Jilong Qin

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, contributing to membrane integrity and environmental interactions. Genome-wide studies defining bacterial gene functions have been extensively performed in the model strain Escherichia coli K-12 which lacks O-antigen (OAg), and therefore does not produce smooth LPS (S-LPS). Consequently, the genetic requirements for S-LPS production in this model system remain incompletely defined. Here, a functional wbbL gene was introduced into the E. coli K-12 KEIO single-gene deletion mutant library to restore OAg synthesis, enabling genome-wide analysis of S-LPS production by screening with colicin E2 (ColE2) and validated with LPS silver staining. This identified 319 mutants with increased sensitivity to ColE2 in the presence of OAg, suggesting broader envelope-associated effects during screening. In addition, 27 mutants showed defects in S-LPS production, corresponding to genes involved in OAg biosynthesis, LPS core and sugar precursor synthesis, OAg ligation and regulation, and enterobacterial common antigen biosynthesis. A further 18 mutants initially appeared defective in S-LPS production but could not be validated upon reconstruction, and whole-genome sequencing revealed secondary mutations responsible for the observed phenotypes. This study provides a validated genetic framework for S-LPS production in E. coli K-12 and highlights the importance of rigorous validation in genome-wide screening approaches.

Bacteria exhibit two lifestyles: planktonic, free-floating individual cells or sessile multicellular aggregates known as biofilms. The biofilm life cycle is characterized by three distinct stages: attachment, maturation and dispersal. Consequently, specific adaptations occur at each stage, governing cellular behaviors such as adhesion and the synthesis and degradation of extracellular matrix components. Characterizing stage-specific bacterial profiles therefore represents a valuable strategy for the development of novel antibiofilm therapies. Here, we used the model biofilm-forming bacterium Pseudomonas aeruginosa PAO1 to characterize the transcriptional profiles of each stage of the biofilm life cycle: attachment, biofilm maturation and spontaneous dispersal in closed cultures. We report that, relative to biofilm dispersal, surface attachment coincided with the upregulation of genes comprising the Pil-Chp mechanosensory system (2.00–7.37-fold), whereas biofilm maturation was characterized by the upregulation of genes involved in Pel polysaccharide synthesis (∼2.5-fold relative to either attachment or dispersal), siaD and PA4396 diguanylate cyclases as well as phosphodiesterases pipA, fimX and PA5442 (2.08–3.73-fold relative to biofilm dispersal). In contrast with cells undergoing biofilm maturation, dispersing cells upregulated genes responsible for the biosynthesis of alginate, rhamnolipid, and extracellular nucleases ( eddA , 3.28-fold; and eddB , 2.95-fold), as well as the transcriptional regulator of dispersal amrZ (6.27-fold). Additionally, genes involved in the biosynthesis and sensing of the dispersal signal cis-2-decenoic acid ( dspS , 2.04-fold; and dspI , 5.59-fold), canonical phosphodiesterases ( nbdA , 7.56-fold; and rbdA , 3.03-fold), four non-canonical HD-GYP phosphodiesterases and seven other c-di-GMP–related enzymes were also upregulated during dispersal. Altogether, this work provides benchmarking stage-specific transcriptional profiles characterizing the biofilm life cycle of P. aerugiosa in closed systems. Furthermore, it allowed the identification of a subset of fourteen genes as transcriptional biomarkers of dispersal, which were used to build reporter plasmids as tools to determine the onset of dispersal.

Background/Objectives: Bacterial biofilms are structured communities of sessile cells embedded in a self-produced extracellular matrix. Within biofilms, bacteria become highly tolerant toenvironmental stressors such as host immune responses and antimicrobial treatments. In response to specific cues, however, biofilm cells can revert to a planktonic free-swimming lifestyle through a process termed biofilm dispersal. When dispersed cells escape the biofilm matrix, they lose biofilm-associated antibiotic tolerance, a major barrier to treating medical biofilms. As such, dispersal-inducing compounds like nitric oxide (NO) are actively investigated as adjuvants to potentiate the biofilm-eradicating activity of existing antibiotics. We recently characterised the transcriptomic responses elicited during spontaneous biofilm dispersal in closed culture-grown Pseudomonas aeruginosa biofilms. Here, we evaluated the transcriptional profiles of P. aeruginosa biofilms treated with the NO donor Spermine-NONOate (SP-NONO) and the nitroxide C-TEMPO, an NO analogue, to determine potential pathways involved in NO-mediated dispersal. Methods: Dispersal activity on P. aeruginosa PAO1 biofilms by SP-NONOate and C-TEMPO was quantified by crystal violet staining. Cellular responses to each compound were profiled by RNA-seq on treated and untreated cells. Results: While both compounds disrupted the transcription of ANR-regulated energy metabolism pathways, only SP-NONO activated canonical NO-regulated responses. Considering that only SP-NONO showed biofilm dispersal activity in this culture system, we investigated shared transcriptional shifts in SP-NONO-treated and spontaneously dispersed biofilms to identify pathways likely involved in central dispersal responses. These mostly included genes involved in the catabolism of branched-chain amino acids (leucine, valine, isoleucine) and lysine, as well as 9 of 14 genes previously defined as transcriptional biomarkers of spontaneous biofilm dispersal. Conclusions: This study suggests that NO disrupts biofilm maturation by prematurely stimulating central pathways of spontaneous biofilm dispersal and highlights this set of biomarkers as robust indicators of dispersal responses.

