Stationary Phase Survival Research Articles

Developmentally Regulated Sphingolipid Degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl−) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl− mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl− promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl−. These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.

The Thermoanaerobacter Glycobiome Reveals Mechanisms of Pentose and Hexose Co-Utilization in Bacteria

Thermoanaerobic bacteria are of interest in cellulosic-biofuel production, due to their simultaneous pentose and hexose utilization (co-utilization) and thermophilic nature. In this study, we experimentally reconstructed the structure and dynamics of the first genome-wide carbon utilization network of thermoanaerobes. The network uncovers numerous novel pathways and identifies previously unrecognized but crucial pathway interactions and the associated key junctions. First, glucose, xylose, fructose, and cellobiose catabolism are each featured in distinct functional modules; the transport systems of hexose and pentose are apparently both regulated by transcriptional antiterminators of the BglG family, which is consistent with pentose and hexose co-utilization. Second, glucose and xylose modules cooperate in that the activity of the former promotes the activity of the latter via activating xylose transport and catabolism, while xylose delays cell lysis by sustaining coenzyme and ion metabolism. Third, the vitamin B12 pathway appears to promote ethanologenesis through ethanolamine and 1, 2-propanediol, while the arginine deiminase pathway probably contributes to cell survival in stationary phase. Moreover, by experimentally validating the distinct yet collaborative nature of glucose and xylose catabolism, we demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, this thermoanaerobic glycobiome reveals novel genetic features in carbon catabolism that may have immediate industrial implications and provides novel strategies and targets for fermentation and genome engineering.

Listeria monocytogenes adapts to long-term stationary phase survival without compromising bacterial virulence

Bacteria withstand starvation during long-term stationary phase through the acquisition of mutations that increase bacterial fitness. The evolution of the growth advantage in stationary phase (GASP) phenotype results in the ability of bacteria from an aged culture to outcompete bacteria from a younger culture when the two are mixed together. The GASP phenotype was first described for Escherichia coli, but has not been examined for an environmental bacterial pathogen, which must balance long-term survival strategies that promote fitness in the outside environment with those that promote fitness within the host. Listeria monocytogenes is an environmental bacterium that lives as a saprophyte in soil, but is capable of replicating within the cytosol of mammalian cells. Herein, we demonstrate the ability of L. monocytogenes to express GASP via the acquisition of mutations during long-term stationary growth. Listeria monocytogenes GASP occurred through mechanisms that were both dependent and independent of the stress-responsive alternative sigma factor SigB. Constitutive activation of the central virulence transcriptional regulator PrfA interfered with the development of GASP; however, L. monocytogenes GASP cultures retained full virulence in mice. These results indicate that L. monocytogenes can accrue mutations that optimize fitness during long-term stationary growth without negatively impacting virulence.

Differential high pressure survival in stationary-phase Escherichia coli MG1655

Hydrostatic pressure exerts a profound influence on nearly all facets of cellular structure and function with exposures to sufficiently high pressure leading to microbial inactivation. We report the first observation of a persistent, pressure-resistant subpopulation within stationary-phase samples of Escherichia coli MG1655, a mesophilic bacterium adapted to surface pressure. This high pressure-resistant subpopulation exhibits pressure survival ranging from 0.6 to 2.0 orders of magnitude greater survival than high pressure treatments at pressures of 225–400 MPa. We also examine some aspects of pressure treatment protocol that may influence the measurements of high pressure survival.

