Flagellar Operon Research Articles

Exploitation of a "hockey-puck" phenotype to identify pilus and biofilm regulators in Serratia marcescens through genetic analysis.

Pili are essential adhesive determinants for many bacterial pathogens. A suppressor mutation screen that takes advantage of a pilus-mediated self-aggregative "hockey-puck" colony phenotype was designed to identify novel regulators of type I pili in Serratia marcescens. Mutations that decreased pilus biosynthesis mapped to the fimABCD operon; to the genes alaT, fkpA, and oxyR; upstream of the flagellar master regulator operon flhDC; and to an uncharacterized gene encoding a predicted DUF1401 domain. Biofilm formation and pilus-dependent agglutination assays were used to characterize the relative importance of the identified genes in pilus biosynthesis. Additional mutagenic or complementation analysis was used to verify the role of candidate genes in pilus biosynthesis. Presented data support a model that CRP negatively regulates pilus biosynthesis through increased expression of flhDC and decreased expression of oxyR. Further studies are warranted to determine the mechanism by which these genes mediate pilus biosynthesis or function.

S-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil.

The Arthrobacter sp. strain AK-YN10 is an s-triazine pesticide degrading bacterium isolated from a sugarcane field in Central India with history of repeated atrazine use. AK-YN10 was shown to degrade 99 % of atrazine in 30 h from media supplemented with 1000 mg L(-1) of the herbicide. Draft genome sequencing revealed similarity to pAO1, TC1, and TC2 catabolic plasmids of the Arthrobacter taxon. Plasmid profiling analyses revealed the presence of four catabolic plasmids. The trzN, atzB, and atzC atrazine-degrading genes were located on a plasmid of approximately 113 kb.The flagellar operon found in the AK-YN10 draft genome suggests motility, an interesting trait for a bioremediation agent, and was homologous to that of Arthrobacter chlorophenolicus. The multiple s-triazines degradation property of this isolate makes it a good candidate for bioremediation of soils contaminated by s-triazine pesticides.

Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence.

Pyrococcus furiosus flagella: biochemical and transcriptional analyses identify the newly detected flaB0 gene to encode the major flagellin.

We have described previously that the flagella of the Euryarchaeon Pyrococcus furiosus are multifunctional cell appendages used for swimming, adhesion to surfaces and formation of cell-cell connections. Here, we characterize these organelles with respect to their biochemistry and transcription. Flagella were purified by shearing from cells followed by CsCl-gradient centrifugation and were found to consist mainly of a ca. 30 kDa glycoprotein. Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments. The N-terminal sequence of the main flagellin was determined by Edman degradation, but none of the genes in the complete genome code for a protein with that N-terminus. Therefore, we resequenced the respective region of the genome, thereby discovering that the published genome sequence is not correct. A total of 771 bp are missing in the data base, resulting in the correction of the previously unusual N-terminal sequence of flagellin FlaB1 and in the identification of a third flagellin. To keep in line with the earlier nomenclature we call this flaB0. Very interestingly, the previously not identified flaB0 codes for the major flagellin. Transcriptional analyses of the revised flagellar operon identified various different cotranscripts encoding only a single protein in case of FlaB0 and FlaJ or up to five proteins (FlaB0-FlaD). Analysing the RNA of cells from different growth phases, we found that the length and number of detected cotranscript increased over time suggesting that the flagellar operon is transcribed mostly in late exponential and stationary growth phase.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

Regulation, Aufbau und Funktion einer bakteriellen Nanomaschine

The bacterial flagellum is a molecular nanomachine used for locomotion. In Salmonella, the assembly of the flagellum is a highly coordinated and regulated process. On top of a complex regulatory network resides the flagellar master operon flhDC. Many environmental signals are integrated on the level of flhDC expression, including transcriptional cross talk between the virulence and flagellar systems that contributes to the correct spatiotemporal expression of the different virulence factors during the infection process.

Genomic analysis ofPseudomonas aeruginosaPA96, the host of carbapenem resistance plasmid pOZ176

Pseudomonas aeruginosa PA96 is a clinical isolate from Guangzhou, China, that is multiresistant to antibiotics. We previously described the 500-kb IncP-2 plasmid, pOZ176 that encodes many resistance genes including the IMP-9 carbapenemase. Whole-genome sequencing of PA96 enabled characterization of its genomic islands, virulence factors, and chromosomal resistance genes. We filled gaps using PCR and used optical mapping to confirm the correct contig order. We automatically annotated the core genome and manually annotated the genomic islands. The genome is 6444091bp and encodes 5853 ORFs. From the whole-genome sequence, we constructed a physical map and constructed a phylogenetic tree for comparison with sequenced P.aeruginosa strains. Analysis of known core genome virulence factors and resistance genes revealed few differences with other strains, but the major virulence island is closer to that of DK2 than to PA14. PA96 most closely resembles the environmental strain M18, and notably shares a common serotype, pyoverdin type, flagellar operon, type IV pilin, and several genomic islands with M18.