Background/Objectives: Bacterial biofilms are structured communities of sessile cells embedded in a self-produced extracellular matrix that protects against environmental stress, host immune responses and antimicrobial treatments. In response to specific cues, biofilm cells can revert to a planktonic free-swimming lifestyle through a process termed biofilm dispersal. When dispersed cells escape the biofilm matrix, they lose bio-film-associated antibiotic tolerance, a major barrier to treating medical biofilms. As such, dispersal-inducing compounds like nitric oxide (NO) are actively investigated as adjuvants to potentiate the biofilm eradicating activity of existing antibiotics. We recently characterised the transcriptomic responses elicited during spontaneous biofilm dispersal in closed culture-grown Pseudomonas aeruginosa biofilms. Here, we evaluated the tran-scriptional profile of P. aeruginosa biofilms treated with the NO donor Spermine-NONOate (SP-NONO) and the nitroxide C-TEMPO, an NO analogue to determine potential pathways involved in NO-mediated dispersal. Methods: Dispersal activity on P. aeruginosa PAO1 biofilms by SP-NONOate and C-TEMPO was quantified by crystal violet staining. Cellular responses to each compound were profiled by RNA-seq on treated and untreated cells. Results: While both compounds disrupted the transcription of ANR-regulated energy metabolism pathways, only SP-NONO activated canonical NO-regulated responses. Considering that only SP-NONO showed biofilm dispersal activity in this culture system, we investigated shared transcriptional shifts in SP-NONO-treated and spontaneously dispersed biofilms to identify pathways likely involved in central dispersal responses. These mostly included genes participating in the catabolism of leucine, valine, isoleucine and lysine, as well as 9 of 14 genes previously defined as transcriptional biomarkers of spontaneous biofilm dispersal. Conclusions: This study suggests that NO disrupts biofilm maturation by prematurely stimulating central pathways of spontaneous biofilm dispersal and highlights this set of biomarkers as robust indicators of dispersal responses.

Bacteria exhibit two lifestyles: planktonic free-floating individual cells or sessile multicellular aggregates known as biofilms. The biofilm lifecycle is characterised by three distinct stages: attachment, maturation and dispersal. Distinct adaptations occur in each stage, determining cellular behaviours such as surface attachment or synthesis and degradation of extracellular matrix components. Characterising stage-specific bacterial profiles therefore represents a valuable strategy for the development of novel antibiofilm therapies. Here, we used the model biofilm-forming bacterium Pseudomonas aeruginosa PAO1 to characterise the transcriptional profiles of each stage of the biofilm life cycle: attachment, biofilm maturation and spontaneous dispersal in closed cultures. We report that surface attachment was accompanied by the upregulation of genes comprising the Pil-Chp mechanosensory system, whereas biofilm maturation was characterised by the upregulation of genes involved in Pel polysaccharide synthesis, siaD and PA4396 diguanylate cyclases as well as pipA, fimX and PA5442. In contrast, dispersing cells upregulated genes responsible for the biosynthesis of alginate, rhamnolipid, and extracellular nucleases ( eddA , eddB ), as well as the transcriptional regulator of dispersal amrZ . Additionally, genes encoding the spontaneous dispersal molecule cis-2-decenoic acid ( dspS and dspI ), canonical phosphodiesterases ( nbdA and rbdA ), four non-canonical HD-GYP phosphodiesterases and seven other c-di-GMP–related enzymes were also upregulated during dispersal. Our comprehensive analysis of transcriptional changes across biofilm stages therefore provides benchmarking stage-specific transcriptional profiles for P. aeruginosa biofilms in closed culture systems. Furthermore, it allowed the identification of a subset of fourteen genes as transcriptional biomarkers of dispersal, which were used to build reporter plasmids as tools to determine the onset of dispersal. Importance: Biofilm infections by P. aeruginosa are a major medical challenge due to the increased tolerance to antimicrobials displayed by bacteria living in sessile communities, which is reduced during spontaneous biofilm dispersal. Attachment, biofilm maturation and dispersal represent the main stages of a dynamic process known as the biofilm lifecycle. However, the global regulatory responses governing transitions between these stages remain understudied. Here, we combine live microscopy and biomass quantification to track the progression of P. aeruginosa cultures through the three main stages of the biofilm lifecycle. We show that cells from each stage recapitulate canonical, stage-specific transcriptional responses and identify a set of biomarkers associated with the onset of dispersal. These biomarkers may offer a practical tool for rapidly screening dispersal-inducing compounds, aiding in the discovery of the next generation of antibiofilm therapeutics.