Lifespan extension and paraquat resistance in a ubiquinone-deficient Escherichia coli mutant depend on transcription factors ArcA and TdcA

We recently reported a genome-wide screen for extended stationary phase survival in Escherichia coli. One of the mutants recovered is deleted for ubiG, which encodes a methyltransferase required for the biosynthesis of ubiquinone. The ubiG mutant exhibits longer lifespan, as well as enhanced resistance to thermal and oxidative stress compared to wt at extracellular pH9. The longevity of the mutant, as well as its resistance to the superoxide-generating agent paraquat, is partially dependent on the hypoxia-inducible transcription factor ArcA. A microarray analysis revealed several genes whose expression is either suppressed or enhanced by ArcA in the ubiG mutant. TdcA is a transcription factor involved in the transport and metabolism of amino acids during anaerobic growth. Its enhanced expression in the ubiG mutant is dependent on ArcA. Our data are consistent with the hypothesis that ArcA and TdcA function in the same genetic pathway to increase lifespan and enhance oxidative stress resistance in the ubiG mutant. Our results might be relevant for the elucidation of the mechanism of lifespan extension in mutant mice and worms bearing mutations in ubiquinone biosynthetic genes.

Staphylococcus aureus ClpC is involved in protection of carbon-metabolizing enzymes from carbonylation during stationary growth phase

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Among them, ClpC, belonging to the Hsp100/Clp ATPase family, seems to play an important role. For instance, we previously demonstrated that a functional clpC deletion resulted in enhanced survival in the late stationary phase (death phase period) compared to the parental S. aureus strain. However, the mechanisms for the enhanced survival of a S. aureus clpC mutant during the death phase period are still elusive. In Escherichia coli, among the factors that might lead to bacterial cell death during stationary phase, the amount of protein aggregates and/or oxidized proteins appears to be of major importance. Thus, in the present study, we have evaluated protein aggregates and carbonylated protein (as a marker of protein oxidation) contents both in the wild type and in an S. aureus clpC mutant during the exponential growth phase and the death phase. Whereas at all time points the tested clpC mutant exhibits the same amount of protein aggregates as the WT strain, the total amount of carbonylated proteins appears to be lower in the clpC mutant. Moreover, we observed that at the entrance of the death phase carbon-metabolizing enzymes [such as the TCA cycle enzymes Mqo2 (malate: quinone oxidoreductase) and FumC/CitG (fumarate hydratase)] albeit not the bulk proteins are carbonylated to a larger extent in the clpC mutant. Reduced activity of the TCA cycle due to specific carbonylation of these proteins will result in a decrease of endogenous oxidative stress which in turn might confer enhanced survival of the clpC mutant during the death phase period thus contributing to bacterial longevity and chronic infection.

Insights into the Extracytoplasmic Stress Response of Xanthomonas campestris pv. campestris : Role and Regulation of σ E -Dependent Activity

Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) σ factors. The alternative sigma factor σ(E), encoded by rpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of σ(E)-dependent activation in X. campestris pv. campestris. We show that the rpoE gene is organized as a single transcription unit with the anti-σ gene rseA and the protease gene mucD and that rpoE transcription is autoregulated. rseA and mucD transcription is also controlled by a highly conserved σ(E)-dependent promoter within the σ(E) gene sequence. The σ(E)-mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the σ(E) regulon of X. campestris pv. campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress σ factor σ(H). The consensus sequence for the predicted σ(E)-regulated promoter elements is GGAACTN(15-17)GTCNNA. Determination of the rpoH transcription start site revealed that rpoH was directly regulated by σ(E) under both normal and heat stress conditions. Finally, σ(E) activity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.

Genome‐wide screen identifies Escherichia coli TCA‐cycle‐related mutants with extended chronological lifespan dependent on acetate metabolism and the hypoxia‐inducible transcription factor ArcA

Single-gene mutants with extended lifespan have been described in several model organisms. We performed a genome-wide screen for long-lived mutants in Escherichia coli, which revealed strains lacking tricarboxylic acid (TCA)-cycle-related genes that exhibit longer stationary-phase survival and increased resistance to heat stress compared to wild-type. Extended lifespan in the sdhA mutant, lacking subunit A of succinate dehydrogenase, is associated with the reduced production of superoxide and increased stress resistance. On the other hand, the longer lifespan of the lipoic acid synthase mutant (lipA) is associated with reduced oxygen consumption and requires the acetate-producing enzyme pyruvate oxidase, as well as acetyl-CoA synthetase, the enzyme that converts extracellular acetate to acetyl-CoA. The hypoxia-inducible transcription factor ArcA, acting independently of acetate metabolism, is also required for maximum lifespan extension in the lipA and lpdA mutants, indicating that these mutations promote entry into a mode normally associated with a low-oxygen environment. Because analogous changes from respiration to fermentation have been observed in long-lived Saccharomyces cerevisiae and Caenorhabditis elegans strains, such metabolic alterations may represent an evolutionarily conserved strategy to extend lifespan.

