{"paper_id":"bf0b8c16-efca-428b-9936-43613e1e487d","body_text":"Pseudomonas aeruginosa  is an opportunistic bacterial pathogen for causing secondary infections in hospital settings, raising significant global public health concerns as highlighted in the WHO’s 2024 bacterial priority pathogens list [ 1 ]. Its ability to form robust biofilms, to adapt swiftly, and to exhibit high levels of inherent resistance to antimicrobial has positioned it as a critical pathogen across a wide variety of natural and artificial environments, together with various indwelling medical devices [ 2 ]. Biofilm-associated infections pose a major threat to human health in the era of rising antimicrobial resistance [ 3 , 4 ]. Although the genetic mutations often underlie antimicrobial resistance, biofilms also offer an adaptive resistance. Even bacteria sensitive to antibiotics could escape the effect in a biofilm state, however, they may return to a susceptible form once the biofilm disperses [ 5 , 6 ].\nPopulation density-dependent cell–cell communication, referred to as quorum sensing (QS), is essential for the formation of biofilms by  P. aeruginosa  [ 7 , 8 ]. QS system plays a pivotal in the pathogenesis of  P. aeruginosa , starting from the initial colonization in the host to subsequent invasion, infection, spread, evasion of the immune response, and development of drug resistance [ 9 ]. The four primary and interconnected QS networks in  P. aeruginosa  include the Las, Rhl, Pqs, and Iqs systems. These systems function through a hierarchical framework, facilitating intricate interactions among various cellular signals [ 10 ]. The Las system occupies the highest position in this hierarchy, overseeing the regulation of the other QS systems, while the Rhl system is positioned beneath. The Pqs system activates the Rhl system and is under the regulation of Las, whereas Iqs governs both the Pqs and Rhl systems and is itself activated by Las [ 8 , 11 ]. Through this complex network, the activated Rhl system controls the production of various QS-related virulence factors [ 10 , 12 ]. The relationship between QS system and biofilm formation is indirectly influenced by the nature of motility, as well as the production of various components of biofilm matrix [ 5 ]. Swarming motility, characterized as a coordinated feature of surface movement, is particularly significant during the initial phase of biofilm development and is regulated by the Rhl system in  P. aeruginosa  [ 13 , 14 ].\nA complex regulatory network, acting via transcriptional, post-transcriptional, and post-translational processes in response to environmental and host-derived signals through its QS-system, modulates  P. aeruginosa ’s adaptability and pathogenicity [ 5 , 8 ]. The AlgU (σ 22 ) sigma factor, a stress response master regulator and functional counterpart of  Escherichia coli  σ E , coordinates nearly 300 regulatory genes and plays a crucial role in regulating synthesis of virulence factors and other infection-related processes by affecting QS network [ 15–18 ]. AlgU enhances alginate production by up-regulating the  algD  operon and activating transcription factors AlgR and AmrZ, those are pivotal for alginate synthesis in mucoid strains [ 16 , 17 ]. The transition from the nonmucoid to the mucoid phenotype is accompanied by a cascade of genetic changes resulting from  mucA  mutations [ 17 , 18 ]. Under stress-free conditions, AlgU is inhibited by MucA, whereas under stress AlgU becomes relieved to trigger alginate production.  mucA- mutant strains of  P. aeruginosa  have been reported to show increased alginate synthesis with increased biofilm forming ability and stress resistance [ 19–21 ]. The Rhl system of  P. aeruginosa  regulates biofilm formation through the RhlA protein, which synthesizes rhamnolipids, a glycolipid essential for maintaining the biofilm matrix [ 5 , 8 , 22 , 23 ]. The autoinducer binds to RhlR, resulting enhanced expression of  rhlA , which is crucial for the production of rhamnolipids [ 24 ]. Rhamnolipids not only support biofilm structure but also facilitate bacterial dispersion, allowing  P. aeruginosa  to occupy new ecological niche [ 25 , 26 ]. RsmA, another key regulator in  P. aeruginosa , belongs to the CsrA family of RNA-binding proteins, which has been reported to control virulence, motility, biofilm formation, and metabolism by interacting with the target mRNAs [ 27–29 ].\nRecently, non-coding small RNAs (sRNAs) have been reported to act as the crucial regulator for the adaptability of  P. aeruginosa , including biofilm development and pathogenesis [ 30–33 ]. The Gac/Rsm signaling cascade promotes the production of  RsmY  and  RsmZ , both subsequently relieve the repression by RsmA on the target genes, and thereby facilitates biofilm development [ 34 ]. Another sRNA,  PhrS , was reported to regulate the QS system regulator PqsR (MvfR), which is essential for biofilm formation via the PQS signaling pathway [ 35 ]. The  CrcZ  sRNA is the part of the carbon catabolite repression system and affects biofilm formation by inhibiting  Crc  expression, especially when carbon sources are limited [ 36 ]. Recently, regulatory role of  PA0730 .1 sRNA on the expression of different traits of  P. aeruginosa  has also been reported to be linked with pathogenicity and biofilm formation [ 32 ]. The  srbA  sRNA was earlier reported to up-regulate during the stationary phase and biofilm formation in  P. aeruginosa  PA14, though its precise function in biofilm regulation remained unclear [ 37 ]. More recently, it has been documented that  srbA  could regulate genes encoding the major enzymes involved in the TCA cycle, thus is responsible in nutritional adaptation in  P. aeruginosa  PAO1, which was further reported to be linked with the production of various virulence factors [ 33 ].\nWith this background, the present study was aimed to investigate the possible role of the  srbA  sRNA in biofilm formation and its regulatory influence on various biofilm-controlling factors by studying  srbA  deletion and overexpression strains to unveil its molecular targets. Understanding the role of  srbA  could reveal new insights into the regulation of biofilm development that could help to manage biofilm-associated infections by  P. aeruginosa .