RelA protein led to the overexpression the genes \u003cem\u003erpoD\u003c/em\u003e (encoding the sigma factor 70), \u003cem\u003esigV\u003c/em\u003e (encoding the sigma factor 5) and \u003cem\u003erpoB\u003c/em\u003e (encoding the \u0026beta; subunit of the bacterial RNA polymerase) relative to its parental strain (Fig. 4). These genes are related to cell maintenance, extracytoplasmic stress and gene transcription, respectively. Conversely, there was a down-expression of \u003cem\u003erpoE\u003c/em\u003e gene that encodes the sigma factor 24 (Fig. 4), which was shown to respond to heat shock. Unexpectedly, it was observed the overexpression of \u003cem\u003emutL\u003c/em\u003e gene, whose protein works to the mismatch repair mechanism, and the down-expression of \u003cem\u003efusA\u003c/em\u003e gene that encodes the elongation factor involved in protein translation (Fig. 4). The altered levels of all these housekeeping genes may have triggered a metabolic disorganization that might account for the phenotypic traits of the \u0026Delta;\u003cem\u003erelA\u003c/em\u003e mutant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbsence of\u003c/strong\u003e \u003cstrong\u003erelA\u003c/strong\u003e \u003cstrong\u003egene affected cell wall homeostasis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe observed increased cell size and the lack of flagellum in \u0026Delta;\u003cem\u003erelA\u003c/em\u003e strain led us to determine the expression of genes involved in cell wall biogenesis (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). It was found that the genes involved in alanine metabolism (\u003cem\u003ealaT\u003c/em\u003e and \u003cem\u003eddl\u003c/em\u003e) were down-expressed, with emphasis on \u003cem\u003eddl\u003c/em\u003e, which was 5 times less expressed in \u0026Delta;\u003cem\u003erelA\u003c/em\u003e compared to the parental strain. The \u003cem\u003ealaR\u003c/em\u003e gene showed equal expression in the parent and the mutant. Genes \u003cem\u003egmlS\u003c/em\u003e and \u003cem\u003egmlU\u003c/em\u003e were over expressed in \u0026Delta;\u003cem\u003erelA\u003c/em\u003e. However, \u003cem\u003emurB\u003c/em\u003e and \u003cem\u003edltD\u003c/em\u003e genes were up-regulated, as were the \u003cem\u003epbp1AB\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003epbp1AB\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/sup\u003e involved in the structuring of the cell wall, On the other hand, \u003cem\u003enagA\u003c/em\u003e and \u003cem\u003epbpX\u003c/em\u003e genes were down-regulated.\u003c/p\u003e\n\u003cp\u003eWe next tested the response of the \u0026Delta;\u003cem\u003erelA\u003c/em\u003e mutant to stressing conditions, since RelA protein would be involved in the SR regulatory mechanism that also involves stress response. During the preliminary assays to set up the conditions for stress tolerance tests, we noticed a constant drop in the population of viable cells of the \u0026Delta;\u003cem\u003erelA\u003c/em\u003e strain when suspended in buffer even before starting exposure to stressors. This observation, together with the fact that genes involved in cell wall maintenance were affected by the absence of RelA, led us to analyse the stability of the bacterial cell wall by quantifying the rate of spontaneous cell lysis of the strains. Cells grown in MRS were collected in the exponential phase of growth, resuspended in STE buffer and the suspension was immediately evaluated for variation in cell density. The data clearly showed that mutant strain cell suspensions presented a significant drop in OD value while the parental strain maintained a practically constant OD throughout 10 h of incubation (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating the indeed the absence of RelA protein fragilizes the bacterial cell wall.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e \u003cstrong\u003erelA\u003c/strong\u003e \u003cstrong\u003estrain is more resistant to \u0026beta;-lactam\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e showed that the absence of RelA protein increases the expression of two PBP proteins involved with transglycosylation and transpeptidation reactions for the construction of the bacterial cell wall. These proteins are the target of \u0026beta;-lactam antibiotics. Thus, MIC values to \u0026beta;-lactam antibiotics for both parental and mutant cells were determined. The results showed very consistent increments in MICs for five different \u0026beta;-Lactams in the mutant cells compared to the parental JP 789, varying from 2x for penicillin to 8x for cephalothin (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the overexpression of PBP gene coincided with the increment of cell resistance to \u0026beta;-Lactams antibiotics.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMinimal Inhibitory Concentration (MIC) of antibiotics affecting the cell wall synthesis of \u003cem\u003eLiquorilactobacillus vini\u003c/em\u003e JP7.8.9 and its isogenic mutant \u0026Delta;\u003cem\u003erelA\u003c/em\u003e mutant. The results represent the values of six experiments for each antibiotic in microtitration plates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAntibiotic\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSubclass\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eJP7.8.9\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003erelA\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePenicillin G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePenicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePenicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmoxicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePenicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCephalothin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCephalosporin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eImipenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarbapenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe SR mediated by the alarmone (p)ppGpp is a survival system under adverse conditions and has been characterized in model organisms such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e. However, the function of these alarmones goes far beyond the SR, they may also control the cell cycle (B\u0026uuml;ke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), growth (Mu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and antibiotic resistance (Salzer and Wolz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In lactic acid bacteria such as \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, the alarmones are involved in the classic SR (Abranches et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and their absence results in changes in the fermentative profile, production of high levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and causes unbalancing of the cell metabolism (Gaca et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In \u003cem\u003eL.vini\u003c/em\u003e strain defective for the \u003cem\u003erelA\u003c/em\u003e gene, it was observed phenotypes under non-limiting conditions that go beyond a SR. The growth rate was slowed down cell size was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). B\u0026uuml;ke et al (B\u0026uuml;ke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) defined in their work with \u003cem\u003eE. coli\u003c/em\u003e that decreasing ppGpp production resulted in progressively slower growth and larger cells and considered that changes in ppGpp concentration dynamically affected cell size and its control.\u003c/p\u003e \u003cp\u003eIn the present work, we showed that the absence of the RelA protein in \u003cem\u003eL.vini\u003c/em\u003e also modulated the expression of genes related to cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). \u003cem\u003efusA\u003c/em\u003e gene was down-regulated in the \u003cem\u003erelA\u003c/em\u003e mutant. This gene encodes the elongation factor G (EF-G) in bacteria which is an essential component of the protein translation machinery (Nor\u0026eacute;n et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and directly impacts growth. In contrast, the gene encoding the sigma 70 factor responsible for the transcription of maintenance genes was overexpressed in \u003cem\u003eL.vini relA\u003c/em\u003e mutant. The Δ\u003cem\u003erelA\u003c/em\u003e strain also showed, even in the absence of DNA damage agents, the overexpression of \u003cem\u003emutL\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Its product MutL is a key component of the DNA mismatch repair system (Putnam \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003emutL\u003c/em\u003e overexpression may be indicative of problems in DNA replication in Δ\u003cem\u003erelA\u003c/em\u003e cells, which might partly explain the low growth of the Δ\u003cem\u003erelA\u003c/em\u003e strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacterial flagella are synthesized by systems strictly controlled by nutritional and environmental conditions (Lemke et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eL. vini\u003c/em\u003e was described as a mobile bacterium (Rodas et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In agreement with this, strain JP 7.8.9 also possesses a flagellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B) that allows the cells to spread on the surface of semi-solid media (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). However, this flagellum was lost or reduced beyond our detection limit when \u003cem\u003erelA\u003c/em\u003e gene was interrupted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D), limiting the mobility capacity of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Studies on \u003cem\u003eE. coli\u003c/em\u003e have shown that motility is closely linked to the cell growth rate (Sim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, it was shown that ppGpp can inhibit the expression of genes in the flagellar building cascade during the stationary phase and after starvation, thus coordinating the assembly of the flagella (Lemke et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, the lack of \u003cem\u003espoT\u003c/em\u003e, which also synthesizes (p)ppGpp, resulted in the loss of the flagellum (Yin et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and a previous study revealed that the swimming ability of \u003cem\u003eV. alginolyticus\u003c/em\u003e Δ\u003cem\u003erelA\u003c/em\u003e Δ\u003cem\u003espoT\u003c/em\u003e double mutant decreased dramatically (Yin et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Since \u003cem\u003eL. vini\u003c/em\u003e apparently does not encode a \u003cem\u003espoT\u003c/em\u003e homolog (de Lucena et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the single Δ\u003cem\u003erelA\u003c/em\u003e mutant should behave like Δ\u003cem\u003erelA\u003c/em\u003e Δ\u003cem\u003espoT\u003c/em\u003e double mutants in other bacterial species. However, the effect of \u003cem\u003erelA\u003c/em\u003e gene interruption in \u003cem\u003eBacillus subitilis\u003c/em\u003e was just the opposite, with the mutant cells showing more flagella and turning more mobile, despite the low growth rate (Ababneh and Herman \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, a classic work published in 1969 by Vaituzis and Doetsch pointed out the relationship between the cell wall, cytoplasmic membrane, and bacterial motility, and explained the importance of the cell wall in anchoring bacterial flagella (Vaituzis and Doetsch \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Hence, the loss of the flagella might also be linked to the apparent weakening of the cell wall also observed in the Δ\u003cem\u003erelA\u003c/em\u003e strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a previous report, we showed that treatment of \u003cem\u003eL. vini\u003c/em\u003e cells with organic acids, as well as with hydrochloric acid, induced a restructuring of the bacterial cell wall as a consequence of the differential regulation of key genes (Mendon\u0026ccedil;a et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Some of these genes were tested in this work for their basal expression in a Δ\u003cem\u003erelA\u003c/em\u003e strain relative to the parental strain, given the suspicion that the absence of the RelA protein weakens the bacterial cell wall. The results obtained showed that \u003cem\u003emurB\u003c/em\u003e, \u003cem\u003ealaT\u003c/em\u003e and \u003cem\u003eddl\u003c/em\u003e genes were under-expressed in the mutant strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), especially \u003cem\u003eddl\u003c/em\u003e. The \u003cem\u003emurB\u003c/em\u003e gene encodes a UDP-N-acetylmuramate dehydrogenase involved in the biosynthesis of N-acetylmuramic acid (Matsuo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e); \u003cem\u003ealaT\u003c/em\u003e encodes the alanine aminotransferase, which catalyses reversible transamination between alanine and α-ketoglutarate, forming pyruvate and glutamate (Welch et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1975\u003c/span\u003e); \u003cem\u003eddl\u003c/em\u003e encodes a D-alanyl-D-alanine ligase involved in the peptidoglycan crossbridge peptide synthesis. Ddl is an essential protein for the proper growth and development of bacterial cell walls (Pederick et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These three enzymes are directly involved in the biogenesis of peptidoglycan. Thus, the observed lysis profile of \u003cem\u003eΔrelA\u003c/em\u003e could be explained by the weakening of the cell wall, although the induction of endogenous lysines or activation of prophages in the mutant strain cannot be discharged. However, the results in expression of several genes involved in cell wall synthesis and maintenance and increased resistance to b-lactam antibiotics were indicative of alterations in the bacterial cell wall.\u003c/p\u003e \u003cp\u003eIn this hypothesis, failure in the correct construction of this cell component by the down-expression of relevant genes would lead to the weakening of the cell wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Besides, it was observed the overexpression of \u003cem\u003egmlS\u003c/em\u003e (encoding a glucosamine synthase), \u003cem\u003egmlU\u003c/em\u003e (glucosamine-1-phosphate N-acetyltransferase) and \u003cem\u003edltD\u003c/em\u003e (in this work representing the operon \u003cem\u003edlt\u003c/em\u003e involved in the biosynthesis of teichoic acid), indicating the synthesis of N-acetylglucosamine. However, the down expression of \u003cem\u003emurB\u003c/em\u003e gene might interfere in the synthesis of UDP-MurNAC as essential component of PG formation. Moreover, overexpression of \u003cem\u003epbp1ABα\u003c/em\u003e and \u003cem\u003epbp1ABb\u003c/em\u003e, especially the second gene, was also observed (Fig.\u0026nbsp;4). These genes encode a transglycosylation and a transpeptidation enzymes, respectively, involved in the cross-linking of the peptides to the glucan chain in the cell wall structure. Together, the overexpression of these genes might represent a counter-action mechanism triggered in the mutant cells to maintain the integrity of the peptidoglycan in face of the shortage of N-acetylmuramic acid and D-alanyl-D-alanyl dipeptide used to build-up a stiff cell wall. A study with \u003cem\u003eStreptococcus pneumonie\u003c/em\u003e, lactic acid bacteria, showed that the enzymes responsible for cell wall biosynthesis are connected to the alarmone through their ability to improve errors in protein translation (Aggarwal et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eIn L.vini\u003c/em\u003e Δ\u003cem\u003erelA\u003c/em\u003e mutant, the protein elongation factor was negatively regulated (Fig.\u0026nbsp;4), but the correlation between these two factors still seems uncertain.\u003c/p\u003e \u003cp\u003eΔ\u003cem\u003erelA\u003c/em\u003e mutant showed resistance to the tested beta-lactams (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and simultaneously, greater expression of the genes encoding PBPs proteins due to defects in the cell wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). There are many mechanisms for bacteria to resist the action of β-lactams, a class of antibiotics that primarily acts on PBPs (Penicillin-Binding Proteins) (Sun et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Interestingly, the inactivation of RelA together with the overexpression of the \u003cem\u003epbp\u003c/em\u003e genes provided a moderate increase in resistance to β-lactam antibiotics, in opposition to what has been seen in other bacteria (Das and Bhadra \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Salzer and Wolz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, the increased number of antibiotic-targeted PBPs substrates would