Production of highly soluble and immuno-reactive recombinant flagellin protein of Clostridium chauvoei.

OBJECTIVE: Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. METHODS: Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. RESULTS: rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). CONCLUSION: The study indicated the production of conformational recombinant flagellin-rFliA(C)-antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

Surgical shortening of lengthened iliac arteries in endurance athletes: Short-term and long-term satisfaction.

OBJECTIVE: Endurance athletes are prone to develop flow limitations in iliac arteries (FLIA). Especially in cyclists and ice speed skaters, excessive hemodynamic loading coupled with hip hyperflexion may cause kinking in lengthened iliac arteries necessitating surgical correction. This study investigated the short-term (≤1.5 years) and long-term (≥5 years) satisfaction of operative shortening of the iliac artery in endurance athletes. METHODS: All patients who were diagnosed and operated for FLIA owing to lengthened and kinked iliac arteries between 1997 and 2015 in one center were analyzed. Short-term follow-up consisted of an incremental maximal cycling test, ankle-brachial index with flexed hips, echo-Doppler examination with peak systolic velocity measurements and contrast-enhanced magnetic resonance angiography before and 6 to 18 months after surgery. Both short- and long-term satisfaction were assessed using questionnaires. RESULTS: A total of 83 patients (90 operated legs; 96.7% males; median age of 34 years at the time of surgery; interquartile range [IQR], 29-47) were analyzed. In the short-term, 87.5% reported symptom reduction with an 86.4% overall satisfaction rate. Symptom-free cycling improved from 272 ± 84 W to 384 ± 101 W (P < .001), whereas the maximal workload increased from 419 ± 72 W to 428 ± 67 W (P = .01). The ankle-brachial index with flexed hips increased from 0.55 (IQR, 0.45-0.65) to 0.62 (IQR, 0.52-0.74; P = .008), and the peak systolic velocity measured with hips flexed decreased from 2.50 m/s (IQR, 1.77-3.13 m/s) to 1.57 m/s (IQR, 1.20-2.04 m/s; P < .001). After a median of 12 years (IQR, 9.0-15.4 years), symptoms were still decreased in 84.1% of patients with an 81.2% overall satisfaction rate (79.5% response rate). Three patients needed a reintervention (recurrent FLIA, n = 2; failure, n = 1). CONCLUSIONS: Operative shortening of a lengthened and kinked iliac artery causing FLIA is successful both in the short- and long-term.

fliA, flrB, and fliR regulate adhesion by controlling the expression of critical virulence genes in Vibrio harveyi.

Bacteria adhesion to fish mucus is a crucial virulence mechanism. As the initial step of bacterial infection, adhesion is impacted by bacterial motility and environmental conditions. However, its molecular mechanism is yet unclear. In this study, a significant decrease in gene expression of adhesion-deficient Vibrio harveyi was observed when the bacteria were subjected by Cu2+(50 mg/L), Pb2+(100 mg/L), Hg2+(25 mg/L), and Zn2+(50 mg/L). The genes fliA, fliR, and flrB were responsible for flagellation; being crucial for adhesion, these genes were identified and silenced via RNAi. After silencing of these genes by RNAi technology, the ability of adhesion, biofilm formation, motility, and flagella synthesis of V. harveyi were considerably reduced. Compared with the control group, it was observed that the expression levels of fliS, fliD, flgH, and flrC were significant down-regulated in fliR-RNAi, flrB-RNAi, and fliA-RNAi. This data indicates that the expression levels of most virulence genes are affected by fliA, fliR, and flrB. Also, the expression of fliA, fliR, and flrB can be influenced by the salinity, temperature, and pH. The results show that: (1) fliA, fliR, and flrB have important roles in the adhesion of V. harveyi; (2) fliA, fliR, and flrB can regulate bacterial adhesion by affecting its motility, and biofilm formation; (3) fliA, fliR, and flrB can regulate adhesion ability of V. harveyi in different environments.

Multiple Copies of flhDC in Paraburkholderia unamae Regulate Flagellar Gene Expression, Motility, and Biofilm Formation.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia, and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1, plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter-lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia. In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2. The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae, and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC. In P. unamae, the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

Temperature-Dependent Influence of FliA Overexpression on PHL628 E. coli Biofilm Growth and Composition.

