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Background Nitrofurantoin has been re-introduced as a first-choice antibiotic to treat uncomplicated acute urinary tract infections in England and Wales. Highly effective against common uropathogens such as Escherichia coli, its use is accompanied by a low incidence ( Objective To explore the in situ evolution of NitR in E. coli isolates from 17 patients participating in AnTIC, a 12-month open label randomized controlled trial assessing the efficacy of antibiotic prophylaxis in reducing urinary tract infections (UTIs) incidence in clean intermittent self-catheterizing patients. Methods The investigation of NitR evolution in E. coli used general microbiology techniques and genetics to model known NitR mutations in NitSE. coli strains. Results Growth rate analysis identified a 2%–10% slower doubling time for nitrofurantoin resistant strains: NitS: 20.8 ± 0.7 min compared to NitR: 23 ± 0.8 min. Statistically, these data indicated no fitness advantage of evolved strains compared to the sensitive predecessor (P-value = 0.13). Genetic manipulation of E. coli to mimic NitR evolution, supported no fitness advantage (P-value = 0.22). In contrast, data argued that a first-step mutant gained a selective advantage, at sub-MIC (4–8 mg/L) nitrofurantoin concentrations. Conclusion Correlation of these findings to nitrofurantoin pharmacokinetic data suggests that the low incidence of E. coli NitR, within the community, is driven by urine-based nitrofurantoin concentrations that selectively inhibit the growth of E. coli strains carrying the key first-step loss-of-function mutation.

The AnTIC trial linked continuous low-dose antibiotic prophylaxis treatments to a lower incidence of symptomatic urinary tract infections (UTIs) among individuals performing clean intermittent self-catheterisation (CISC).To explore potential mechanisms underlying the protective effects of low-dose antibiotic prophylaxis treatments, blood and urine samples and uro-associatedBlood samples (Urine samples from CISC users, irrespective of antibiotic treatment regimens, were associated with robust urothelial innate responses. No links were identified betweenAntibiotic treatments did not impact urogenital responses to infection in CISC users. Host genetics in terms ofOur findings show that the natural urogenital defences of clean intermittent self-catheterisation (CISC) users were not impacted by antibiotic treatments. For some CISC users, prophylaxis with low-dose antibiotics selected for a stable, predominantly

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 40 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NFκB luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NFκB signalling. Although two isolates were associated with swarm sizes of >7 cm and NFκB activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NFκB signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4±1.8 to 46.1±2.5 fold, P = NS) NFκB signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

The flagellar systems of Escherichia coli and Salmonella enterica exhibit a significant level of genetic and functional synteny. Both systems are controlled by the flagellar specific master regulator FlhD4C2. Since the early days of genetic analyses of flagellar systems it has been known that E. coli flhDC can complement a ∆flhDC mutant in S. enterica. The genomic revolution has identified how genetic changes to transcription factors and/or DNA binding sites can impact the phenotypic outcome across related species. We were therefore interested in asking: using modern tools to interrogate flagellar gene expression and assembly, what would the impact be of replacing the flhDC coding sequences in S. enterica for the E. coli genes at the flhDC S. entercia chromosomal locus? We show that even though all strains created are motile, flagellar gene expression is measurably lower when flhDCEC are present. These changes can be attributed to the impact of FlhD4C2 DNA recognition and the protein-protein interactions required to generate a stable FlhD4C2 complex. Furthermore, our data suggests that in E. coli the internal flagellar FliT regulatory feedback loop has a marked difference with respect to output of the flagellar systems. We argue due diligence is required in making assumptions based on heterologous expression of regulators and that even systems showing significant synteny may not behave in exactly the same manner.

