Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the σ 54-dependent regulon in Campylobacter jejuni

N, Kamal; Dorrell, Nick; Jagannathan, Aparna; Turner, Susan M.; Constantinidou, Chrystala; Studholme, David J.; Marsden, Gemma; Hinds, Jason; Laing, Ken; Wren, Brendan W.; Penn, Charles W.

doi:10.1099/mic.0.2007/007401-0

Cited by 27 publications

(34 citation statements)

References 55 publications

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“…Disrupting fliK in H. pylori or C. jejuni stimulates expression of RpoN-dependent genes, further suggesting a link between the export apparatus and RpoN regulons in these bacteria (Kamal et al, 2007;Ryan et al, 2005;.…”

Section: Introductionmentioning

confidence: 99%

Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression

et al. 2013

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Flagellar biogenesis in the gastric pathogen Helicobacter pylori involves a transcriptional hierarchy that utilizes all three sigma factors found in this bacterium (RpoD, RpoN and FliA). Transcription of the RpoN-dependent genes requires the sensor kinase FlgS and response regulator FlgR. It is thought that FlgS senses some cellular cue to regulate transcription of the RpoN-dependent flagellar genes, but this signal has yet to be identified. Previous studies showed that transcription of the RpoN-dependent genes is inhibited by mutations in flhA, which encodes a membrane-bound component of the flagellar protein export apparatus. We found that depending on the H. pylori strain used, insertion mutations in flhA had different effects on expression of RpoN-dependent genes. Mutations in flhA in H. pylori strains B128 and ATCC 43504 (the type strain) were generated by inserting a chloramphenicol resistance cassette so as to effectively eliminate expression of the gene (DflhA), or within the gene following codon 77 (designated flhA77) or codon 454 (designated flhA454), which could allow expression of truncated FlhA proteins. All three flhA mutations severely inhibited transcription of the RpoN-dependent genes flaB and flgE in H. pylori B128. In contrast, levels of flaB and flgE transcripts in H. pylori ATCC 43504 bearing either flhA77 or flhA454, but not DflhA, were~60 % of wild-type levels. The FlhA 454 variant was detected in membrane fractions prepared from H. pylori ATCC 43504 but not H. pylori B128, which may account for the phenotypic differences in the flhA mutations of the two strains. Taken together, these findings suggest that only the N-terminal region of FlhA is needed for transcription of the RpoN regulon. Interestingly, expression of an flaB9-9xylE reporter gene in H. pylori ATCC 43504 bearing the flhA77 allele was about eightfold higher than that of a strain with the wild-type allele, suggesting that expression of flaB is not only regulated at the level of transcription but also regulated post-transcriptionally.

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Section: Introductionmentioning

confidence: 99%

Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression

et al. 2013

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“…Significant expression changes greater than the 1.5-fold change threshold are indicated (*). The predicted sigma factors for the genes are indicated: s 70 (X), s 54 ($), s 28 (&) (Carrillo et al, 2004;Hendrixson & DiRita, 2003;Kamal et al, 2007;Petersen et al, 2003).…”

Section: Jejuni Exhibits a Preferential Order Of Amino Acid Utilizmentioning

confidence: 99%

“…All three sigma factors are involved in the regulation of flagellar genes, with s 70 believed to regulate early 'class I' genes that are involved in assembly of the basal body and export apparatus, s 54 regulating 'class II' genes that are involved in basal body, hook and filament biosynthesis, and s 28 regulating 'class III' genes that are involved in filament biosynthesis and capping and flagellin post-translational modification (Carrillo et al, 2004;Kamal et al, 2007). In order to examine whether bacterial motility correlates with the transcription of flagellar genes, we compared expression data with the motility of C. jejuni throughout the growth curve and identified two main relationships.…”

Section: Bacterial Motility Peaks During Stationary Phasementioning

confidence: 99%

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch

et al. 2009

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Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic 'stationary phase', characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR ( 1 H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival. INTRODUCTIONCampylobacter jejuni is the major cause of food-borne bacterial gastroenteritis worldwide, with an estimated 1 in 100 individuals in both the USA and the UK developing Campylobacter-related illness each year (Gaynor et al., 2004;Gillespie et al., 2002). Although most cases are selflimiting, C. jejuni can cause severe post-infection complications including the peripheral neuropathies GuillainBarré and Miller-Fisher syndromes (Nachamkin et al., 2000). Despite the significance of C. jejuni as a food-borne pathogen, our understanding of its basic biochemistry and physiology, gene regulation, colonization, virulence and environmental survival mechanisms lags behind that of many other pathogenic and non-pathogenic bacteria, and many questions regarding C. jejuni physiology and pathogenesis remain (Young et al., 2007). Research has been hampered by a lack of genetic tools, the difficulty of growing the bacteria in the laboratory and the absence of a suitable animal model that mimics human disease.C. jejuni is a fastidious bacterium, with a limited capacity for biosynthesis, and requires complex growth media (Kelly, 2001). Establishing the metabolic capabilities of C. jejuni is central to comprehension of environmental persistence and host colonization. The microaerophilic nature, complex nutritional requirements and relative difficulty of culturing C. jejuni have all contributed to the limited understanding of metabolism (Kelly, 2001(Kelly, , 2005Sellars et al., 2002). C. jejuni is unable to catabolize glucose and other hexose sugars due to the absence of the key glycolytic enzyme 6-phosphofructokinase (Parkhill et al., 2000;Velayudhan & Kelly, 2002 The importance of each amino acid in C. jejuni metabolism is yet to be fully established, however, and i...

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“…Inactivation of fliK in H. pylori and C. jejuni stimulates expression of RpoN-dependent flagellar genes (Kamal et al, 2007;Ryan et al, 2005). Based on the role of FliK and FlhB in substrate switching, we wished to determine how processingdeficient FlhB variants would affect expression of the H. pylori RpoN and FliA regulons.…”

Section: Introductionmentioning

confidence: 99%

Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

2009

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Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors s 54 (RpoN), employed for expression of genes required midway through flagellar assembly, and s 28 (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhB C ). The hook length control protein FliK interacts with FlhB C to modulate the substrate specificity of the export apparatus. FlhB C undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the DflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the DflhB mutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN-and FliA-dependent reporter genes. In the strain producing FlhB N265A , expression of RpoN-and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhB P266G. H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhB C (FlhB CC ) and can substitute for FlhB CC in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the DfliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhB CC in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus. INTRODUCTIONBacterial flagellar biosynthesis involves transcriptional hierarchies that coordinate flagellar gene expression with assembly. Flagellar gene hierarchies are well characterized in a few bacteria, including Salmonella enterica serovar Typhimurium (S. typhimurium) and Caulobacter crescentus (Chilcott & Hughes, 2000;Wu & Newton, 1997). Flagellar gene regulation in e-Proteobacteria, such as Helicobacter pylori and Campylobacter jejuni, shares some similarities with these established paradigms but also differs significantly. As in S. typhimurium, H. pylori flagellar genes required late in assembly, such as the major flagellin (FlaA) and filament cap protein, are transcribed by s 28 (FliA)-RNA polymerase holoenzyme (Kim et al., 1999;Leying et al., 1992), and, as in C....

show abstract

Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the σ 54-dependent regulon in Campylobacter jejuni

Cited by 27 publications

References 55 publications

Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression

Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch

Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

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