\n\nPseudomonas  genome database reveals the existence of a 239 bp span encoding  srbA  sRNA, resulting from the transcription of the reverse strand of a locus in between  aceA  and  PA2633  genes of  P. aeruginosa  PAO1 [ 33 , 38 ], that was earlier coined as  pant235  [ 39 ] and  PA2633 .1 [ 40 ]. Additionally,  srbA  sequence data obtained from PAO1 were reported as conserved among other strains of  P. aeruginosa , including that in PA14, termed as  PA14sr_067  [ 33 , 41 ]. Higher expression level of  srbA  was earlier documented under biofilm state of  P. aeruginosa  PA14 [ 37 ]; however, the regulatory role of  srbA  on biofilm development is still unclear.\nIn this study, expression levels of  srbA  sRNA in  P. aeruginosa  PAO1 were studied during substratum-attached biofilm and colony biofilm states and were compared with that in the mid-log planktonic stage. The RT-qPCR data revealed increase level of  srbA  by ~5.5-fold in colony biofilm and ~9.7-fold in substratum biofilm states in comparison with that in the planktonic cells ( Figure 1A ). Additionally, the abundance of  srbA  was quantitatively analyzed using  srbA  overexpression strain SrbA + ,  srbA  deleted strain ΔSrbA, and complementation of deletion strain ΔSrbApSrbA, and their respective control strains, pEV, wildtype (WT), and ΔSrbApEV. In planktonic state, the expression of the  srbA  was found to be ~5.1 and ~3.6-fold higher in SrbA +  and ΔSrbApSrbA, respectively, compared with the WT strain.\n( A ) Comparative  srbA  expression among the substratum-attached biofilm, colony biofilm, and mid-log phase planktonic growth. ( B )  srbA  expression in wildtype (WT), empty vector control (pEV), overexpression (SrbA + ), deletion (ΔSrbA), deletion empty vector control (ΔSrbApEV), and complementation of  srbA  deletion (ΔSrbApSrbA) strains under mid-log planktonic, substratum attached biofilm, and colony biofilm conditions. Fold changes in expression were calculated relative to the WT strain grown in planktonic conditions, regardless of growth state. The level of quantitative expression of  srbA  was determined using  rpoD  as the reference house-keeping gene for the normalization. Statistical analysis was performed using one-way ANOVA for each condition, comparing the mean of each group with that of the WT strain. Additionally, the expression levels of the WT strain in different conditions were compared across all groups. Data presented are the mean of three replicates with ±SEM. ‘Ud’ and ‘ns’ stand for undetermined and non-significant, respectively. Statistical significance is indicated by ** P <0.01 and **** P <0.0001.\nIn substratum-attached biofilm state,  srbA  levels were elevated by ~29.9 and ~19.1-fold in SrbA +  and ΔSrbApSrbA, respectively, compared with the WT strain. In contrast, abundance of  srbA  was increased by ~229.9 and ~211-fold in SrbA +  and ΔSrbApSrbA, respectively, in comparison with the WT strain under colony biofilm state. Within each growth condition, WT and pEV strains showed similar levels of  srbA  ( Figure 1B ).\nThe contribution of  srbA  on the biofilm formation in  P. aeruginosa  was analyzed using SrbA + , ΔSrbA, and ΔSrbApSrbA, and respective control strains pEV, WT, and ΔSrbApEV. Crystal violet assay for biofilm formation revealed that the SrbA +  produced significantly ~27% more biofilm compared to the WT, whereas deletion of  srbA  caused a significant decrease in biofilm formation by ~46%. Biofilm forming ability restored in ΔSrbApSrbA construct after plasmid-mediated reintroduction of  srbA  in deletion strain ( Figure 2A ). Viable cell mass within the biofilms, as measured by MTT assay, significantly declined (~37%) in ∆SrbA which was restored in ΔSrbApSrbA strain; however, no significant alteration in cell viability was observed due to overexpression of  srbA  compared with the WT strain ( Figure 2B ). The EPS content could indicate the amount of biofilm formed and was quantified using Congo red binding method. Overexpression of the  srbA  resulted ~15% increase in EPS production; on the contrary, it was sharply decreased (~65%) due to the deletion of  srbA ; however, it was found to be restored in the ΔSrbApSrbA strain, resembling that in the SrbA +  ( Figure 2C ). Biofilm maturation and its structural integrity largely depend on the amount of alginate present in the matrix; thus, the alginate was quantified in all the test strains. The SrbA +  strain exhibited an ~38% increase in alginate production compared with the pEV strain. In contrast, the ∆SrbA showed about a ~46% reduction in alginate levels in comparison with the WT strain. However, the complementation of deletion strain ΔSrbApSrbA was able to restore alginate production to a level comparable with those of the WT strain ( Figure 2D ).\n( A ) Biofilm forming capability of WT was measured by CV assay and compared with that of the pEV, SrbA + , ΔSrbApSrbA, and ΔSrbA strains. ( B ) The cell viability of WT strains in the biofilm, as measured by MTT assay, was compared with the pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains. ( C ) Amount of EPS in the biofilm matrix of WT strains, as determined by Congo red staining, was compared with the pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains. ( D ) Amount of alginate determined by carbazole assay in the biofilm of pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains in comparison with the WT strain. ( E ) Comparative morphological nature of the colonies as appeared on the Congo red agar plates of WT, pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains, with a quantitative illustration of respective colony diameters. Data presented are the mean of three replications with ±SEM; statistical analysis was performed using one-way ANOVA for each condition, comparing the mean of each group with that of the WT strain. ‘ns’ indicates non-significance, and *, **, ***, and **** correspond to significance at  P <0.1, 0.01, 0.001, and 0.0001, respectively. sRNA, small RNA; WT, wildtype.