Flagellar gene expression is bimodal inSalmonella enterica. Under certain growth conditions, some cells express the flagellar genes whereas others do not. This results in mixed populations of motile and nonmotile cells. In the present study, we found that two independent mechanisms control bimodal expression of the flagellar genes. One was previously found to result from a double negative-feedback loop involving the flagellar regulators RflP and FliZ. This feedback loop governs bimodal expression of class 2 genes. In this work, a second mechanism was found to govern bimodal expression of class 3 genes. In particular, class 3 gene expression is still bimodal, even when class 2 gene expression is not. Using a combination of experimental and modeling approaches, we found that class 3 bimodality results from the σ28-FlgM developmental checkpoint.IMPORTANCEMany bacterial use flagella to swim in liquids and swarm over surface. InSalmonella enterica, over 50 genes are required to assemble flagella. The expression of these genes is tightly regulated. Previous studies have found that flagellar gene expression is bimodal inS. enterica, which means that only a fraction of cells express flagellar genes and are motile. In the present study, we found that two separate mechanisms induce this bimodal response. One mechanism, which was previously identified, tunes the fraction of motile cells in response to nutrients. The other results from a developmental checkpoint that couples flagellar gene expression to flagellar assembly. Collectively, these results further our understanding of how flagellar gene expression is regulated inS. enterica.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background Age is a significant risk factor for recurrent urinary tract (rUTI) infections, but the clinical picture is often confused in older patients who also present with asymptomatic bacteriuria (ASB). Yet, how bacteriuria establishes in such patients and the factors underpinning and/or driving symptomatic UTI episodes are still not understood. To explore this further a pilot study was completed in which 30 male and female community based older patients (mean age 75y) presenting clinically with ASB / rUTIs and 15 control volunteers (72y) were recruited and monitored for up to 6 months. During this period symptomatic UTI episodes were recorded and urines collected for urinary cytokine and uropathogenic Escherichia coli (UPEC) analyses. Results Eighty-six per cent of patients carried E. coli (102 ≥ 105 CFU/ml urine) at some point throughout the study and molecular typing identified 26 different E. coli strains in total. Analyses of urine samples for ten different cytokines identified substantial patient variability. However, when examined longitudinally the pro-inflammatory markers, IL-1 and IL-8, and the anti-inflammatory markers, IL-5 and IL-10, were significantly different in the patient urines compared to those of the controls (P

Pancreas transplantation restores insulin secretion in type 1 diabetes mellitus. The graft also produces exocrine secretions that can be drained enterically (enteric drainage [ED]) or via the bladder (bladder drainage [BD]). We suggest that in BD transplants, such secretions destroy bladder innate immunity, specifically host defense peptides/proteins (HDPs), which increases patient susceptibility to recurrent urinary tract infections (rUTIs).BD and ED patient records were reviewed retrospectively for UTIs. Urine samples from ED and BD transplant recipients were analyzed for pH, the HDPs β-defensin 2 (HBD2) and lipocalin-2, and amylase concentrations. In vitro, bacterial growth curves and antimicrobial assays were used to evaluate the effects of pH, HBD2, and HBD2 + pancreatic digestive enzymes (pancreatin) on uropathogenic Escherichia coli (UPEC) survival and growth.Urinalysis revealed a significant difference in pH between the BD and ED cohorts (7.2 ± 0.8 versus 6.7 ± 0.8; P = 0.012). Urinary HDPs were measured and BD, but not ED, lipocalin-2 concentrations were significantly decreased compared with those of diabetics awaiting transplant (P 0.05). In vitro, an alkaline environment, pH 8.0, concomitant with the urine of the patient who underwent BD transplantation, significantly reduced UPEC growth (P 0.05); addition of pancreatin to the growth medium was associated with a significant increase (P 0.001) in growth rate. Antimicrobial data suggested significant UPEC killing in the presence of HBD2 (P 0.01), but not in the presence of HBD2 + pancreatin (12,500 amylase units).These in vivo and in vitro data suggest that BD pancreatic exocrine secretions inactivate the bladder innate defenses, which facilitate UPEC growth and underpins the increased susceptibility of patients who underwent BD pancreas transplantation to rUTIs.