Biofilm growth and survival pose a problem in both medical and industrial fields. Bacteria in biofilms are more tolerant to antibiotic treatment due to the inability of antibiotics to permeate to the bottom layers of cells in a biofilm and the creation of altered microenvironments of bacteria deep within the biofilm. Despite the abundance of information we have about E. coli biofilm growth and maturation, we are still learning how manipulating different signaling pathways influences the formation and fitness of biofilm. Understanding the impact of signaling pathways on biofilm formation may narrow the search for novel small molecule inhibitors or activators that affect biofilm production and stability. Here, we study the influence of the minor sigma transcription factor FliA (RpoF, sigma-28), which controls late-stage flagellar assembly and chemotaxis, on biofilm production and composition at various temperatures in the E. coli strain PHL628, which abundantly produces the extracellular structural protein curli. We examined FliA's influence on external cellular structures like curli and flagella and the biomolecular composition of the biofilm's extracellular polymeric substance (EPS) using biochemical assays, immunoblotting, and confocal laser scanning microscopy (CLSM). At 37°C, FliA overexpression results in the dramatic growth of biofilm in polystyrene plates and more modest yet significant biofilm growth on silica slides. We observed no significant differences in curli concentration and carbohydrate concentration in the EPS with FliA overexpression. Still, we did see significant changes in the abundance of EPS protein using CLSM at higher growth temperatures. We also noticed increased flagellin concentration, a major structural protein in flagella, occurred with FliA overexpression, specifically in planktonic cultures. These experiments have aided in narrowing our focus to FliA's role in changing the protein composition of the EPS, which we will examine in future endeavors.

Regulatory Cross Talk between Motility and Interbacterial Communication in Salmonella enterica Serovar Typhimurium.

FliA is a broadly conserved σ factor that directs transcription of genes involved in flagellar motility. We previously identified FliA-transcribed genes in Escherichia coli and Salmonella enterica serovar Typhimurium, and we showed that E. coli FliA transcribes many unstable, noncoding RNAs from intragenic promoters. Here, we show that FliA in S Typhimurium also directs the transcription of large numbers of unstable, noncoding RNAs from intragenic promoters, and we identify two previously unreported FliA-transcribed protein-coding genes. One of these genes, sdiA, encodes a transcription factor that responds to quorum-sensing signals produced by other bacteria. We show that FliA-dependent transcription of sdiA is required for SdiA activity, highlighting a regulatory link between flagellar motility and intercellular communication.IMPORTANCE Initiation of bacterial transcription requires association of a σ factor with the core RNA polymerase to facilitate sequence-specific recognition of promoter elements. FliA is a widely conserved σ factor that directs transcription of genes involved in flagellar motility. We previously showed that Escherichia coli FliA transcribes many unstable, noncoding RNAs from promoters within genes. Here, we demonstrate the same phenomenon in Salmonella Typhimurium. We also show that S Typhimurium FliA directs transcription of the sdiA gene, which encodes a transcription factor that responds to quorum-sensing signals produced by other bacteria. FliA-dependent transcription of sdiA is required for transcriptional control of SdiA target genes, highlighting a regulatory link between flagellar motility and intercellular communication.

Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain.

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

Involvement of three FliA-family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA-family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1-fliA2 double-deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild-type and mutant strains of fliA1 and/or fliA2. Comparative RNA-Seq analyses among the wild-type and gene deletion mutant strains showed probable targets of each FliA-family sigma factor, indicating that FliA1- and FliA2-dependent promoters are quite similar to each other, while the FliA3-dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA-family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence.

Dual RNA-Seq Unveils the Role of the Pseudomonas plecoglossicida fliA Gene in Pathogen-Host Interaction with Larimichthys crocea.

In the present study, Larimichthys crocea and Pseudomonas plecoglossicida were selected as a host-pathogen interaction model for teleosts and prokaryotic pathogens. Five shRNAs were designed and synthesized to silence the fliA gene, all of which resulted in pronounced reductions in fliA mRNA; the mutant strain with the best silencing efficiency of 92.16% was chosen for subsequent analysis. A significant decrease in motility, intracellular survival and escape was observed for the fliA-RNAi strain of P. plecoglossicida, whereby silencing of the fliA gene led to a 30% decrease in mortality and a four-day delay in the onset of infection in L. crocea. Moreover, silencing of P. plecoglossicida fliA resulted in a significant change in both the pathogen and host transcriptome in the spleens of infected L. crocea. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of pathogen transcriptome data showed that silencing fliA resulted in downregulation of 18 flagellum-related genes; KEGG analysis of host transcriptome data revealed that infection with the fliA-RNAi strain caused upregulation of 47 and downregulation of 106 immune-related genes. These pathogen-host interactions might facilitate clearance of P. plecoglossicida by L. crocea, with a significant decrease in fliA-RNAi P. plecoglossicida strain virulence in L. crocea.

Management of Endurance Athletes with Flow Limitation in the Iliac Arteries: A Case Series.