\nApparently, slime layer in the bacterial colonies reflects the quantitative level of EPS in the colony biofilm, which was visualized on tryptone agar plates having Congo red, that binds to EPS within the slime layer resulting characteristic colony morphology with red ring around, after an incubation period of 24 h at 37°C. Visibly, SrbA +  colony showed higher density of bound dye, suggesting presence of more EPS; on the contrary, ∆SrbA exhibited different colony morphology with smaller diameter and absence of red ring that might be due to the less amount EPS ( Figure 2E ). Effect of deletion was found to be reversed in ΔSrbApSrbA and resembled the colony morphology and diameter like the WT, and SrbA +  strains. Overall, WT and pEV strains showed similar features as far as their EPS and biofilm production abilities are concerned.\nBiofilm formation capacity and architectural feature of both of SrbA +  and ΔSrbA were visualized by confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM), and comparative analysis was done with their respective control strains, pEV and WT. For CSLM study,  P. aeruginosa  harboring pUCP30T-eCFP was used to qualitative measurement of cell density in the biofilm by eCFP fluorescence. CSLM images revealed almost similar biofilm thickness in WT and pEV strains, and overexpression  srbA  resulted thicker and dense biofilm in SrbA +  strain with higher Z stack; on the contrary, deletion of  srbA  resulted significant decrease in bio-volume and biofilm thickness with thinner Z stack ( Figure 3A  and  Table 1 ). Having consistency with CLSM study, SEM images of both WT and pEV also showed biofilm architecture with highly populated bacterial cells in the well-organized matrix. SEM image of the SrbA +  strain showed cells with slightly larger in length, embedded in higher amount of EPS matrix. In contrast, the ΔSrbA strain failed to develop such a complex biofilm architecture, indicating that the absence of  srbA  severely affected the formation structured biofilms ( Figure 3B ).\n( A ) Confocal laser scanning micrographs of the strains harboring pUCP30T-eCFP, were captured for the analysis of biofilm thickness. ( B ) Scanning electron microscopic images of the strains for visualization of biofilm architecture. Data are representative of three observations. WT, wildtype.\nData are the mean from three replications with ±SD.\nObserving the involvement of  srbA  in the biofilm formation by  P. aeruginosa , emphasis was given to find out the genetic target of  srbA  among different biofilm regulatory genes. Accordingly, base-pair complementarity analyses of  srbA  sRNA with mRNA region containing Shine–Dalgarno (SD) sequence and start codon of different biofilm regulatory genes, such as  algD  (-23 to +52),  algU  (-22 to +6),  fimX  (-54 to +21),  fleQ  (-57 to +18),  mucA  (-19 to +8),  pelA  (-40 to +35),  pslA  (-37 to +38),  pslB  (-54 to +21),  rhlA  (-21 to +11), and  rsmA  (-27 to +6), were done using IntaRNA 2.0 using the default settings ( Supplementary Figure S1 ) [ 8 , 10 , 14 , 15 , 18 , 21 , 24 , 27 , 42 ]. Based on significant matching with the regulatory region near the start codon and SD-sequence,  algU ,  mucA ,  rhlA , and  rsmA  were selected for their expressional study ( Supplementary Figure S2  and  S3 ). The sequence complementarity analysis showed that 84–93 bp, 45–52 bp, 89–113 bp, and 146–156 bp sequences of  srbA  paired with the transcript regions of  algU  (-22 to -12),  mucA  ( + 1 to + 8),  rhlA  (-12 to +11), and  rsmA  (-16 to -6), respectively, suggesting a possible function of  srbA  in regulating the expression of these genes ( Figure 4A ). The expression levels of  algU ,  mucA ,  rhlA , and  rsmA  were measured in the WT, pEV, SrbA + , ΔSrbA, ΔSrbApEV, and ΔSrbApSrbA strains using RT-qPCR under mid-log planktonic, substratum-attached biofilm, and colony biofilm conditions.\n( A ) Interaction of  srbA  sRNA with the regulatory region near the start codon and Shine-Dalgarno sequence of  algU ,  mucA ,  rhlA , and  rsmA , as analyzed by IntaRNA software. RT-qPCR analysis of WT, pEV, SrbA + , ΔSrbA, ΔSrbApEV, and ΔSrbApSrbA cells grown in planktonic, substratum-attached biofilm, and colony biofilm states for the expression study of ( B )  algU , ( C )  mucA , ( D )  rhlA , and ( E )  rsmA , taking  rpoD  as a reference gene. Statistical analysis was performed by one-way ANOVA for each gene under specified condition, comparing the mean of each condition with that of the WT strain. Additionally, the expression levels of the WT strain in different conditions were compared across all groups. Data presented are the mean of three replications with ±SEM; ‘ns’ indicates non-significance, and *, **, ***, and **** correspond to significance at  P <0.1, 0.01, 0.001, 0.0001, respectively. sRNA, small RNA; WT, wildtype.\nRelative abundance of  algU  was found to be ~2.8-fold higher in the SrbA +  strain under planktonic conditions as observed from RT-qPCR analysis, while a ~1.3-fold lower expression level was observed in the ΔSrbA strain in comparison with the WT strain. However, in ΔSrbApSrbA,  algU  expression was ~2.2-fold higher than the WT, restoring the effect of  srbA  deletion. Under substratum-attached biofilm conditions,  algU  expression was ~2.3-fold higher in the SrbA + , while ~1.7-fold lower in ΔSrbA with respect to the WT strain. In ΔSrbApSrbA,  algU  expression was ~1.56-fold higher compared with the WT, effectively compensating the effect of deletion. In the colony biofilm state,  algU  expression was up-regulated by ~2.2-fold in the SrbA + , while a ~2.25-fold decrease was observed in ΔSrbA in comparison with the WT strain. However, in ΔSrbApSrbA,  algU  expression was ~1.71-fold higher in comparison to the WT, overcoming the effect of  srbA  deletion. Interestingly, the expression of  algU  was significantly elevated in WT cells at both the substratum-attached biofilm and colony biofilm states compared with its planktonic counterpart, by ~50.9 and ~68.12-fold, respectively. In each growth condition, the WT and ΔSrbA strains exhibited similar expression levels, when compared with their respective empty vector control strains, pEV and ΔSrbApEV ( Figure 4B ). It seems that the copy number of  srbA  sRNA might contribute a regulatory role in the relative abundance of  algU , possibly through direct or indirect effect on  algU  mRNA stability. Moreover, the substantial up-regulation of  algU  in both biofilm states suggests its functional importance in biofilm formation and its maintenance.