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Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG - mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG - mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG - and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG - mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes., (© 2021 John Wiley & Sons Ltd.)

Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuni FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output - precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility., (© 2015 John Wiley & Sons Ltd.)

The Campylobacter jejuni flagellum exports both proteins that form the flagellar organelle for swimming motility and colonization and virulence factors that promote commensal colonization of the avian intestinal tract or invasion of human intestinal cells respectively. We explored how the C. jejuni flagellum is a versatile secretory organelle by examining molecular determinants that allow colonization and virulence factors to exploit the flagellum for their own secretion. Flagellar biogenesis was observed to exert temporal control of secretion of these proteins, indicating that a bolus of secretion of colonization and virulence factors occurs during hook biogenesis with filament polymerization itself reducing secretion of these factors. Furthermore, we found that intramolecular and intermolecular requirements for flagellar-dependent secretion of these proteins were most reminiscent to those for flagellin secretion. Importantly, we discovered that secretion of one colonization and virulence factor, CiaI, was not required for invasion of human colonic cells, which counters previous hypotheses for how this protein functions during invasion. Instead, secretion of CiaI was essential for C. jejuni to facilitate commensal colonization of the natural avian host. Our work provides insight into the versatility of the bacterial flagellum as a secretory machine that can export proteins promoting diverse biological processes., (© 2014 John Wiley & Sons Ltd.)

Control of surface organelle number and placement is a crucial aspect of the cell biology of many Gram-positive and Gram-negative bacteria, yet mechanistic insights into how bacteria spatially and numerically organize organelles are lacking. Many surface structures and internal complexes are spatially restricted in the bacterial cell (e.g. type IV pili, holdfasts, chemoreceptors), but perhaps none show so many distinct patterns in terms of number and localization as the flagellum. In this review, we discuss two proteins, FlhF and FlhG (also annotated FleN/YlxH), which control aspects of flagellar assembly, placement and number in polar flagellates, and may influence flagellation in some bacteria that produce peritrichous flagella. Experimental data obtained in a number of bacterial species suggest that these proteins may have acquired distinct attributes influencing flagellar assembly that reflect the diversity of flagellation patterns seen in different polar flagellates. Recent findings also suggest FlhF and FlhG are involved in other processes, such as influencing the rotation of flagella and proper cell division. Continued examination of these proteins in polar flagellates is expected to reveal how different bacteria have adapted FlhF or FlhG with specific activities to tailor flagellar biosynthesis and motility to fit the needs of each species., (© 2013 John Wiley & Sons Ltd.)

The flagellum of Campylobacter jejuni provides motility essential for commensal colonization of the intestinal tract of avian species and infection of humans resulting in diarrhoeal disease. Additionally, the flagellar type III secretion system has been reported to secrete proteins such as CiaI that influence invasion of human intestinal cells and possibly pathogenesis. The flagellar regulatory system ultimately influences σ(28) activity required for expression of the FlaA major flagellin and other flagellar filament proteins. In this work, we discovered that transcription of ciaI and four genes we propose annotating as feds (for flagellar coexpressed determinants) is dependent upon σ(28) , but these genes are not required for motility. Instead, the Feds and CiaI are involved in commensal colonization of chicks, with FedA additionally involved in promoting invasion of human intestinal cells. We also discovered that the major flagellin influences production, stability or secretion of σ(28) -dependent proteins. Specific transcriptional and translational mechanisms affecting CiaI were identified and domains of CiaI were analysed for importance in commensalism or invasion. Our work broadens the genes controlled by the flagellar regulatory system and implicates this system in co-ordinating production of colonization and virulence determinants with flagella, which together are required for optimal interactions with diverse hosts., (© 2012 Blackwell Publishing Ltd.)

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.

Phase variation of genes in bacteria enables phenotypic alteration to modulate interactions within a host as conditions change. To promote commensalism in animals and disease in humans, Campylobacter jejuni produces a flagellar organelle for motility. In addition to tight transcriptional regulation of flagellar genes, C. jejuni also controls flagellar biosynthesis by phase variation. In this study, an unusual phase-variable mechanism controlling production of FlgR, the response regulator of the FlgSR two-component system required for transcription of sigma54-dependent flagellar genes, is identified. Phase variation of FlgR production is due to loss or gain of a nucleotide in homopolymeric adenine or thymine tracts within flgR. This mechanism occurs during commensalism in poultry to alter the colonization capacity of C. jejuni, presumably by influencing the motility phenotype of the bacterium. These findings provide more understanding into the genetic and colonization strategies C. jejuni employs to achieve commensalism in a natural host. Second, due to the richness of the C. jejuni genome in adenine or thymine residues and the apparent lack of the normal set of mismatch repair enzymes, the results from this study may suggest that the C. jejuni genome is more unstable and variable than previously realized. Furthermore, phase variation of flagellar motility by targeting flgR may be a phenomenon specific to C. jejuni that is absent in other Campylobacter species and contribute to reasons why C. jejuni is more frequently found as a commensal organism in poultry and as the cause of disease in humans.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature-tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81-176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl-accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP-domain containing proteins of C. jejuni revealed one other required for wild-type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.

We performed a genetic analysis of flagellar regulation in Campylobacter jejuni, from which we elucidated key portions of the flagellar transcriptional cascade in this bacterium. For this study, we developed a reporter gene system for C. jejuni involving astA, encoding arylsulphatase, and placed astA under control of the sigma 54-regulated flgDE2 promoter in C. jejuni strain 81-176. The astA reporter fusion combined with transposon mutagenesis allowed us to identify genes in which insertions abolished flgDE2 expression; genes identified were on both the chromosome and the plasmid pVir. Included among the chromosomal genes were genes encoding a putative sensor kinase and the sigma 54-dependent transcriptional activator, FlgR. In addition, we identified specific flagellar genes, including flhA, flhB, fliP, fliR and flhF, that are also required for transcription of flgDE2 and are presumably at the beginning of the C. jejuni flagellar transcriptional cascade. Deletion of any of these genes reduced transcription of both flgDE2 and another sigma 54-dependent flagellar gene, flaB, encoding a minor flagellin. Transcription of the sigma 28-dependent gene flaA, encoding the major flagellin, was largely unaffected in the mutants. Further examination of flaA transcription revealed significant sigma 28-independent transcription and only weak repressive activity of the putative anti-sigma 28 factor FlgM. Our study suggests that sigma 54-dependent transcription of flagellar genes in C. jejuni is linked to the formation of the flagellar secretory apparatus. A key difference in the C. jejuni flagellar transcriptional cascade compared with other bacteria that use sigma 28 for transcription of flagellar genes is that a mechanism to repress significantly sigma 28-dependent transcription of flaA in flagellar assembly mutants is absent in C. jejuni.

Lactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 A cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its long-recognized antimicrobial properties.

Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni.

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Haps). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C-terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap beta) predicted formation of a beta-barrel with an N-terminal transmembrane alpha-helix followed by 14 transmembrane beta-strands. Additional analysis revealed that the final beta-strand contains an amino acid motif common to other beta-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Haps was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Haps. These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of beta-barrel proteins to the bacterial outer membrane.