Bile salts (BS) are antimicrobials that disrupt bacterial cell membranes and induce oxidative stress. The gut bacterium Escherichia coli is naturally resistant to BS, including the model strain K12 MG1655 that produces a lipopolysaccharide (LPS) without O-antigen (OAg) on the cell surface. Paradoxically, we have previously shown that restoring a wild-type like LPS with attached OAg (MG1655-S) sensitizes E. coli K12 to exogenous BS. In this study, we investigate this phenomenon. We show that mutations causing truncation of the LPS core oligosaccharide render MG1655-S strains even more susceptible to BS compared to MG1655. These mutants phenocopy a K-12 MG1655-S Δ waaL mutant, which is defective in OAg ligase, primarily due to periplasmic accumulation of the unligated lipid-linked UndPP-OAg. Through the characterization of BS-resistant suppressor mutants of MG1655-S Δ waaL , we identify key genetic disruptions involved in resistance. Notably, we observed the highest BS resistance in strains with a weaker connection between the outer membrane (OM) and peptidoglycan (PG), including strains lacking the major OM-anchored, PG-binding proteins OmpA or Lpp. Expressing versions of OmpA and Lpp that lack PG-binding capacity also enhanced the BS resistance. Our data suggest that BS-induced stress in OAg-producing E. coli is due to the spatial constraints between OM and PG and that mutations disrupting OM–PG interactions alleviate this stress, thereby enhancing BS resistance. These findings provide new insights into a major challenge E. coli faces in the gut environment, where it needs to produce OAg for stable colonization and immune evasion while resisting the antimicrobial activity of BS. IMPORTANCE: Enteric bacteria residing in the human gut must withstand host-derived antimicrobial bile salts, but resistance mechanisms are not fully elucidated. In this study, we investigate bile salt resistance mechanisms in O-antigen (OAg)-producing Escherichia coli K-12. We show that the accumulation of carrier lipid-linked OAg in the periplasm of strains with truncated lipopolysaccharide (LPS) core oligosaccharide or defects in OAg ligase can sensitize E. coli more to bile salt, unless the physical links between outer membrane and peptidoglycan are disrupted, highlighting that bile salt-induced stress is attributed to spatial constraints between the outer membrane and peptidoglycan layer. Our work uncovers a previously unappreciated envelope stress response mechanism in E. coli , where reducing outer membrane–peptidoglycan connectivity mitigates bile salt-induced damage arising from OAg production. These findings reshape our understanding of how physical architecture and biosynthetic intermediates intersect to influence bacterial survival in hostile environments.

Biofilm dispersal agents, like nitric oxide (NO), restore antimicrobial effectiveness against biofilm infections by inducing bacteria to shift from a biofilm to a planktonic state, thereby overcoming the antimicrobial tolerance typically associated with biofilms. Sodium nitroprusside (SNP) is a widely used NO donor for investigating the molecular mechanisms underlying NO-mediated biofilm dispersal in the nosocomial pathogen Pseudomonas aeruginosa . However, the biofilm effects of SNP are variable depending on the in vitro experimental conditions, with some studies reporting enhanced growth in both planktonic and biofilm forms instead of dispersal. These discrepancies suggest that SNP affects P. aeruginosa biofilm-residing cells beyond the release of NO. In this study, we compared SNP with another NO donor, Spermine NONOate, to systematically contrast their effects on biofilm and planktonic cultures of P. aeruginosa . We found that SNP, but not Spermine NONOate, increased the biomass of P. aeruginosa biofilms in microplate cultures. This effect was also observed when biofilm cultures were supplemented with iron. Additionally, supplementation with SNP rescued the planktonic growth of P. aeruginosa in iron-depleted media similarly to FeSO₄, suggesting that SNP may serve as an iron source. Our findings indicate that the use of SNP as an NO donor in biofilm dispersal may be compromised by its role in promoting both biofilm and planktonic growth through its iron center. Our study cautions investigators using SNP for studying NO-mediated biofilm dispersal. IMPORTANCE: Research into biofilm dispersal agent nitric oxide (NO) holds promise for treating biofilm-associated infections. Sodium nitroprusside (SNP), an NO donor widely used in antibiofilm research, has been shown in this study to enhance cell growth and biofilm formation in Pseudomonas aeruginosa by acting as a source of iron. Our results suggest that SNP functions both as an NO and an iron donor, with its iron-releasing properties playing a more dominant role in promoting biofilm growth in closed culture systems. This study underscores the dual but conflicting roles of SNP in biofilm growth, which caution its future development as an NO-based therapeutic strategy for biofilm-associated infections.