\nUnder planktonic conditions, the expression level of  mucA  was observed to be ~1.4-fold lower in the SrbA + , and ~1.15-fold higher in the ΔSrbA with respect to the WT strain. Restoration of the  srbA  deletion effect was observed in ΔSrbApSrbA, where  mucA  expression was found to be ~1.11-fold higher compared to the WT. At substratum-adhered biofilm state,  mucA  expression levels were found to be ~1.67-fold lower in SrbA + , and ~1.14-fold higher in ΔSrbA in comparison with the WT strain. ΔSrbApSrbA strain guided to restore the  mucA  expression level as affected by the  srbA  deletion. Under colony biofilm condition,  mucA  expression decreased ~2.9-fold in SrbA +  and increased by ~2.6-fold in ΔSrbA with respect to the WT strain. A lower expression level  mucA  by ~1.4-fold in ΔSrbApSrbA in comparison with the WT suggested reversal of the deletion effect of  srbA . Interestingly, the expression of  mucA  was found to be similar in WT strain both under planktonic and substratum-attached biofilm conditions, whereas at colony biofilm state,  mucA  expression level decreased by ~28.6-fold in comparison with the planktonic WT cells ( Figure 4C ). Both the WT and ΔSrbA strains showed identical levels of  mucA  expression in comparison with their respective empty vector control strains, pEV and ΔSrbApEV, in each growth condition. It could be apprehended that the copy number of  srbA  might contribute in regulating  mucA  mRNA stability.\nExpressional analysis revealed an increase of  rhlA  level by ~12-fold in SrbA +  strain and a decrease of ~4.7-fold in ΔSrbA with respect to that in the WT strain under planktonic growth condition, whereas a ~4.5-fold higher  rhlA  expression level was noted in the ΔSrbApSrbA in comparison with the WT strain, indicated restoration of the effect of  srbA  deletion. At substratum-adhered biofilm state,  rhlA  expression became ~3.3-fold increase in SrbA +  and ~1.7-fold decrease in ΔSrbA as compared with that in the WT strain. The complementation of  srbA  in deletion strain resulted a ~2.4-fold higher  rhlA  expression with respective to the WT strain, suggesting the recovery of the deletion effect. In the colony biofilm state, the expression of  rhlA  became ~3.0-fold increase in the SrbA +  strain and ~1.7-fold decrease in ΔSrbA with respect to the WT strain. The ΔSrbApSrbA strain recovered the effect of  srbA  deletion and exhibited ~2.1-fold higher compared with the WT strain. Interestingly, the expression of  rhlA  significantly increased in WT strain both under substratum-attached and colony biofilm conditions in comparison with its planktonic counterpart by ~34.0 and ~28.8-fold, respectively. At each growth condition, the WT and ΔSrbA strains showed apparently the same levels of expression compared with their respective empty vector control strains, pEV and ΔSrbApEV ( Figure 4D ). It is apparent from the result that the copy number of  srbA  sRNA might play a contributory function in regulating  rhlA  expression or its stability. Additionally, significant higher expression level of  rhlA  in both biofilm conditions suggests its regulatory role in biofilm development and maintenance.\nIn the planktonic growth condition, the  rsmA  expression levels decreased by ~2.3-fold in the SrbA +  and increased by ~2.5-fold in ΔSrbA in comparison with that in the WT strain. The expression of  rsmA  was found ~1.5-fold lower in ΔSrbApSrbA with respect to the WT strain, indicating reversal of deletion effect. Under substratum-attached biofilm condition,  rsmA  expression became ~1.6-fold decrease in SrbA +  and ~3.4-fold increase in ΔSrbA as compared with that in the WT strain. Notably, the complementation of  srbA  deletion lowered the expression level of  rsmA  by ~1.7-fold with respective to the WT strain, suggesting the recovery of the deletion effect. In the colony biofilm, the expression of  rsmA  became approximately four-fold decrease in the SrbA +  strain and ~4.3-fold increase in ΔSrbA compared with the WT strain. The  srbA  deletion effect on the expression level was found to be reversed in the ΔSrbApSrbA strain and showed approximately four-fold lower with respect to the WT strain. The expression of  rsmA  significantly decreased in the WT strain both under substratum-attached and colony biofilm states in comparison with its planktonic counterpart, by ~20 and ~28-fold, respectively. In each separate growth condition, the WT and ΔSrbA strains showed relatively the similar expression levels of  rsmA  compared with the corresponding empty vector control strains, pEV and ΔSrbApEV ( Figure 4E ). It seems that the copy number of  srbA  sRNA might contribute in the regulation of  rsmA  expression or its stability. The significant decreased expression level of  rsmA  in both biofilm states suggests its possible regulatory role in biofilm formation and maintenance.\nSequence alignment analysis indicated that  srbA  may influence the translational efficiency of certain biofilm-related genes by interacting with the ribosomal binding sites of  algU ,  mucA ,  rhlA , and  rsmA  transcripts ( Figure 4A ). To explore this possibility, a translational fusion assay was performed using pUCP30T-eCFP, a plasmid containing an eCFP reporter gene. The selected gene regions, including their start codons and SD sequences, were fused at the upstream of the eCFP gene ( Supplementary Figure S4-S7 ). These constructs were co-transformed into  E. coli  DH5α along with either the empty vector pUCP18 (EV) or pUCP18 containing  srbA +  ( Supplementary Figure S8  and  S9 ). The translational efficiency of each fused gene was assessed by measuring the fluorescence intensity of cell suspensions using fluorescence microscope and a spectrofluorometer.