Identification of flgZ as a Flagellar Gene Encoding a PilZ Domain Protein That Regulates Swimming Motility and Biofilm Formation in Pseudomonas

Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

Inhibition of Escherichia coli O157:H7 motility and biofilm by β-Sitosterol glucoside

BackgroundEscherichia coli O157:H7 (EHEC) is a food borne pathogen, which causes diarrhea and hemolytic uremic syndrome (HUS). There is an urgent need of novel antimicrobials for treatment of EHEC as conventional antibiotics enhance shiga toxin production and potentiate morbidity and mortality. MethodsSix bioactive compounds were isolated, identified from citrus and evaluated for the effect on EHEC biofilm and motility. To determine the possible mode of action, a series of genes known to affect biofilm and motility were overexpressed and the effect on biofilm/motility was assessed. Furthermore, the relative expression of genes involved in motility and biofilm formation was measured by qRT-PCR in presence and absence of phytochemicals, to examine the repression caused by test compounds. ResultsThe β-sitosterol glucoside (SG) was identified as the most potent inhibitor of EHEC biofilm formation and motility without affecting the cell viability. Furthermore, SG appears to inhibit the biofilm and motility through rssAB and hns mediated repression of flagellar master operon flhDC. ConclusionSG may serve as novel lead compound for further development of anti-virulence drugs. General significancePlant sterols constitute significant part of diet and impart various health benefits. Here we present the first evidence that SG, a plant sterol has significant effect on EHEC motility, a critical virulence factor, and may have potential application as antivirulence strategy.

Cranberry impairs selected behaviors essential for virulence inProteus mirabilisHI4320

Proteus mirabilis is an etiological agent of complicated urinary tract infections. North American cranberries (Vaccinium macrocarpon) have long been considered to have protective properties against urinary tract infections. This work reports the effects of cranberry powder (CP) on the motility of P. mirabilis HI4320 and its expression of flaA, flhD, and ureD. Our results show that swimming and swarming motilities and swarmer-cell differentiation were inhibited by CP. Additionally, transcription of the flagellin gene flaA and of flhD, the first gene of the flagellar master operon flhDC, decreased during exposure of P. mirabilis to various concentrations of CP. Moreover, using ureD-gfp, a fusion of the urease accessory gene ureD with gfp, we show that CP inhibits urease expression. Because we demonstrate that CP does not inhibit the growth of P. mirabilis, the observed effects are not attributable to toxicity. Taken together, our results demonstrate that CP hinders motility of P. mirabilis and reduces the expression of important virulence factors.

Xenocin Export by the Flagellar Type III Pathway in Xenorhabdus nematophila

The xenocin operon of Xenorhabdus nematophila consists of xciA and ximB genes encoding a 64-kDa xenocin and 42-kDa immunity protein to kill competing microbes in the insect larva. The catalytic domain of xenocin has RNase activity and is responsible for its cytotoxicity. Under SOS conditions, xenocin is produced with immunity protein as a complex. Here, we show that xenocin and immunity protein complex are exported through the flagellar type III system of X. nematophila. Secretion of xenocin complex was abolished in an flhA strain but not in an fliC strain. The xenocin operon is not linked to the flagellar operon transcriptionally. The immunity protein is produced alone from a second, constitutive promoter and is targeted to the periplasm in a flagellum-independent manner. For stable expression of xenocin, coexpression of immunity protein was necessary. To examine the role of immunity protein in xenocin export, an enzymatically inactive protein was produced by site-directed mutagenesis in the active site of the catalytic domain. Toxicity was abolished in D535A and H538A variants of xenocin, which were expressed alone without an immunity domain and secreted in the culture supernatant through flagellar export. Secretion of xenocin through the flagellar pathway has important implications in the evolutionary success of X. nematophila.

Flagellum Density Regulates Proteus mirabilis Swarmer Cell Motility in Viscous Environments

Proteus mirabilis is an opportunistic pathogen that is frequently associated with urinary tract infections. In the lab, P. mirabilis cells become long and multinucleate and increase their number of flagella as they colonize agar surfaces during swarming. Swarming has been implicated in pathogenesis; however, it is unclear how energetically costly changes in P. mirabilis cell morphology translate into an advantage for adapting to environmental changes. We investigated two morphological changes that occur during swarming--increases in cell length and flagellum density--and discovered that an increase in the surface density of flagella enabled cells to translate rapidly through fluids of increasing viscosity; in contrast, cell length had a small effect on motility. We found that swarm cells had a surface density of flagella that was ∼5 times larger than that of vegetative cells and were motile in fluids with a viscosity that inhibits vegetative cell motility. To test the relationship between flagellum density and velocity, we overexpressed FlhD(4)C(2), the master regulator of the flagellar operon, in vegetative cells of P. mirabilis and found that increased flagellum density produced an increase in cell velocity. Our results establish a relationship between P. mirabilis flagellum density and cell motility in viscous environments that may be relevant to its adaptation during the infection of mammalian urinary tracts and movement in contact with indwelling catheters.