Objectives Recurrent urinary tract infections are associated with uropathogenic Escherichia coli (UPEC) ascending and infecting the urinary tract. Antibiotics provide only symptomatic relief, not prevent recurrence. Clinical evidence suggests that intravesical glycosaminoglycan therapy, such as hyaluronic acid (HA), helps reduce UTI recurrence. This has been investigated here using in vitro systems modelling the urogenital tract tissues. Methods RT4 bladder cells were preconditioned with high molecular weight HA (> 1500 kDa) at 2 mg mL-1 and challenged with UPEC to analyse barrier protection and bacterial adherence. Untreated and HA-preconditioned VK2 E6/E7 vaginal cells were challenged with E. coli flagellin (50 ng mL-1) to mimic bacterial challenge, and media analysed for lipocalin-2, human β-defensin 2 and interleukin-8 by ELISA. Experiments were repeated after siRNA knockdown of Toll-like receptors 2, 4 and 5, and CD44 to investigate signalling. Results Microscopic analyses showed reduced bacterial adherence and urothelial disruption with HA, suggesting that HA functions as a barrier protecting the epithelium from bacterial infection. Cells treated with HA and flagellin simultaneously produced more of the host antimicrobial peptide LCN2 and pro-inflammatory IL-8 (P

Objectifs L’Escherichia coli uropathogene (UPEC) est responsable de 60 a 80 % des cas d’infections urinaires. Notre objectif etait de definir genetiquement les UPEC d’AnTIC et de correler avec celle-ci la mobilite bacterienne, connue comme etant un facteur de virulence, et l’utilisation des antibiotiques afin de savoir si l’antibioprophylaxie selectionne des sous-groupes bacteriens genetiques specifiques et comment se developpe la multiresistance. Methodes L’etude AnTIC etait une etude de superiorite ouverte et randomisee qui comparait l’antibioprophylaxie prolongee a un traitement antibiotique curatif pour la prevention des infections urinaires recidivantes chez des patients aux auto-sondages intermittents. Nous avons selectionne de maniere randomisee 96 echantillons d’UPEC, puis nous les avons genotype afin de creer un arbre phylogenetique en utilisant le systeme MLST d’Achtmann. La mobilite a ensuite ete evaluee sur boite de Petri et correlee aux donnees genetiques. Enfin, nous avons utilise la technologie du « next-generation sequencing » pour sequencer l’ensemble du genome des echantillons d’AnTIC, selectionnes sur la base de profils de mobilite et de resistances aux antibiotiques. Resultats Parmi les echantillons d’E. coli, 44,8 % et 20,8 % appartenaient respectivement aux groupes B2 et D ( Fig. 1 ). Quarante-quatre pour cent de tous les UPEC etaient non mobiles et, s’ils etaient mobiles, 63 % presentaient une mobilite faible (≤ 1 cm ; p Fig. 2 ). L’analyse de la multiresistance parmi les souches d’UPEC a conduit au sequencage complet du genome de 50 autres souches d’AnTIC et de toutes les souches bacterienne du groupe D. Cette analyse nous a permis de mettre en evidence des mutations de genes connues ou inconnues associes aux resistances aux antibiotiques comme les fluoroquinolones ( Fig. 3 ), la nitrofurantoine, le trimethoprime et les B-lactamines. Ces mutations etaient retrouvees chez des patients exposes a ces antibiotiques durant l’etude AnTIC. Conclusion Les UPEC habituellement retrouves au cours des episodes infectieux appartiennent au groupe B2. Notre proportion plus elevee d’UPEC du groupe D est probablement liee au fait qu’il s’agit de patients aux auto-sondages intermittents. La mobilite ne semble pas etre necessaire a la pathogenicite des UPEC. L’analyse genetique permet de mieux apprehender la genese de l’antibioresistance et notamment concernant la nitrofurantoine.