UspB, a member of the sigma-S regulon, facilitates RuvC resolvase function

A growing body of evidence shows that there is an intimate connection between proteins required for genome stability and stationary phase survival. In this work we show that the integral membrane protein UspB, a member of the RpoS regulon, is required for proper DNA repair as mutants lacking uspB are hypersensitive to several DNA damaging agents including ultraviolet light, mitomycin C, bleomycin and ciprofloxacin. Genetic and physical studies demonstrate that UspB acts in the RuvABC recombination repair pathway and removing uspB creates a phenocopy of the Holliday junction resolvase mutant, ruvC. Further, we show that the uspB mutant phenotype can be suppressed by ectopic overproduction of RuvC and that both ruvC and uspB mutants can be suppressed by inactivating recD. The fact that RuvABC-dependent repair requires UspB for proper activity suggests that the sigma-S regulon works together with DNA repair pathways under stress conditions to defend the cell against genotoxic stress.

CspC and CspD are essential for Caulobacter crescentus stationary phase survival

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.

The genetic basis of laboratory adaptation in Caulobacter crescentus.

The dimorphic bacterium Caulobacter crescentus has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between C. crescentus strain CB15 and its derivative NA1000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus, zwf, between lab-adapted C. crescentus and clinical isolates of Pseudomonas aeruginosa that have adapted to a human host during chronic infection.

A Role for the Class A Penicillin-Binding Protein PonA2 in the Survival of Mycobacterium smegmatis under Conditions of Nonreplication

Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis Delta ponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a Delta ponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria.

Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation

To transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen's life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV-secretion components, secreted host effectors and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating that ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (sigma(28)) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to co-ordinate L. pneumophila differentiation.

Structure of SurE protein fromAquifex aeolicusVF5 at 1.5 Å resolution

SurE is a stationary-phase survival protein found in bacteria, eukaryotes and archaea that exhibits a divalent-metal-ion-dependent phosphatase activity and acts as a nucleotidase and polyphosphate phosphohydrolase. The structure of the SurE protein from the hyperthermophile Aquifex aeolicus has been solved at 1.5 A resolution using molecular replacement with one dimer in the asymmetric unit and refined to an R factor of 15.6%. The crystal packing reveals that two dimers assemble to form a tetramer, although gel-filtration chromatography showed the presence of only a dimer in solution. The phosphatase active-site pocket was occupied by sulfate ions from the crystallization medium.

Functional and small-angle X-ray scattering studies of a new stationary phase survival protein E (SurE) from Xylella fastidiosa- evidence of allosteric behaviour

The genome data of bacterium Xylella fastidiosa strain 9a5c has identified several orfs related to its phytopathogenic adaptation and survival. Among these genes, the surE codifies a survival protein E (XfSurE) whose function is not so well understood, but functional assays in Escherichia coli revealed nucleotidase and exopolyphosphate activity. In the present study, we report the XfSurE protein overexpression in E. coli and its purification. The overall secondary structure was analyzed by CD. Small-angle X-ray scattering and gel filtration techniques demonstrated that the oligomeric state of the protein in solution is a tetramer. In addition, functional kinetics experiments were carried out with several monophosphate nucleoside substrates and revealed a highly positive cooperativity. An allosteric mechanism involving torsion movements in solution is proposed to explain the cooperative behaviour of XfSurE. This is the first characterization of a SurE enzyme from a phytopathogen organism and, to our knowledge, the first solution structure of a SurE protein to be described.