INTRODUCTION: Vascular surgeons increasingly encounter flow limitation of iliac arteries (FLIA) in endurance athletes. An experience of managing this condition is reported. REPORT: This is a retrospective cohort analysis of prospectively collected data at a single vascular centre. Between 2001 and 2017, 12 athletes with exercise induced pain underwent investigation and assessment. Patients with significant radiological findings (iliac kinking ± stenosis demonstrated on duplex ultrasound or catheter angiography) and dynamic flow changes (marked reduction in ankle brachial pressure indices following exertion, or increase in the common iliac artery peak systolic velocity during hip flexion on duplex) underwent surgery after trialling conservative management; the majority were open iliac shortening procedures. Patients with radiological findings, but no dynamic flow changes were managed conservatively. All patients were followed up. DISCUSSION: There were 10 men and two women with a median age of 40 years. Nine patients had iliac kinking (five in isolation, four associated with stenosis), two had stenosis, and one had no iliac disease. Eight patients had severe symptoms (absolute loss of power on maximal exertion) demonstrated dynamic post-exertional flow changes. Seven patients successfully underwent surgery, returning to their sport at similar intensity. One procedure was abandoned owing to severe adhesions from a prior procedure. This patient subsequently changed sport. Three patients with mild symptoms (two had reduction in power at maximal intensity, one was an incidental finding) and who demonstrated no clinical signs of FLIA continued their sport at a lower intensity. Kinking of the iliac arteries in athletes can occur with or without of iliac stenosis. Patients with the most severe iliac symptoms demonstrate dynamic post-exertional flow limitation and may benefit from surgery following a period of conservative management. Patients who have milder symptoms and no dynamic exercise flow limitations can be managed conservatively.

Characterization of Flagellum and Toxin Phase Variation in Clostridioides difficile Ribotype 012 Isolates.

Clostridioides difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum expression and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a flagellar switch upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting that the strain is phase locked. To determine whether a phase-locked flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase-off, nonmotile, and attenuated for toxin production. We isolated low-frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase-on derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains and perhaps other ribotypes, which could impact disease progression and diagnosis.IMPORTANCEC. difficile produces flagella that enhance bacterial motility and secretes toxins that promote diarrheal disease symptoms. Previously, we found that production of flagella and toxins is coregulated via a flippable DNA element termed the flagellar switch, which mediates the phase-variable production of these factors. Here, we evaluate multiple isolates of C. difficile ribotype 012 strains and find them to be primarily flagellar phase off (flg-off state). Some, but not all, of these isolates showed the ability to switch between flg-on and -off states. These findings suggest heterogeneity in the ability of C. difficile ribotype 012 strains to phase-vary flagellum and toxin production, which may broadly apply to pathogenic C. difficile.

Characterization of the role of global regulator FliA in the pathophysiology of Pseudomonas aeruginosa infection.

FliA is known to be a sigma factor that regulates bacterial flagella gene expression. Accumulating evidence suggests that FliA is involved in bacterial behavior other than motility. To elucidate the contribution of FliA to Pseudomonas aeruginosa pathophysiology, we analyzed the biological properties and gene expression profiles of a ΔfliA mutant. Transcriptome analysis results demonstrated that the expression levels of flagella biogenesis genes decreased dramatically in the mutant; consequently, the ΔfliA mutant failed to synthesize flagella and exhibited reduced motility. The ΔfliA mutant displayed stronger hemolytic and caseinolytic activities, as well as pyocyanin production. The expression of type 6 secretion system-II genes and interbacterial competition activity was decreased in the ΔfliA mutant. Direct evidence of fliA participation in virulence was obtained from analysis of hypervirulent strain B136-33. Adhesion to and cytotoxicity toward mammalian cells and penetration through cell layers were noted; furthermore, the colonization ability of the fliA::Tn5 mutant in the intestines of laboratory mice was compromised. Notably, the fliA-overexpressing strain displayed phenotypes similar to that of the fliA-defective strain, indicating that optimal FliA levels are critical to bacterial physiology. Our findings indicate that FliA plays diverse roles in P. aeruginosa, not only in flagella biosynthesis, but also in pathophysiology.

The Flagellar Regulon of Legionella-A Review.

The Legionella genus comprises more than 60 species. In particular, Legionella pneumophila is known to cause severe illnesses in humans. Legionellaceae are ubiquitous inhabitants of aquatic environments. Some Legionellaceae are motile and their motility is important to move around in habitats. Motility can be considered as a potential virulence factor as already shown for various human pathogens. The genes of the flagellar system, regulator and structural genes, are structured in hierarchical levels described as the flagellar regulon. Their expression is modulated by various environmental factors. For L. pneumophila it was shown that the expression of genes of the flagellar regulon is modulated by the actual growth phase and temperature. Especially, flagellated Legionella are known to express genes during the transmissive phase of growth that are involved in the expression of virulence traits. It has been demonstrated that the alternative sigma-28 factor is part of the link between virulence expression and motility. In the following review, the structure of the flagellar regulon of L. pneumophila is discussed and compared to other flagellar systems of different Legionella species. Recently, it has been described that Legionella micdadei and Legionella fallonii contain a second putative partial flagellar system. Hence, the report will focus on flagellated and non-flagellated Legionella strains, phylogenetic relationships, the role and function of the alternative sigma factor (FliA) and its anti-sigma-28 factor (FlgM).