The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P 392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p

The flagellum is a rotary motor that enables bacteria to swim in liquids and swarm over surfaces. Numerous global regulators control flagellar assembly in response to cellular and environmental factors. Previous studies have also shown that flagellar assembly is affected by the growth-rate of the cell. However, a systematic study has not yet been described under controlled growth conditions. Here, we investigated the effect of growth rate on flagellar assembly in Escherichia coli using steady-state chemostat cultures where we could precisely control the cell growth-rate. Our results demonstrate that flagellar abundance correlates with growth rate, where faster growing cells produce more flagella. They also demonstrate that this growth-rate dependent control occurs through the expression of the flagellar master regulator, FlhD4C2. Collectively, our results demonstrate that motility is intimately coupled to the growth-rate of the cell.

FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a ΔfliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A ΔfliH-fliI flhB(P28T) ΔflgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhA(C) ). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhA(C) . The FlgN-FlgK complex bound to FlhA(C) and FliJ to form the FlgN-FlgK-FliJ-FlhA(C) complex. The FlgN-FlhA(C) interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues still bound to FlgK and FliJ but not to FlhA(C) . A highly conserved Tyr-122 residue was required for the interaction with FlhA(C) . These results suggest that FlgN efficiently transfers FlgK/L subunits to FlhA(C) to promote their export.

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica . To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.

Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD(4) C(2) activity. FlhD(4) C(2) activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti-regulator, in a manner similar to FlgM inhibition of σ(28) activity. We have found that efficient growth-dependent regulation of FlhD(4) C(2) requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar-specific regulatory circuits, deletion of fliT or fliD did not lead to an all-or-nothing response in FlhD(4) C(2) activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhD(4) C(2) circuit. When FlhD(4) C(2) was not bound to DNA, FliT disrupted the FlhD(4) C(2) complex. Interestingly, when FlhD(4) C(2) was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD(4) C(2) activity by preventing the formation of the FlhD(4) C(2) :DNA complex. Our data would suggest that this level of endogenous regulation of FlhD(4) C(2) activity allows the flagellar system to efficiently respond to external signals.

Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the σ 28 -FlgM checkpoint, which couples the expression of late flagellar (P class3 ) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (P class2 ) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD 4 C 2 -dependent activator of P class2 /middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD 4 C 2 posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor σ 28 and the filament-cap chaperone/FlhD 4 C 2 inhibitor FliT. Furthermore, we show that the previously described ability of σ 28 to activate P class2 /middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping P class2 and P class3 promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ's role in swimming and swarming motility.

The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5′ untranslated region (5′UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5′UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5′ end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC -dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the −10 and −35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5′ end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5′UTR. Motility-specific mutants resulted from base substitutions only in the fliC -coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P- and l-ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti-sigma28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic-facing protein anchored to the inner membrane by a single, C-terminal transmembrane-spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti-sigma28 factor FlgM was increased in flk mutant strains. Using FlgM-beta-lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar-associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes.

The small basic histone-like protein H-NS is known for bacteria to attenuate virulence of several animal pathogens. An hns homologue from E. amylovora was identified by complementing an E. coli hns-mutant strain with a cosmid library from E. amylovora. A 1.6 kb EcoRI-fragment complemented the mucoid phenotype and repressed the ss-glucosidase activity of E. coli PD32. The open reading frame encoding an H-NS-like protein of 134 amino acid was later shown to be located on plasmid pEA29 (McGhee and Jones 2000). A chromosomal hns gene was amplified with PCR consensus primers and localized near galU of E. amylovora. E. amylovora mutants were created by insertion of a resistance cassette, and the intact gene was inserted into a high copy number plasmid for constitutive expression. Purified chromosomal H-NS protein preferentially bound to a DNA fragment from the lsc region and bending was predicted for an adjacent fragment with the rlsB-promoter. Levan production was significantly increased by hns mutations. Synthesis of the capsular exopolysaccharide amylovoran and of levan were reduced, when hns from the E. amylovora plasmid was overexpressed. A mutation in chromosomal hns of E. amylovora increased amylovoran synthesis, and both mutations retarded symptom formation on immature pears.