Generation of Metabolically Diverse Strains ofStreptococcus pyogenesduring Survival in Stationary Phase

Streptococcus pyogenes, in addition to causing fulminant disease, can be carried asymptomatically and may survive in the host without causing disease. Long-term stationary-phase cultures were used to characterize the metabolism of cultures surviving after glucose depletion. Survival of stationary-phase cultures in glucose-depleted rich medium was truncated by switching the cells to phosphate-buffered saline or by the addition of antibiotics, suggesting that survival depended on the presence of nutrients and metabolic activity. The metabolites of the pyruvate-to-acetate (PA) pathway (acetate and formate) and amino acid catabolic pathways (ammonia) accumulated throughout long-term stationary phase (12 weeks). Acid and ammonia production was balanced so that the culture pH was maintained above pH 5.6. Strains isolated from long-term stationary-phase cultures accumulated mutations that resulted in unique exponential-phase metabolisms, with some strains expressing the PA pathway, some strains producing ammonia, and some strains expressing both in the presence of glucose. Strains expressing high levels of PA pathway activity during exponential growth were unable to survive when regrown in pure culture due to the production of excess acid. These data suggest that S. pyogenes diversifies during survival in stationary phase into distinct strains with different metabolisms and that complementary metabolism is required to control the pH in stationary-phase cultures. One of three survivor strains isolated from tonsillar discard material from patients expressed high levels of the PA pathway during exponential growth. Sequencing of multiple group A streptococcus regulators revealed two different mutations in two different strains, suggesting that random mutation occurs during survival.

The Staphylococcus aureus LytSR Two-Component Regulatory System Affects Biofilm Formation

Studies of the Staphylococcus aureus LytSR two-component regulatory system have led to the identification of the cid and lrg operons, which affect murein hydrolase activity, stationary-phase survival, antibiotic tolerance, and biofilm formation. The cid gene products enhance murein hydrolase activity and antibiotic tolerance whereas the lrg gene products inhibit these processes in a manner believed to be analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of genomic DNA, which then becomes an important structural component of the biofilm matrix. To determine the role of LytSR in biofilm development, a lytS knockout mutant was generated from a clinical S. aureus isolate (UAMS-1) and the effects on gene expression and biofilm formation were examined. As observed in laboratory isolates, LytSR was found to be required for lrgAB expression. Furthermore, the lytS mutant formed a more adherent biofilm than the wild-type and complemented strains. Consistent with previous findings, the increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the lrgAB operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of a wide variety of genes involved in basic metabolism. Overall, the results of these studies demonstrate that the LytSR two-component regulatory system plays an important role in S. aureus biofilm development, likely as a result of its direct influence on lrgAB expression.

An Overlap between the Control of Programmed Cell Death in Bacillus anthracis and Sporulation

The Staphylococcus aureus cid and lrg operons have been shown to control cell death and lysis in a manner thought to be analogous to programmed cell death (apoptosis) in eukaryotic organisms. Although orthologous operons are present in a wide variety of bacterial species, members of the Bacillus cereus group are unique in that they have a total of four cid-/lrg-like operons. Two of these operons are similar to the S. aureus cid and lrg operons, while the other two (designated clhAB(1) and clhAB(2)) are unique to this group. In the present study, the functions and regulation of these loci were examined. Interestingly, the Bacillus anthracis lrgAB mutant displayed decreased stationary-phase survival, whereas the clhAB(2) mutant exhibited increased stationary-phase survival compared to the parental and complementation strains. However, neither mutation had a dramatic effect on murein hydrolase activity or autolysis. Furthermore, a quantitative analysis of the sporulation efficiency revealed that both mutants formed fewer spores than did the parental strain. Similar to S. aureus, B. anthracis lrgAB transcription was shown to be induced by gramicidin and CCCP, agents known to dissipate the proton motive force, in a lytSR-dependent manner. Northern blot analyses also demonstrated a positive role for lytSR in the clhAB(2) transcription. Taken together, the results of the present study demonstrate that B. anthracis lrgAB and clhAB(2) play important roles in the control of cell death and lysis and reveal a previously unrecognized role of this system in sporulation.

Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population.