\nFor the  algU  fusion, forward and reverse primers were designed from 43 bp upstream and 187 bp downstream of the start codon, respectively ( Supplementary Figure S4 ). Fluorescence microscopy showed a definite increase in translation efficiency of  algU  in the presence of  srbA  ( Figure 5A ). A fluorescence reporter assay further confirmed this finding that showed a ~3.1-fold increase in fluorescence intensity when pUCP30T- algU -eCFP was co-transformed with pUCP18- srbA +  in comparison with the empty vector control ( Figure 5B ). The results suggest that the  srbA  sRNA have regulatory role on the expression of the  algU  transcript possibly by facilitating ribosome binding. Bioinformatics analysis was further done to unveil the possible RNA–RNA interactions and the stability of the secondary structures involved. The analysis predicted that the ribosome binding site, SD sequence, and start codon of  algU  formed a highly structured, intramolecular base-paired region, which might hinder ribosome binding and proper translation initiation. However, binding of  srbA  sRNA with the  algU  RNA resulted RNA duplex structure, which was appeared to be more stable, with the SD sequence and start codon positioned favorably for ribosome binding. This structural rearrangement seems to enhance the translational initiation of  algU  by the  srbA  sRNA ( Figure 5C ).\n( A ) Fluorescence micrographs and bright field images of  E. coli  DH5α cells co-transformed with pUCP30T- algU -eCFP and either pUCP18 (EV) or pUCP18-SrbA +  and ( B ) fluorescence intensity of the respective cell suspensions. ( C ) Bioinformatics-derived secondary structures of  algU  mRNA (1–75 bp) alone and the duplex secondary structure of  algU  mRNA (1–75 bp), and  srbA  sRNA (24–119 bp) for visualizing the probable effect of RNA–RNA interaction on the stability of the secondary structure and  algU  mRNA translation. Values are the mean of three replications with ±SEM, and ** stands significance at  P <0.01.\nFor the  mucA  fusion, forward and reverse primers were designed 41 bp upstream and 93 bp downstream of the start codon, respectively ( Supplementary Figure S7 ). Fluorescence microscopy showed a notable reduction in the translation efficiency of  mucA  when  srbA  sRNA was present ( Figure 6A ). This was further supported by the fluorescence reporter assay, which demonstrated a six-fold reduction in fluorescence intensity when pUCP30T- mucA -eCFP was co-transformed with pUCP18- srbA +  in comparison with the empty vector control ( Figure 6B ). Results suggest that  srbA  sRNA might directly interact with the  mucA  transcript, thus inhibiting ribosome binding, and consequently lowering  mucA  translation. Further bioinformatics analysis examined the potential RNA–RNA interactions and secondary structure stability of  mucA . The analysis revealed that the ribosome binding site, SD sequence, and start codon of  mucA  were properly aligned for ribosome binding and translation initiation under normal conditions. However, when  srbA  sRNA binds to  mucA  RNA, the formation of a more stable RNA duplex structure was observed ( Figure 6C ). The results suggest that  srbA  sRNA regulates  mucA  transcript expression possibly by inhibiting ribosome binding and thereby reducing its translation.\n( A ) Fluorescence micrographs and bright field images of  E. coli  DH5α cells co-transformed with pUCP30T- mucA -eCFP and either pUCP18 (EV) or pUCP18-SrbA + , and ( B ) fluorescence intensity of the respective cell suspensions. ( C ) Bioinformatics-derived secondary structures of  mucA  mRNA (1–75 bp) alone and the duplex secondary structure of  mucA  mRNA (1–75 bp), and  srbA  sRNA (10–95 bp) for visualizing the probable effect of RNA–RNA interaction on the stability of the secondary structure and  mucA  mRNA translation. Values are the mean of three replications with ±SEM, and ** stands significance at  P <0.01.\nFor the  rhlA  fusion, primers were designed from 27 bp upstream and 103 bp downstream of the start codon ( Supplementary Figure S5 ). Fluorescence microscopy showed a notable increase in the translation efficiency of  rhlA  when  srbA  sRNA was present ( Figure 7A ). This observation was further confirmed by fluorescence reporter assay, which showed a ~3.7-fold increase in fluorescence intensity when pUCP30T- rhlA -eCFP was co-transformed with pUCP18- srbA +  in comparison with the empty vector control ( Figure 7B ). Bioinformatics analysis was explored to find the possible RNA–RNA interactions and the stability of the secondary structures involved. The analysis predicted that the ribosome binding site, SD sequence, and start codon of  rhlA  formed a highly structured region with intramolecular base pairing, which could hinder ribosome binding and translation initiation. However, when  srbA  sRNA binds to  rhlA  RNA, a more stable RNA duplex structure forms, positioning the SD sequence and start codon in a way that facilitates ribosome binding ( Figure 7C ). The results indicate that  srbA  sRNA enhance the expression of the  rhlA  transcript likely by promoting ribosome binding and increasing its translation efficiency.\n( A ) Fluorescence micrographs and bright field images of  E. coli  DH5α cells co-transformed with pUCP30T- rhlA -eCFP and either pUCP18 (EV) or pUCP18-SrbA +  and ( B ) fluorescence intensity of the respective cell suspensions. ( C ) Bioinformatics-derived secondary structures of  rhlA  mRNA (1–75 bp) alone and the duplex secondary structure of  rhlA  mRNA (1–75 bp), and  srbA  sRNA (1–85 bp) for visualizing the probable effect of RNA–RNA interaction on the stability of the secondary structure and  rhlA  mRNA translation. Values are the mean of three replications with ±SEM, and ** stands significance at  P <0.01.