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook-associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti-sigma28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half-life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK-lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility-defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.

Several members of the two-component signal transduction family have been implicated in the control of polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer-to-stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant-negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD approximately P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer-to-stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole-specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C-terminal output domain in modulating the polar cell surface of C. crescentus are discussed.

The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor sigma32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat-induced sigma32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both sigma32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.

Research on the molecular mechanism of bacterial flagellar assembly has been an ongoing study that spans three decades. Early work showed that regulation of flagellar gene transcription was coupled to the assembly process. Recent advances in the understanding of the regulation of flagellar assembly have shown that translational and post-translational regulation also plays a significant role in flagellar assembly. In both Salmonella and Caulobacter crescentus, translational regulation influences the secretion of the anti-sigma(28) factor FlgM and the flagellin fljK, respectively. Post-translational regulatory mechanisms also control the length of the hook and the ability of the type III secretion system to discriminate between middle and late secretion substrates. The flagellum provides a model system for understanding how gene regulation functions to ensure the efficient assembly of a complex structure and fundamental mechanisms common to all type III secretion systems.

For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

This chapter provides an understanding of the general developmental concepts associated with cyclic di-GMP (c-di-GMP) regulation. It also provides a broad understanding of the concepts of planktonic and biofilm lifestyles and the modes utilized by bacteria to achieve directed movement. There are distinct advantages to both free-swimming and biofilm lifestyles. In general, free-swimming bacteria may be better suited to identifying new environments, permitting colonization, and proliferation. The authors discuss the two lifestyles by defining the regulatory principles associated with c-di-GMP important for the transition between these lifestyles. They then discuss the biofilm lifestyle and the transition to this lifestyle from a planktonic state. A central concept of c-di-GMP regulation is that its accumulation favors biofilm formation and inhibits motility and the planktonic lifestyle. Importantly, the ability to utilize one or more of the modes of movement can dictate the efficiency of the transition from the planktonic to biofilm lifestyle. Furthermore, it is not only the expression of these systems that is being controlled by c-di-GMP. Another exciting area of exploration is identifying the environmental signals that control c-di-GMP signaling. The fascinating flexibility of c-di-GMP has the net result of a versatile signaling molecule that can induce a gradual response, through changes in gene expression, as well as an immediate response by directly altering the functionality of macromolecular assembly pathways.

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C-terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators. The corresponding gene was subsequently named rcsV (regulator of capsular synthesis affecting viscosity). A mutant of rcsV in wild-type Erwinia amylovora had no detectable phenotype and produced typical levels of amylovoran under laboratory conditions. The rcsV gene on a high copy number plasmid under the control of its own promoter did not alter amylovoran production, in contrast to in-frame fusions of the structural gene in expression vectors. Since even the lac promoter was inert in the expression of rcsV, a DNA-binding protein could inhibit transcription of the gene in Erwinia amylovora. On the other hand, an Erwinia amylovora rcsA mutant was suppressed by rcsV when its promoter was replaced and the structural gene fused in-frame with lacZ' or malE. Northern blots, with total RNA from Erwinia amylovora, or promoter analysis using the GUS reporter gene did not show expression of rcsV in Erwinia amylovora, although primer extension analysis did. RcsV could be a component involved in the regulation of amylovoran synthesis, and gene expression may require an unknown external signal during the life cycle or pathogenesis of Erwinia amylovora.

Several Erwinia amylovora strains isolated in the Netherlands from apple and pear trees and other hosts for fireblight were found to be conditionally deficient in levan synthesis. The levansucrase genes of five strains were cloned via PCR amplification, and the encoded enzyme was expressed in Escherichia coli. Levan was also produced from the cloned genes in high copy number plasmids, when they were transformed into the corresponding levan-deficient strains. The expression was not driven by the promoter in the cloning vector. An lsc transcript was detected by northern blot analysis with an intermediate signal for strain PD207 and, surprisingly, with a strong signal for PD576. The other four strains did not produce a hybridization signal. The nucleotide sequence in front of the promoter region, when assayed for all five deficient strains, was identical with the sequence previously determined from wild type strain Ea7/74. Also strain E8, which is deficient in levan and in amylovoran synthesis, carries an intact lsc -gene. At 28 °C the expression of lsc from the deficient strains was low in several media with various nutrients and sugars. However, when the strains were grown at 18 °C, levan synthesis was partially restored, indicating a temperature-dependent regulation of lsc -expression.