account for this antibiotic resistance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe role of (p)ppGpp alarmones in controlling cell cycle, cell growth and stress response in connection with the nutritional and physic-chemical conditions of the environment is well documented for Gram negative and positive bacteria. However, little information is reported for \u003cem\u003eLactobacillus\u003c/em\u003e sensu lato, including the remaining species of old genus \u003cem\u003eLactobacillus\u003c/em\u003e and the derived species recently allocated in new genera, as it is the case of \u003cem\u003eL. vini\u003c/em\u003e. In the present work, we revealed for the first time the relevance of this sensor for the maintenance of the cell wall structure and for the synchrony between cell cycle and cell division in \u003cem\u003eL. vini\u003c/em\u003e. However, the question that remains open is whether cell wall structural alterations are affecting cell division or whether metabolic deficiency due to the absence of RelA would affect both cell wall structure and cell division regulation. Experiments are underway to determine the influence of the \u003cem\u003erelA\u003c/em\u003e gene, and consequently the production of alarmone, on the carbohydrate and energy metabolism of \u003cem\u003eL. vini\u003c/em\u003e, which will contribute to the understanding of this sensor in the control of the cell cycle.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information.\u003c/strong\u003e The online version contains supplementary material available at https://doi.org/\u0026hellip;\u0026hellip;...\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e DSS and AAM carried out the experiment, analysed the data, prepared the draft version of the manuscript and prepared figures and tables. CE carried out the experiment and analysed the data. MZC analysed the data and revised the final version of the manuscript. MAMJ analysed the data, prepared the draft version and revised the final version of the manuscript. All authors reviewed and approved the manuscript for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e DSS acknowledge the Funda\u0026ccedil;\u0026atilde;o de Amparo a Ci\u0026ecirc;ncia e Tecnologia do Estado de Pernambuco (FACEPE) for the PHD scholarship support (grant IBPG-0670-2.02/22). This research was funded with resouces from Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (grant 441640/2023-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This research article does not include any human or animal studies performed by any author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e Informed consent is not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbabneh QO, Herman JK (2015) RelA Inhibits Bacillus subtilis Motility and Chaining. 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Microbiol Res 253:126883. https://doi.org/10.1016/j.micres.2021.126883\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Stringent response, (p)ppGpp, Lactic acid bacteria","lastPublishedDoi":"10.21203/rs.3.rs-4252796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4252796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of (p)ppGpp alarmone produced by RelA protein is well documented as a response to stress and nutritional deficiency in bacteria. However, little information is reported for \u003cem\u003eLactobacillus\u003c/em\u003e sensu lato, including the remaining species of old genus \u003cem\u003eLactobacillus\u003c/em\u003e and the derived species recently allocated in new genera. The present study aimed to characterize for the first time the effect of the inactivation of \u003cem\u003erelA\u003c/em\u003e in a representative of the \u003cem\u003eLiquorilactobacillus\u003c/em\u003e genus. Results obtained have revealed an unexpected role of RelA protein on the cell wall homeostasis and cell size. \u003cem\u003eL.vini ΔrelA\u003c/em\u003e showed low growth and increased cell size, related to overexpression of genes responsible for DNA transcription and repair and down-expression of the \u003cem\u003efusA\u003c/em\u003e gene. The low growth also resulted in the loss of the flagellum in Δ\u003cem\u003erelA\u003c/em\u003e, which may also be associated with the fragility of the cell wall, ease of lysis and resistance to beta-lactams of the Δ\u003cem\u003erelA\u003c/em\u003e mutant. Cell wall homeostasis was deregulated mainly by the down-expression of the gene encoding alanine ligase and \u003cem\u003emurB\u003c/em\u003e, which may have triggered the overexpression of the genes encoding PBPs proteins. Growth data suggest that the absence of the RelA protein promoted a disconnection between the cell cycle and cell division in \u003cem\u003eL. vini\u003c/em\u003e, and a consequent reduction in the growth rate of the culture, cell wall fragility and beta-lactam resistance, expanding the RelA function in \u003cem\u003eLiquorilactobacillus\u003c/em\u003e beyond the stringent response.\u003c/p\u003e","manuscriptTitle":"Beyond the stress response: absence of RelA protein hampers the cell wall structuring and cell size in Liquorilactobacillus vini","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 17:57:56","doi":"10.21203/rs.3.rs-4252796/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d3dcd45-38b5-449e-b1a0-9a99b39c5061","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-15T13:21:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 17:57:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4252796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4252796","identity":"rs-4252796","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}