\nFor the  rsmA  fusion, forward and reverse primers were designed from 41 bp upstream and 91 bp downstream of the start codon, respectively ( Supplementary Figure S6 ). Fluorescence microscopy indicated a substantial decrease in the translation efficiency of  rsmA  when  srbA  sRNA was present ( Figure 8A ). This was further validated by fluorescence reporter assay, which showed a ~2.4-fold reduction in fluorescence intensity when pUCP30T- rsmA -eCFP was co-transformed with pUCP18- srbA +  in comparison with the empty vector control ( Figure 8B ). Further bioinformatics analysis was investigated to find possible RNA–RNA interactions and secondary structure stability for  rsmA . The analysis revealed that, under normal conditions, the ribosome binding site, SD sequence, and start codon of  rsmA  are well-positioned for ribosome binding and translation initiation. However, when  srbA  sRNA binds to  rsmA  RNA, a more stable RNA duplex structure forms, negating ribosome binding through intermolecular base pairing, which likely results to the decrease of  rsmA  translation in the presence of  srbA  sRNA ( Figure 8C ). The finding suggests that  srbA  sRNA may down-regulate  rsmA  transcript expression probably by preventing ribosome binding, leading to reduced translation.\n( A ) Fluorescence micrographs and bright field images of  E. coli  DH5α cells co-transformed with pUCP30T- rsmA -eCFP and either pUCP18 (EV) or pUCP18-SrbA +  and ( B ) fluorescence intensity of the respective cell suspensions. ( C ) Bioinformatics-derived secondary structures of  rsmA  mRNA (1–75 bp) alone and the duplex secondary structure of  rsmA  mRNA (1–75 bp), and  srbA  sRNA (31–132 bp) for visualizing the probable effect of RNA–RNA interaction on the stability of the secondary structure and  rsmA  mRNA translation. Values are the mean of three replications with ±SEM, and *** stands significance at  P  < 0.001.\n\nWith the alarming emergence of multidrug-resistant  P. aeruginosa  strains and its versatile adaptability, understanding the regulatory network that allows this bacterium to adapt and thrive in diverse environments is crucial for combating infections. Approximately one tenth of  P. aeruginosa  genome is dedicated for encoding different transcriptional modulators, along with abundant sRNAs dispersed throughout its genome. In many cases, sRNAs exert their influence through base-pairing with target mRNAs, thus modulate the expressions of the genes. This interaction may occur either in untranslated regulatory regions or coding regions of the mRNA, thereby activating or repressing the translation [ 43–45 ]. The regulatory role of sRNAs in controlling stress adaptation and virulence is well established in  P. aeruginosa  [ 32 , 33 , 46–48 ]. A sophisticated regulatory machinery, operating via transcriptional, post-transcriptional, and post-translational courses in response to the environmental and host-derived signals through the QS system, modulates adaptability, biofilm formation, motility, and pathogenicity of  P. aeruginosa  [ 5 , 8 , 10 , 49–52 ]. Understanding these genetic regulatory mechanisms is essential for addressing the molecular basis of pathogenicity and developing strategies to combat the opportunistic infections caused by  P. aeruginosa .\nThe  srbA  sRNA was earlier documented to be up-regulated at the stationary growth phase and biofilm state of  P. aeruginosa  PA14, though its exact molecular regulatory role in biofilm formation remained unclear yet [ 37 ]. In this study,  srbA  was found to be up-regulated during biofilm and colony biofilm growth phases in  P. aeruginosa  PAO1 strains, suggesting its involvement in the development of biofilm. The differences in  srbA  expression levels observed among the planktonic, substratum-attached biofilm, and colony biofilm states in either SrbA +  or ΔSrbApSrbA are intriguing, even though the  srbA  is overexpressed from the same plasmid ( Figure 1B ). Generally, various factors collectively affect the overall expression profile of genes, be it on bacterial chromosomes or plasmids, depending on their growth conditions. Alteration in critical or limiting factors under different growth conditions may regulate the copy number of  srbA  through various mechanisms, such as plasmid copy number, promoter activity, and RNA stability. The regulatory role of  srbA  in biofilm development was further investigated using  srbA  deletion and overexpression strains. The results revealed that  srbA  overexpression enhanced biofilm formation, while its deletion reduced biofilm development. The study of colony morphology revealed that the SrbA +  colonies exhibited a higher density of EPS, whereas the ∆SrbA colonies displayed a distinct morphology, characterized by a smaller colony diameter and reduced quantity of EPS ( Figure 2E ). This observation aligns with earlier findings on the role of  srbA  in regulating motility in  P. aeruginosa  [ 33 ]. Earlier studies on  MacS  sRNA of  E. coli  suggested its regulatory role on biofilm formation by controlling bacterial motility and polysaccharide production [ 50 ]. Similarly, in  P. aeruginosa ,  RsmZ  and  RsmY  sRNAs stimulate biofilm formation by inhibiting RsmA activity [ 34 , 53 ], while  ErsA  regulates biofilm formation by modulating AmrZ at the post-transcriptional level [ 46 ]. Additionally,  PA0730.1  sRNA has been reported to regulate biofilm formation through its control over the  mucA  and  rpoS  genes in  P. aeruginosa  [ 32 ]. These findings suggest that  srbA  might also target specific genes to play diverse roles in biofilm development.\nAlgU is a critical σ-factor that regulates the expression of numerous genes involved in survival of  P. aeruginosa  under diverse environmental situations and the production of various virulence factors [ 54 ]. AlgU has an autoregulatory function through its interaction with anti-sigma factor MucA and controls the production of biofilm architectural components, particularly alginate [ 55 ]. MucA is responsible for regulating the mucoid phenotype often associated with chronic lung infections caused by  P. aeruginosa  [ 21 , 56 , 57 ]. Both  algU  and  mucA  are components of the same operon (algUmucABCD), which is transcribed from shared promoters (P1–P5) under the regulatory control of the sigma factor AlgT/U [ 58 , 59 ]. The differential effects of  srbA  on the transcript levels of  algU  and  mucA  suggest that its regulatory role is more likely to occur at a post-transcriptional level, rather than at the transcriptional level. It seems that the observed differences may arise from selective modulation of mRNA stability or processing by  srbA . Overexpression of  srbA  resulted in increased expression of  algU , which may explain the enhanced biofilm formation observed in  srbA  overexpression strains. Furthermore, a significant reduction in  mucA  translation was detected in strains with higher  srbA  copy numbers, leading to increased levels of active AlgU. Thus, the combined positive regulation of  algU  and negative regulation of  mucA  by  srbA  likely contributes to its role in biofilm formation by  P. aeruginosa . Furthermore, an increase in AlgU levels coupled with a decrease in MucA levels is expected to lead to enhanced mucoid phenotype, which correlates with the findings from the alginate production and colony morphological nature ( Figure 2D and E ).