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration., Author Summary For construction of the bacterial flagellum beyond the inner and outer membranes, the flagellar type III export apparatus transports fourteen flagellar proteins with their copy numbers ranging from a few to tens of thousands to the distal growing end of the flagellar structure. The export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. Here, we show that the export engine of the flagellar type III export apparatus is robust in maintaining its export activity against internal and external perturbations arising from genetic variations and/or environmental changes. When the cytoplasmic ATPase complex is absent, the export gate complex is able to utilize sodium motive force (SMF) across the cytoplasmic membrane as a fuel in addition to proton motive force (PMF). However, the export gate utilizes only PMF as the energy source when the ATPase complex is active. An export gate protein FlhA shows an intrinsic ion channel activity. These observations suggest that the export gate intrinsically uses both PMF and SMF for protein export and that the ATPase complex switches the export gate into a highly efficient PMF-driven export engine to become much more robust against environmental perturbations.

FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a ΔfliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A ΔfliH-fliI flhB(P28T) ΔflgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhA(C) ). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhA(C) . The FlgN-FlgK complex bound to FlhA(C) and FliJ to form the FlgN-FlgK-FliJ-FlhA(C) complex. The FlgN-FlhA(C) interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues still bound to FlgK and FliJ but not to FlhA(C) . A highly conserved Tyr-122 residue was required for the interaction with FlhA(C) . These results suggest that FlgN efficiently transfers FlgK/L subunits to FlhA(C) to promote their export.

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in Δ fljK Δ fljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.

The endospore-forming Gram-positive pathogen Bacillus anthracis is responsible for the usually fatal disease, inhalational anthrax. The success of this pathogen is dependent on its ability to subvert elements of the innate immune system of its animal hosts. B. anthracis spores, which are the main infective agent, are engulfed and germinate in patrolling alveolar macrophages. In order for the infection to progress, the resulting vegetative cells must resist the antimicrobial oxidative burst mounted by the host NADPH oxidase complex. The response of B. anthracis to this and other macrophage-related stresses is therefore of major importance to the success of this pathogen, and consequently we have analysed the superoxide and peroxide stress stimulons of B. anthracis strain UM23C1-2 by means of a combined transcriptomics and proteomics approach. The results show distinct patterns of expression in response to paraquat (endogenous superoxide) and hydrogen peroxide stress. While the main response to paraquat is the induction of iron uptake pathways, the response to peroxide predominantly involves the induction of protection and repair mechanisms. Comparisons between the responses of B. anthracis and related soil bacterium, B. subtilis, reveal differences that are likely to be relevant to their respective habitats.

Salmonella enterica , a common food-borne pathogen, differentially regulates the expression of multiple genes during the infection cycle. These genes encode systems related to motility, adhesion, invasion, and intestinal persistence. Key among them is a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). In addition to the SPI1 T3SS, other systems, including flagella and type 1 fimbriae, have been implicated in Salmonella pathogenesis. In this study, we investigated the dynamic expression of the flagellar, SPI1, and type 1 fimbrial genes. We demonstrate that these genes are expressed in a temporal hierarchy, beginning with the flagellar genes, followed by the SPI1 genes, and ending with the type 1 fimbrial genes. This hierarchy could mirror the roles of these three systems during the infection cycle. As multiple studies have shown that extensive regulatory cross talk exists between these three systems, we also tested how removing different regulatory links between them affects gene expression dynamics. These results indicate that cross talk is critical for regulating gene expression during transitional phases in the gene expression hierarchy. In addition, we identified a novel regulatory link between flagellar and type 1 fimbrial gene expression dynamics, where we found that the flagellar regulator, FliZ, represses type 1 fimbrial gene expression through the posttranscriptional regulation of FimZ. The significance of these results is that they provide the first systematic study of the effect of regulatory cross talk on the expression dynamics of flagellar, SPI1, and type 1 fimbrial genes.

