Restoration of flagellar biosynthesis by varied mutational events in Campylobacter jejuni

Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas, TX, USA.
Molecular Microbiology (Impact Factor: 4.42). 09/2008; 70(2):519-36. DOI: 10.1111/j.1365-2958.2008.06428.x
Source: PubMed


Both a complex regulatory cascade involving the FlgSR two-component system and phase variation control expression of sigma(54)-dependent flagellar genes in Campylobacter jejuni. In this study, mutational mechanisms influencing production of the FlgS histidine kinase were discovered. Random non-motile, non-flagellated flgS variants were impaired for growth in the chick intestinal tract. Spontaneous revertants restored for flagellar biosynthesis, gene expression, and motility identified by in vivo and in vitro studies had undergone diverse intragenic and extragenic mutational events relative to flgS. Restorative intragenic events included true phase variation, second-site intragenic reversion, and insertion and deletion of short DNA segments within flgS. In vivo-isolated motile revertants possessed an identical, single extragenic mutation to create a partially constitutively active FlgR protein in the absence of FlgS. Considering that FlgR production is also influenced by phase variation, these new findings suggest that the FlgSR two-component system is unique in that each protein is controlled by phase variation and phosphorylation. In addition, this study highlights the mutational activities of C. jejuni and suggests that the bacterium may possess a repertoire of mutational mechanisms to overcome genetic lesions that impair production of virulence and colonization determinants while lacking a normal mismatch repair system.

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Available from: David Hendrixson, Aug 26, 2014
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    • "Flagellar gene expression is tightly controlled and requires the alternative d 54 and d 28 factors, the FlgSR twocomponent system, the FlhF GTPase and the flagellar secretion system (Balaban et al., 2009; Hendrixson & DiRita, 2003; Joslin & Hendrixson, 2009). Phase variation mechanisms acting on the FlgSR system that affect motility in C. jejuni 81-176 include reversible phase variation in (i) homopolymeric poly-A and poly-T tracts within the flgR response regulator (Hendrixson, 2006) and (ii) poly-A tracts and heteropolymeric repeats located in the flgS sensor histidine kinase (Hendrixson, 2008). It is noteworthy that the C. jejuni flagellar filament is extensively glycosylated, predominantly with pseudaminic acid and legionaminic acid and derivatives thereof. "
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    ABSTRACT: Genetic variation due to mutation and phase-variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility, and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome re-sequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of wild-type fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of i) screening multiple defined gene deletion mutant clones, ii) genetic complementation of the gene deletion, and ideally iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.
    Full-text · Article · Sep 2015 · Microbiology
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    • "Alternatively, pseudorevertants can arise in which nucleotides are removed or added close to the original mutated homopolymeric nucleotide tract to restore the correct reading frame but which result in changes in the amino acid sequence of the protein [113]. flgS is also regulated by phase variation in C. jejuni, providing multiple levels of phase-variable control for the RpoN regulon [114, 115]. Similar mechanisms of phase variation also exist for flhA in C. coli [116] and fliP in H. pylori [117] which leads to the formation of truncated proteins that prevent flagellar biogenesis. "
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    ABSTRACT: Flagellar biogenesis in bacteria is a complex process in which the transcription of dozens of structural and regulatory genes is coordinated with the assembly of the flagellum. Although the overall process of flagellar biogenesis is conserved among bacteria, the mechanisms used to regulate flagellar gene expression vary greatly among different bacterial species. Many bacteria use the alternative sigma factor σ 54 (also known as RpoN) to transcribe specific sets of flagellar genes. These bacteria include members of the Epsilonproteobacteria (e.g., Helicobacter pylori and Campylobacter jejuni), Gammaproteobacteria (e.g., Vibrio and Pseudomonas species), and Alphaproteobacteria (e.g., Caulobacter crescentus). This review characterizes the flagellar transcriptional hierarchies in these bacteria and examines what is known about how flagellar gene regulation is linked with other processes including growth phase, quorum sensing, and host colonization.
    Full-text · Article · Jan 2014
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    • "Flagellar expression is subject to phase variation and contributes to the unstable motile phenotype in Campylobacter. Phase variation between motile and non‐motile phenotypes occurs frequently in vitro and in vivo (Caldwell et al., 1985; Karlyshev et al., 2002; Hendrixson, 2006; 2008), and is mediated by slipped‐strand mispairing primarily in homopolymeric or occasionally heteropolymeric tracts, resulting in frameshift mutations in the genes associated with flagellar biosynthesis and regulation (flhA, flgS, flgR and maf) (Park et al., 2000; Karlyshev et al., 2002; Hendrixson, 2006; 2008). The high‐frequency phase variation was attributed to the A/T‐rich genetic content of the C. jejuni chromosome and the absence of an intact mismatch repair system (Hendrixson, 2006). "
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    ABSTRACT: Campylobacter jejuni is a major foodborne pathogen of animal origin and a leading cause of bacterial gastroenteritis in humans. During the past decade, especially since the publication of the first C. jejuni genome sequence, major advances have been made in understanding the pathobiology and physiology of this organism. It is apparent that C. jejuni utilizes sophisticated mechanisms for effective colonization of the intestinal tracts in various animal species. Although Campylobacter is fragile in the environment and requires fastidious growth conditions, it exhibits great flexibility in the adaptation to various habitats including the gastrointestinal tract. This high adaptability is attributable to its genetically, metabolically and phenotypically diverse population structure and its ability to change in response to various challenges. Unlike other enteric pathogens, such as Escherichia coli and Salmonella, Campylobacter is unable to utilize exogenous glucose and mainly depends on the catabolism of amino acids as a carbon source. Campylobacter proves highly mutable in response to antibiotic treatments and possesses eukaryote-like dual protein glycosylation systems, which modify flagella and other surface proteins with specific sugar structures. In this review we will summarize the distinct biological traits of Campylobacter and discuss the potential biotechnological approaches that can be developed to control this enteric pathogen.
    Full-text · Article · May 2010 · Microbial Biotechnology
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