\nIn  P. aeruginosa , rhamnolipid production is crucial for maintaining biofilm structure, as it modulates the biofilm surface properties and facilitates microcolony formation [ 8 ]. The enzyme RhlA, which plays a key role in rhamnolipid biosynthesis, is modulated by the Rhl system [ 24 ]. Overexpression of  srbA  leads to increased expression of  rhlA  at both the transcriptional and translational levels, which likely explains the enhanced biofilm formation observed in  srbA  overexpressing strains. Additionally, SrbA +  strains were found to produce higher levels of rhamnolipids than WT  P. aeruginosa , both in nutrient-rich and nutrient-limited conditions [ 33 ]. Increased rhamnolipid production also correlates with the previous findings on enhanced swarming motility of SrbA +  strain [ 33 ].\nRsmA, a critical regulator in  P. aeruginosa , is part of the CsrA family of RNA-binding proteins and has been shown to control virulence, motility, biofilm formation, and metabolism by interacting with target mRNAs [ 27–29 ]. RsmA inhibits biofilm formation by suppressing the production of EPS such as Psl and Pel, key components of the biofilm matrix, and modulates flagellar motility, promoting bacterial dispersal [ 29 , 60 ]. In  srbA  overexpressing strains,  rsmA  expression was reduced at both the transcriptional and translational levels, contributing to the enhanced biofilm formation in these strains. Moreover, changes in  rsmA  levels could affect virulence traits, which corroborates with previous observations on the role of  srbA  in regulating the synthesis of various virulence factors in  P. aeruginosa  [ 33 ].\nThese findings have unveiled the pivotal role  srbA  as a modulator of various genes, such as  algU ,  mucA ,  rhlA , and  rsmA , involved in regulatory network for biofilm development in  P. aeruginosa  ( Figure 9 ). However, to verify a direct interaction, nucleotide substitutions should be performed both in the sRNA and the predicted mRNA interaction region. The functional intricacy of sRNA is seemed to be greater than previously thought about, and it warrants in-depth genetic and structural analyses to understand such regulatory networks controlled by  srbA , especially in host-pathogen interactions. The outcome of this study suggests  srbA  as a promising drug target and to develop oligonucleotide-based therapeutic strategies for combating the threat of multidrug-resistant  P. aeruginosa .\n\nPseudomonas aeruginosa  PAO1 (ATCC 15692) used as the experimental model organism in this study. For cloning and translational fusion experiments,  Escherichia coli  DH5α was used. Details of the plasmids and bacterial strains used in this study are given in  Supplementary Table S1  and  S2 , respectively. The  srbA  overexpression (SrbA + ),  srbA  deletion (ΔSrbA), and plasmid-mediated complementation of  srbA  deletion (ΔSrbApSrbA) strains, and their respective control, empty vector (pEV), WT, and complementation of deletion strain by empty vector (ΔSrbApEV) strains were constructed earlier [ 33 ] and were used for studying the effect of  srbA  sRNA on biofilm development in  P. aeruginosa  PAO1. For the planktonic culture, cells were grown in the LB (Luria Bertani) broth for 3 h in 37°C at 200 rpm shaking condition or till OD 600  of 0.8. For developing substratum-attached biofilm, cells were grown in LB broth and kept at 37°C under static condition for 24 h. The colony biofilm was prepared in LB agar plate, where 5 µl of mid-log phase culture was spot inoculated and was kept for 48 h at 37°C. For the translation fusion analysis, the  E. coli  cells were grown in the LB broth. Selective antibiotics were supplemented in the specified media as per the experimental requirements and the strains used. The antibiotics were used in this study were carbenicillin 150 µg/ml, gentamycin 100 µg/ml, tetracycline 100 µg/ml for strains of  P. aeruginosa , and ampicillin 50 µg/ml, gentamycin 50 µg/ml for the strains of  E. coli .\nThe strains pEV, SrbA + , ΔSrbA, ΔSrbApEV, and ΔSrbApSrbA were earlier constructed in the laboratory and were used in this study [ 33 ]. For confocal microscopy, the eCFP reporter-based plasmid pUCP30T-eCFP was electroporated into the WT PAO1 strain, along with pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains. For the translational fusion assay,  srbA  was inserted into the pUCP18 plasmid [ 33 ], and its target mRNA was cloned into the pUCP30T-eCFP plasmid containing an eCFP reporter gene [ 61 ]. Specified regions of the selected gene, including start codons and SD sequences, were fused upstream of the eCFP gene. For  mucA  translation fusion, the pUCP30T- mucA -eCFP construct was used [ 32 ]. The pUCP30T- algU -eCFP construct was developed using forward and reverse primers designed from the transcription initiation site and downstream regions of the  algU  gene, respectively. Similarly, pUCP30T- rhlA -eCFP and pUCP30T- rsmA -eCFP were constructed with corresponding primers for the  rhlA  and  rsmA  genes. All primers used for the strain construction are presented in  Supplementary Table S3 .\nBiofilm quantification using the crystal violet assay was conducted as described earlier [ 62 ]. Bacterial cultures (OD 600  = 0.4) from mid-exponential phase of WT, pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains were inoculated in a 96-well microtiter plate and incubated at 37°C without any shaking. After incubation, the culture broth was carefully pipetted out, and the adhered biofilm was gently rinsed with sterile PBS. Biofilms were then fixed with methanol, air-dried, and were stained with 0.1% Hucker crystal violet. Excess dye was removed, and the crystal violet retained by the biofilm was dissolved with 33% glacial acetic acid. The absorbance was measured at 570 nm using a microtiter plate reader (iMARK, Bio-Rad, Japan).