Bacterial sensing of environmental signals plays a key role in regulating virulence and mediating bacterium-host interactions. The sensing of the neuroendocrine stress hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) plays an important role in modulating bacterial virulence. We used Mu d J transposon mutagenesis to globally screen for genes regulated by neuroendocrine stress hormones in Salmonella enterica serovar Typhimurium. We identified eight hormone-regulated genes, including yhaK , iroC , nrdF , accC , yedP , STM3081, and the virulence-related genes virK and mig14 . The mammalian α-adrenergic receptor antagonist phentolamine reversed the hormone-mediated effects on yhaK , virK , and mig14 but did not affect the other genes. The β-adrenergic receptor antagonist propranolol had no activity in these assays. The virK and mig14 genes are involved in antimicrobial peptide resistance, and phenotypic screens revealed that exposure to neuroendocrine hormones increased the sensitivity of S . Typhimurium to the antimicrobial peptide LL-37. A virK mutant and a virK mig14 double mutant also displayed increased sensitivity to LL-37. In contrast to enterohemorrhagic Escherichia coli (EHEC), we have found no role for the two-component systems QseBC and QseEF in the adrenergic regulation of any of the identified genes. Furthermore, hormone-regulated gene expression could not be blocked by the QseC inhibitor LED209, suggesting that sensing of hormones is mediated through alternative signaling pathways in S . Typhimurium. This study has identified a role for host-derived neuroendocrine stress hormones in downregulating S . Typhimurium virulence gene expression to the benefit of the host, thus providing further insights into the field of host-pathogen communication.

This past January, in Cuernavaca Mexico, a conglomerate of scientists met to discuss the contemporary view of Bacterial Locomotion and Signal Transduction (BLAST). The BLAST meetings represent a field that has its roots in chemotaxis and the flagellum-based motility but now encompass all types of cellular movement and signalling. The topics varied from the interactions between molecules to the interactions between species. We heard about 3D reconstructions of transmembrane chemoreceptors within cells, new biophysical methods for understanding cellular engines, intricate phosphorelays, elaborate gene networks, new messenger molecules, and emerging behaviours within complex populations of cells. At BLAST X we gained an appreciation for the lifestyle choices bacteria make, how they get to where they are going, and the molecular mechanisms that underlie their decisions. Herein we review the highlights of the meeting.

Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the sigma(28)-FlgM checkpoint, which couples the expression of the late flagellar (P(class3)) genes to the completion of the hook-basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the sigma(28)-FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (P(class2)) and late (P(class3)) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of P(class2) activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in P(class2) activity were not correlated with the associated changes in P(class3) activity, which was found to be proportional to late protein secretion rates. Our data indicate that both P(class2) and P(class3) promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.

The σ28 protein is a member of the bacterial σ70-family of transcription factors that directs RNA polymerase to flagellar late (class 3) promoters. The σ28 protein is regulated in response to flagellar assembly by the anti-σ28 factor FlgM. FlgM inhibits σ28-dependent transcription of genes whose products are needed late in assembly until the flagellar basal motor structure, the hook-basal body (HBB), is constructed. A second function for the σ28 transcription factor has been discovered: σ28 facilitates the secretion of FlgM through the HBB, acting as the FlgM Type III secretion chaperone. Transcription-specific mutants in σ28 were isolated that remained competent for FlgM-facilitated secretion separating the transcription and secretion-facilitation activities of σ 28. Conversely, we also describe the isolation of mutants in σ28 that are specific for FlgM-facilitated secretion. The data demonstrate that σ28 is the Type III secretion chaperone for its own anti-sigma factor FlgM. Thus, a novel role for a σ70-family transcription factor is described.