The Rhizome of the Multidrug-Resistant Enterobacter aerogenes Genome Reveals How New “Killer Bugs” Are Created because of a Sympatric Lifestyle

Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of foreign languages.

Structural insight of a concentration-dependent mechanism by which YdiV inhibits Escherichia coli flagellum biogenesis and motility

YdiV is a negative regulator of cell motility. It interacts with FlhD4C2 complex, a product of flagellar master operon, which works as the transcription activator of all other flagellar operons. Here, we report the crystal structures of YdiV and YdiV2–FlhD2 complex at 1.9 Å and 2.9 Å resolutions, respectively. Interestingly, YdiV formed multiple types of complexes with FlhD4C2. YdiV1–FlhD4C2 and YdiV2–FlhD4C2 still bound to DNA, while YdiV3–FlhD4C2 and YdiV4–FlhD4C2 did not. DNA bound FlhD4C2 through wrapping around the FlhC subunit rather than the FlhD subunit. Structural analysis showed that only two peripheral FlhD subunits were accessible for YdiV binding, forming the YdiV2–FlhD4C2 complex without affecting the integrity of ring-like structure. YdiV2–FlhD2 structure and the negative staining electron microscopy reconstruction of YdiV4–FlhD4C2 suggested that the third and fourth YdiV molecule bound to the FlhD4C2 complex through squeezing into the ring-like structure of FlhD4C2 between the two internal D subunits. Consequently, the ring-like structure opened up, and the complex lost DNA-binding ability. Thus, YdiV inhibits FlhD4C2 only at relatively high concentrations.

Characterization of the RpoN regulon reveals differential regulation of T6SS and new flagellar operons in Vibrio cholerae O37 strain V52

The alternative sigma factor RpoN is an essential colonization factor of Vibrio cholerae and controls important cellular functions including motility and type VI secretion (T6SS). The RpoN regulon has yet to be clearly defined in T6SS-active V. cholerae isolates, which use T6SS to target both bacterial competitors and eukaryotic cells. We hypothesize that T6SS-dependent secreted effectors are co-regulated by RpoN. To systemically identify RpoN-controlled genes, we used chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to determine RpoN-binding sites and RpoN-controlled gene expression. There were 68 RpoN-binding sites and 82 operons positively controlled by RpoN, among which 37 operons had ChIP-identified binding sites. A consensus RpoN-binding motif was identified with a highly conserved thymine (−14) and an AT-rich region in the middle between the hallmark RpoN-recognized motif GG(−24)/GC(−12). There were seven new RpoN-dependent promoters in the flagellar regions. We identified a small RNA, flaX, downstream of the major flagellin gene flaA. Mutation of flaX substantially reduced motility. In contrast to previous results, we report that RpoN positively regulates the expression of hcp operons and vgrG3 that encode T6SS secreted proteins but has no effect on the expression of the main T6SS cluster encoding sheath and other structural components.

Loss of FlhE in the flagellar Type III secretion system allows proton influx into Salmonella and Escherichia coli

flhE belongs to the flhBAE flagellar operon in Enterobacteria, whose first two members function in Type III secretion (T3S). In Salmonella enterica, absence of FlhE affects swarming, but not swimming, motility. Based on a chance observation of a 'green' colony phenotype of flhE mutants on pH indicator plates containing glucose, we have established that this phenotype is associated with lysis of flagellated cells in an acidic environment created by glucose metabolism. The flhE mutant phenotype of Escherichia coli is similar overall to that of S. enterica but is seen in the absence of glucose and, unlike in S. enterica, causes a substantial growth defect. flhE mutants have a lowered cytoplasmic pH in both bacteria, indicative of a proton leak. GFP reporter assays indicate that the leak is dependent on the flagellar system, is present before the T3S system switches to secretion of late substrates, and gets worse after the switch and upon filament assembly, leading to cell lysis. We show that FlhE is a periplasmic protein that co-purifies with flagellar basal bodies. FlhE may act as a plug or a chaperone to regulate proton flow through the flagellar T3S system.