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis.

Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophilaL. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.

Expression of flagellin and key regulatory flagellar genes in the non-motile bacterium Piscirickettsia salmonis.

The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.

Expression of the phosphodiesterase BifA facilitating swimming motility is partly controlled by FliA in Pseudomonas putida KT2440.

Flagella-mediated motility is an important capability of many bacteria to survive in nutrient-depleted and harsh environments. Decreasing the intracellular cyclic di-GMP (c-di-GMP) level by overexpression of phosphodiesterase BifA promotes flagellar-mediated motility and induces planktonic lifestyle in Pseudomonas. The mechanism that regulates expression of bifA gene was poorly studied. Here we showed that expression of BifA was partly controlled by flagellar sigma factor FliA (σ28 ) in Pseudomonas putidaKT2440. FliA deletion led to an approximately twofold decrease in transcription of bifA. 5' race assay revealed two transcription start points in bifA promoter region, with the putative σ70 and σ28 promoter sequences upstream, respectively. Point mutation in σ28 promoter region reduced transcriptional activity of the promoter in wild-type KT2440, but showed no influence on that in fliA deletion mutant. FliA overexpression decreased the intracellular c-di-GMP level in a BifA-dependent way, suggesting that FliA was able to modulate the intracellular c-di-GMP level and BifA function was required for the modulation. Besides, FliA overexpression enhanced swimming ability of wild-type strain, while made no difference to the bifA mutant. Our results suggest that FliA acts as a negative regulator to modulate the c-di-GMP level via controlling transcription of bifA to facilitate swimming motility.

Expression of fljB: z66 on a linear plasmid of Salmonella enterica serovar typhi is dependent on FliA and FlhDC and regulated by OmpR

Salmonella enterica serovar Typhi z66-positive strains have two different flagellin genes, fliC:d/j and fljB:z66, located on the chromosome and on a linear plasmid, respectively. To investigate the mechanism underlying the expressional regulation of fljB:z66, gene deletion mutants of the regulators FliA, FlhDC, and OmpR were constructed in this study. The expression levels of fliC and fljB:z66 were analyzed by qRT-PCR in the wild-type strain and mutants at high and low osmolarity. The results show that the expression levels of both fljB:z66 and fliC were greatly reduced in fliA and flhDC mutants under both high and low osmotic conditions. In the ompR mutant, the expression levels of fljB:z66, fliC, fliA, and flhD were increased at low osmotic conditions. SDS-PAGE and western blotting analysis of the secreted proteins revealed that the FljB:z66 was almost absent in the fliA and flhDC mutants at both high and low osmolarity. In the wild-type strain, the fljB:z66 was more highly expressed under high-osmolarity conditions than under low-osmolarity conditions. However, this difference in expression disappeared in the ompR mutant. Translational expression assay of FljB:z66 showed that the FljB:z66 expression was decreased in ompR mutant at both low and high osmolarity. These results suggest that the expression of fljB:z66 in S. enterica serovar Typhi is dependent on FliA and FlihDC, and OmpR can regulate the expression and secretion of FljB:z66 in different osmolarity.

Expression of FLJB:Z66 on a Linear Plasmid of Salmonella Enterica Serovar Typhi is Dependent on FLIA and FLHDC and Regulated By OMPR.

Salmonella enterica serovar Typhi z66-positive strains have two different flagellin genes, fliC:d/j and fljB:z66, located on the chromosome and on a linear plasmid, respectively. To investigate the mechanism underlying the expressional regulation of fljB:z66, gene deletion mutants of the regulators FliA, FlhDC, and OmpR were constructed in this study. The expression levels of fliC and fljB:z66 were analyzed by qRT-PCR in the wild-type strain and mutants at high and low osmolarity. The results show that the expression levels of both fljB:z66 and fliC were greatly reduced in fliA and flhDC mutants under both high and low osmotic conditions. In the ompR mutant, the expression levels of fljB:z66, fliC, fliA, and flhD were increased at low osmotic conditions. SDS-PAGE and western blotting analysis of the secreted proteins revealed that the FljB:z66 was almost absent in the fliA and flhDC mutants at both high and low osmolarity. In the wild-type strain, the fljB:z66 was more highly expressed under high-osmolarity conditions than under low-osmolarity conditions. However, this difference in expression disappeared in the ompR mutant. Translational expression assay of FljB:z66 showed that the FljB:z66 expression was decreased in ompR mutant at both low and high osmolarity. These results suggest that the expression of fljB:z66 in S. enterica serovar Typhi is dependent on FliA and FlihDC, and OmpR can regulate the expression and secretion of FljB:z66 in different osmolarity.