\nCell viability in biofilms was determined by the MTT assay [ 63 ]. The biofilms formed by WT, pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA in the 96-well microtiter plate were washed with PBS, and 200 μl of LB containing 0.5 mg/ml MTT reagent was poured to each well. After incubation of 2 h at 37°C in the dark, the formazan crystals formed were dissolved with DMSO, and the A 570  was measured by the microtiter plate reader.\nThe amount of EPS in the biofilms was quantified by Congo red binding assay [ 64 ]. Biofilms formed by WT, pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA strains in a 96-well plate were stained with 1% Congo red, incubated in the dark for 30 min, and were then washed with PBS. Bound dye was dissolved in DMSO, and absorbance was recorded at 490 nm.\nThe alginate production was determined following the method described earlier [ 65 ]. Cells of WT, pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA from the substratum-attached biofilm were suspended in 500 μl PBS. The suspension was then mixed with 500 μl of 1 M NaCl and was vortexed to extract the alginate bound to the cell surface. The resulting mixture was then centrifuged at 8000 ×  g  for 20 min. The supernatant was mixed with cetylpyridinium chloride (2% w/v) and was kept overnight at 4°C. The alginate–cetylpyridinium chloride complex was subsequently collected by centrifugation at 8000 ×  g  for 10 min at 4°C. The supernatant was discarded, and the pellet was dissolved in 500 μL of chilled isopropanol. This mixture was then centrifuged again at 8000 ×  g  for 10 min at 4°C. The pellet was resuspended in 1 M NaCl and was kept overnight at 4°C. The alginate content was quantified using the carbazole method [ 66 ].\nFor colony morphology, mid-log phase cultures of WT, pEV, SrbA + , ΔSrbA, and ΔSrbApSrbA were point inoculated onto tryptone agar plates having Congo red and Coomassie brilliant blue. After an incubation of 24 h at 37°C, morphological features of the colonies were visualized under a stereo microscope (Zoomstar-II, Dewinter).\nCLSM and SEM were used to observe biofilm architecture. For both microscopic studies, WT, pEV, SrbA + , and ΔSrbA strains transformed with pUCP30T-eCFP were grown on coverslips in a 24-well plate and were incubated for 24 h. Biofilms were washed to remove unadhered cells, fixed with 2.5% glutaraldehyde, then again washed with PBS, and were air-dried. For CSLM, the images were captured using a confocal microscope (Zeiss LSM 800, Carl Zeiss, Germany) with a 434 nm excitation and 477 nm emission wavelengths to detect eCFP fluorescence in the cells embedded within the biofilm matrices. COMSTAT analysis was performed for measuring the average bio-volume and thickness of the biofilm images. For SEM, the fixed-dried biofilm on the coverslips was dehydrated with a gradient of ethanol and was then dehydrated thrice with absolute ethanol for 10 min each. After platinum coating, the images were captured by a SEM (Evo LS10, Carl Zeiss, Germany).\nThe involvement of  srbA  on the translational efficiency of selected genes was assessed using translational fusion assay by transforming  E. coli  DH5α with eCFP reporter plasmid constructs [pUCP30T- algU (-43 to +187)-eCFP, pUCP30T- mucA (-41 to +91)-eCFP, pUCP30T- rhlA (-27 to +103)-eCFP, pUCP30T- rsmA (-41 to +91)-eCFP] co-transformed with SrbA +  or EV strains [ 32 , 33 ]. Cells from mid-log phase culture of each strain were inoculated to LB and grown at 37°C to attain an OD 600  of ~0.2. Isopropyl β-d-1-thiogalactopyranoside (IPTG) of a final concentration of 50 µg/ml was added to the growing culture and was kept further for 2 h. The cell pellets were washed using PBS by centrifugation, and the cell suspension in PBS was put on glass slide, and finally the fluorescence was measured using fluorescence microscope (Axio Vert.A1 FL-LED, Carl Zeiss, Germany). The fluorescence intensity of eCFP in the cell suspensions of each strain was determined using an excitation and emission wavelength of 434 nm and 477 nm, respectively, by a fluorescence spectrophotometer (F-7100, Hitachi, Japan).\nTotal RNA from biofilm and planktonic cultures was extracted by the TRIzol MAX Bacterial RNA isolation kit (Ambion Lifetechnology, U.S.A.) and was digested with DNase to remove any DNA contamination. The cDNA was synthesized using 2 µg of RNA and random hexamers, following the manufacturer’s manual (Applied Biosystems, U.S.A.). The real-time PCR (RT-qPCR) was carried out using a thermal cycler (StepOne Real-Time PCR System, Applied Biosystem, U.S.A.). A reaction mixture of 20 μl was prepared using 10 μl of Power UP™ SYBR™ Green Master Mix (Applied Biosystems, U.S.A.), 0.4 μl both of forward and reverse primers (20 μM), 7.2 μL nuclease-free water, and 2 μl cDNA. The cycle of reaction was set with an initial holding stage at 50°C for 2 min and then at 95°C for 2 min, denaturation was done at 95°C for 15 sec. Annealing temperature and extension time were set according to the Tm of the respective primer and amplicon size. Each RT-qPCR study was performed in triplicate. The level of quantitative expression for each selected gene was analyzed comparing that in the respective control set, and  rpoD  was considered as the reference house-keeping gene for the normalization. The primers used for this RT-qPCR study are listed in  Supplementary Table S3 .\nPseudomonas  PAO1 gene sequences were obtained from the  Pseudomonas  genome sequence database [ 38 ]. Complementary base-pair analysis of  srbA  with biofilm regulatory transcripts was carried out using IntaRNA 2.0 [ 42 ], using default set up ( Supplementary Figure S1 ). The RNA secondary structure prediction was done using RNAStructure 6.0.1 [ 67 ].\nData were analyzed for all experiments by descriptive statistics and one-way ANOVA using GraphPad Prism version 9.0. All experiments were done in triplicate, and results are showed as the mean ± SEM.","source_license":"CC-BY-4.0","license_restricted":false}