Flagellin is a major surface antigen for many bacterial species. The pathogen Salmonella enterica switches between two alternative, antigenic forms of its flagellin filament protein, either type B or C. This switching (flagellar phase variation) is achieved by stochastic inversion of a promoter that produces both type B flagellin (FljB) and an inhibitor (FljA) of type C flagellin formation. When the fljB-fljA operon is expressed, only type B flagella are produced; when the operon is not transcribed, the gene for type C flagellin ( fliC ) is released from inhibition and forms type C flagella. Long thought to be a transcription repressor, the FljA inhibitor is shown here to block both translation and use of the FliC protein by binding to an mRNA region upstream from the translation start codon. Bypass mutants resistant to this inhibition alter this mRNA region, and some prevent FljA–RNA binding. Other bypass mutations are duplications within the leader mRNA that make FljA essential for FliC assembly. Certain bypass mutations allow FljA to block FliC-dependent motility without blocking production of the FliC protein, per se . Other mutations in the FliC mRNA leader block expression of the unlinked fljB gene. Results suggest that mRNAs for types B and C flagellin compete for occupancy of a site that directs the product toward assembly and that FljA influences this competition. This mechanism may serve to prevent assembly of flagella with a mixture of subunit types, especially during periods of switching from one type to the other.

Our perception that host-bacterial interactions lead to disease comes from rare, unsuccessful interactions resulting in the development of detectable symptoms. In contrast, the majority of host-bacterial interactions go unnoticed as the host and bacteria perceive each other to be no threat. In July 2004, a focused international symposium on epithelial-bacterial pathogen interactions was held in Newcastle upon Tyne (UK). The symposium concentrated on recent advances in our understanding of bacterial interactions at respiratory and gastrointestinal mucosal epithelial layers. For the host these epithelial tissues represent a first line of defence against invading bacterial pathogens. Through the discovery that the innate immune system plays a pivotal role during host-bacterial interactions, it has become clear that epithelia are being utilized by the host to monitor or communicate with both pathogenic and commensal bacteria. Interest in understanding the bacterial perspective of these interactions has lead researchers to realize that the bacteria utilize the same factors associated with disease to establish successful long-term interactions. Here we discuss several common themes and concepts that emerged from recent studies that have allowed physiologists and microbiologists to interact at a common interface similar to their counterparts -- epithelia and bacterial pathogens. These studies highlight the need for further multidisciplinary studies into how the host differentiates between pathogenic and commensal bacteria.

A chromosomal DNA fragment from Erwinia amylovora was identified that complemented a deletion mutant in the gut(srl) operon of Escherichia coli. The E. amylovora srl operon on the cloned fragment was localized by transposon mutagenesis. A DNA fragment including the srl genes of E. amylovora was sequenced and found to contain six open reading frames (ORFs). These ORFs were highly homologous to genes of the gut operon of E. coli. No large gene was found that encoded a protein equivalent to GutA of E. coli; instead two ORFs with extensive similarity to GutA were identified in the E. amylovora srl operon. All transposon insertions were mapped by PCR analysis, and several insertions in a plasmid bearing the srl operon were unable to complement a mutation in the E. coli gutD gene. All E. amylovora srl mutants could be complemented by introducing the sorbitol operon from E. coli. The direction of transcription was confirmed by analysis of lacZ fusions. Expression of the srl operon in E. amylovora was high in the presence of sorbitol in the medium and was repressed by glucose. Mutants with a sorbitol deficiency were still virulent on slices of immature pears, but were unable to cause significant fire blight symptoms on apple shoots. Since sorbitol is used for carbohydrate transport in host plants of E. amylovora, this sugar alcohol may be an important factor in determining host specificity for the fire blight pathogen.

A cap at the tip of the bacterial flagellum uses a dynamic differential binding of individual subunits to allow the filament tip to grow, achieving control of assembly far from the point of protein translation.