The Flagellar Regulator fliT Represses Salmonella Pathogenicity Island 1 through flhDC and fliZ

Salmonella pathogenicity island 1 (SPI1), comprising a type III section system that translocates effector proteins into host cells, is essential for the enteric pathogen Salmonella to penetrate the intestinal epithelium and subsequently to cause disease. Using random transposon mutagenesis, we found that a Tn10 disruption in the flagellar fliDST operon induced SPI1 expression when the strain was grown under conditions designed to repress SPI1, by mimicking the environment of the large intestine through the use of the intestinal fatty acid butyrate. Our genetic studies showed that only fliT within this operon was required for this effect, and that exogenous over-expression of fliT alone significantly reduced the expression of SPI1 genes, including the invasion regulator hilA and the sipBCDA operon, encoding type III section system effector proteins, and Salmonella invasion of cultured epithelial cells. fliT has been known to inhibit the flagellar machinery through repression of the flagellar master regulator flhDC. We found that the repressive effect of fliT on invasion genes was completely abolished in the absence of flhDC or fliZ, the latter previously shown to induce SPI1, indicating that this regulatory pathway is required for invasion control by fliT. Although this flhDC-fliZ pathway was necessary for fliT to negatively control invasion genes, fliZ was not essential for the repressive effect of fliT on motility, placing fliT high in the regulatory cascade for both invasion and motility.

The fimbriae activator MatA switches off motility in Escherichia coli by repression of the flagellar master operon flhDC

Flagella provide advantages to Escherichia coli by facilitating taxis towards nutrients and away from unfavourable niches. On the other hand, flagellation is an energy sink to the bacterial cell, and flagella also stimulate host innate inflammatory responses against infecting bacteria. The flagellar assembly pathway is ordered and under a complex regulatory circuit that involves three classes of temporally regulated promoters as well as the flagellar master regulator FlhD(4)C(2). We report here that transcription of the flhDC operon from the class 1 promoter is under negative regulation by MatA, a key activator of the common mat (or ecp) fimbria operon that enhances biofilm formation by E. coli. Ectopic expression of MatA completely precluded motility and flagellar synthesis in the meningitis-associated E. coli isolate IHE 3034. Northern blotting, analysis of chromosomal promoter-lacZ fusions and electrophoretic mobility shift assays revealed an interaction between MatA and the flhDC promoter region that apparently repressed flagellum biosynthesis. However, inactivation of matA in the chromosome of IHE 3034 had only a minor effect on flagellation, which underlines the complexity of regulatory signals that promote flagellation in E. coli. We propose that the opposite regulatory actions of MatA on mat and on flhDC promoters advance the adaptation of E. coli from a planktonic to an adhesive lifestyle.

The Transcript from the σ 28 -Dependent Promoter Is Translationally Inert in the Expression of the σ 28 -Encoding Gene fliA in the fliAZ Operon of Salmonella enterica Serovar Typhimurium

There are three classes of promoters for flagellar operons in Salmonella. Class 2 promoters are transcribed by σ(70) RNA polymerase in the presence of an essential activator, FlhD(4)C(2), and activated by an auxiliary regulator, FliZ. Class 3 promoters are transcribed by σ(28) RNA polymerase and repressed by an anti-σ(28) factor, FlgM. σ(28) (FliA) and FliZ are encoded by the fliA and fliZ genes, respectively, which together constitute an operon transcribed in this order. This operon is transcribed from both class 2 and class 3 promoters, suggesting that it should be activated by its own product, σ(28), even in the absence of FlhD(4)C(2). However, σ(28)-dependent transcription occurs in vivo only in the presence of FlhD(4)C(2), indicating that transcription from the class 2 promoter is a prerequisite to that from the class 3 promoter. In this study, we examined the effects of variously modified versions of the fliA regulatory region on transcription and translation of the fliA gene. We showed that FliA is not significantly translated from the class 3 transcript. In contrast, the 5'-terminal AU-rich sequence found in the class 2 transcript confers efficient fliA translation. Replacement of the Shine-Dalgarno sequence of the fliA gene with a better one improved fliA translation from the class 3 transcript. These results suggest that the 5'-terminal AU-rich sequence of the class 2 transcript may assist ribosome binding. FliZ was shown to be expressed from both the class 2 and class 3 transcripts.

FliZ Acts as a Repressor of the ydiV Gene, Which Encodes an Anti-FlhD 4 C 2 Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium

YdiV acts as an anti-FlhD4C2 factor, which negatively regulates the class 2 flagellar operons in poor medium in Salmonella enterica serovar Typhimurium. On the other hand, one of the class 2 flagellar genes, fliZ, encodes a positive regulator of the class 2 operons. In this study, we found that the FliZ-dependent activation of class 2 operon expression was more profound in poor medium than in rich medium and not observed in the ydiV mutant background. Transcription of the ydiV gene was shown to increase in the fliZ mutant. Purified FliZ protein was shown in vitro to bind to the promoter region of the nlpC gene, which is located just upstream of the ydiV gene, and to repress its transcription. These results indicate that FliZ is a repressor of the nlpC-ydiV operon and activates the class 2 operons by repressing ydiV expression. Therefore, the fliZ and ydiV genes form a regulatory loop.