Biofilm dispersal agents, like nitric oxide (NO), restore antimicrobial effectiveness against biofilm infections by inducing bacteria to shift from a biofilm to a planktonic state, thereby overcoming the antimicrobial tolerance typically associated with biofilms. Sodium nitroprusside (SNP) is a widely used NO donor for investigating the molecular mechanisms underlying NO-mediated biofilm dispersal in the nosocomial pathogen Pseudomonas aeruginosa . However, the biofilm effects of SNP are variable depending on the in vitro experimental conditions, with some studies reporting enhanced growth in both planktonic and biofilm forms instead of dispersal. These discrepancies suggest that SNP affects P. aeruginosa biofilm-residing cells beyond the release of NO. In this study, we compared SNP with another NO donor, Spermine NONOate, to systematically contrast their effects on biofilm and planktonic cultures of P. aeruginosa . We found that SNP, but not Spermine NONOate, increased the biomass of P. aeruginosa biofilms in microplate cultures. This effect was also observed when biofilms were supplemented with iron. Additionally, supplementation with SNP rescued the planktonic growth of P. aeruginosa in iron-depleted media, similar to FeSO ₄ supplementation, suggesting that SNP may serve as an iron source. Our findings suggest that SNP’s potential as an NO agent used for biofilm dispersal may be confounded by its role in promoting both biofilm and planktonic growth through its iron centre. Our study cautions investigators using SNP for studying NO-mediated biofilm dispersal.

Bile salts (BS) are derived from cholesterol in the liver and act as antimicrobial agents in the intestines by disrupting bacterial cell membranes and inducing oxidative stress. The gut bacterium E. coli is naturally resistant to BS, including the model strain K12 that produces a truncated LPS without O-antigen (OAg). Paradoxically, restoring a wild-type like LPS with OAg sensitises E. coli K12 to exogenous BS. In this study, we investigate this phenomenon. We show that mutations causing truncation of the LPS core oligosaccharide render these strains even more susceptible to BS, similar to the mutant strain MG1655-SΔ waaL defective in OAg ligase, primarily due to the accumulation of the lipid-linked intermediate UndPP-OAg. Through the characterisation of BS-resistant suppressor mutants of MG1655-SΔ waaL , we identify key genetic disruptions involved in resistance. Notably, we observed the highest BS resistance in strains with a weaker connection between the outer membrane (OM) and peptidoglycan (PG), including strains lacking the major OM-anchored, PG-binding proteins OmpA or Lpp, or expressing versions of these that lack PG-binding. Our data suggest that BS-induced stress in OAg-producing E. coli is due to the spatial constraints between OM and PG, and that mutations disrupting OM-PG interactions alleviate this stress, enhancing BS resistance. These findings provide new insights into a major challenge E. coli faces in the gut environment where it needs to produce OAg for stable colonisation and resists BS. E. coli can only survive BS exposure by fine-tuning the connectivity between its cell envelope layers, which highlights a potential target for modulating bacterial responses to BS in the gut. Author summary: Enteric bacteria residing in the human gut must withstand the host-derived antimicrobial agents, bile salts (BS), but the underlying resistance mechanisms are not fully elucidated. This study investigates the BS resistance mechanisms in O-antigen (OAg)-producing Escherichia coli K-12. We show that truncation of lipopolysaccharide (LPS) core oligosaccharides or restoration of OAg production increases BS sensitivity due to the accumulation of UndPP-OAg intermediates. By analysing suppressor mutants, we identify key genetic disruptions, particularly affecting the level of outer membrane-peptidoglycan (OM-PG) interactions involving OmpA and Lpp, which confer heightened BS resistance. Our findings highlight how BS-induced stress is linked to spatial constraints between the OM and PG layer, offering new insights into bacterial adaptation to BS stress. This research may provide new targets for therapeutic interventions to modulate gut microbial responses to BS.

IMPACT STATEMENT: Membrane fluidity plays a crucial role in bacterial fitness and adaptation to cope rapid environmental changes. While high membrane fluidity promotes robust biofilm formation in Klebsiella pneumoniae , studies in several other species, including Salmonella enterica , suggest that biofilm formation is associated with reduced fluidity. This paradox may reflect the complex relationship between lipid composition and biofilm formation. Our findings demonstrated that both low and high extremes of lipid fluidity restrict biofilm formation. We propose that the required fluidity for biofilm growth, relative to that required for planktonic growth, may differ between species and are readily adjusted during lifestyle transitions.