Flagellar Gene Expression Research Articles

Pseudomonas Flagella: Generalities and Specificities.

Flagella-driven motility is an important trait for bacterial colonization and virulence. Flagella rotate and propel bacteria in liquid or semi-liquid media to ensure such bacterial fitness. Bacterial flagella are composed of three parts: a membrane complex, a flexible-hook, and a flagellin filament. The most widely studied models in terms of the flagellar apparatus are E. coli and Salmonella. However, there are many differences between these enteric bacteria and the bacteria of the Pseudomonas genus. Enteric bacteria possess peritrichous flagella, in contrast to Pseudomonads, which possess polar flagella. In addition, flagellar gene expression in Pseudomonas is under a four-tiered regulatory circuit, whereas enteric bacteria express flagellar genes in a three-step manner. Here, we use knowledge of E. coli and Salmonella flagella to describe the general properties of flagella and then focus on the specificities of Pseudomonas flagella. After a description of flagellar structure, which is highly conserved among Gram-negative bacteria, we focus on the steps of flagellar assembly that differ between enteric and polar-flagellated bacteria. In addition, we summarize generalities concerning the fuel used for the production and rotation of the flagellar macromolecular complex. The last part summarizes known regulatory pathways and potential links with the type-six secretion system (T6SS).

Regulatory Effects of CsrA in Vibrio cholerae.

CsrA is a posttranscriptional global regulator in Vibrio cholerae Although CsrA is critical for V. cholerae survival within the mammalian host, the regulatory targets of CsrA remain mostly unknown. To identify pathways controlled by CsrA, RNA-seq transcriptome analysis was carried out by comparing the wild type and the csrA mutant grown to early exponential, mid-exponential, and stationary phases of growth. This enabled us to identify the global effects of CsrA-mediated regulation throughout the V. cholerae growth cycle. We found that CsrA regulates 22% of the V. cholerae transcriptome, with significant regulation within the gene ontology (GO) processes that involve amino acid transport and metabolism, central carbon metabolism, lipid metabolism, iron uptake, and flagellum-dependent motility. Through CsrA-RNA coimmunoprecipitation experiments, we found that CsrA binds to multiple mRNAs that encode regulatory proteins. These include transcripts encoding the major sigma factors RpoS and RpoE, which may explain how CsrA regulation affects such a large proportion of the V. cholerae transcriptome. Other direct targets include flrC, encoding a central regulator in flagellar gene expression, and aphA, encoding the virulence gene transcription factor AphA. We found that CsrA binds to the aphA mRNA both in vivo and in vitro, and CsrA significantly increases AphA protein synthesis. The increase in AphA was due to increased translation, not transcription, in the presence of CsrA, consistent with CsrA binding to the aphA transcript and enhancing its translation. CsrA is required for the virulence of V. cholerae and this study illustrates the central role of CsrA in virulence gene regulation.IMPORTANCEVibrio cholerae, a Gram-negative bacterium, is a natural inhabitant of the aqueous environment. However, once ingested, this bacterium can colonize the human host and cause the disease cholera. In order to successfully transition between its aqueous habitat and the human host, the bacterium must sense changes in its environment and rapidly alter gene expression. Global regulators, including CsrA, play an integral role in altering the expression of a large number of genes to promote adaptation and survival, which is required for intestinal colonization. We used transcriptomics and a directed CsrA-RNA coimmunoprecipitation to characterize the CsrA regulon and found that CsrA alters the expression of more than 800 transcripts in V. cholerae Processes regulated by CsrA include motility, the rugose phenotype, and virulence pathways. CsrA directly binds to the aphA transcript and positively regulates the production of the virulence regulator AphA. Thus, CsrA regulates multiple processes that have been linked to pathogenesis.

Downregulation of a novel flagellar synthesis regulator AsiR promotes intracellular replication and systemic pathogenicity of Salmonella Typhimurium

ABSTRACT The intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits host macrophage as a crucial survival and replicative niche. To minimize host immune response stimulated by flagellin, the expression of flagellar genes is downregulated during S. Typhimurium growth within host macrophages. However, the underlying mechanisms are largely unknown. In this study, we show that STM14_1285 (named AsiR), a putative RpiR-family transcriptional regulator, which is downregulated within macrophages as previously reported and also confirmed here, positively regulates the expression of flagellar genes by directly binding to the promoter of flhDC. By generating an asiR mutant strain and a strain that persistently expresses asiR gene within macrophages, we confirmed that the downregulation of asiR contributes positively to S. Typhimurium replication in macrophages and systemic infection in mice, which could be attributed to decreased flagellar gene expression and therefore reduced flagellin-stimulated secretion of pro-inflammatory cytokines IL-1β and TNF-α. Furthermore, the acidic pH in macrophages is identified as a signal for the downregulation of asiR and therefore flagellar genes. Collectively, our results reveal a novel acidic pH signal-mediated regulatory pathway that is utilized by S. Typhimurium to promote intracellular replication and systemic pathogenesis by repressing flagellar gene expression.

Comparative Omics Analysis of Historic and Recent Isolates of Bordetella pertussis and Effects of Genome Rearrangements on Evolution.

Despite high vaccination coverage, pertussis is increasing in many industrialized countries, including the Czech Republic. To better understand Bordetella pertussis resurgence, we analyzed historic strains and recent clinical isolates by using a comparative omics approach. Whole-genome sequencing showed that historic and recent isolates of B. pertussis have substantial variation in genome organization and form separate phylogenetic clusters. Subsequent RNA sequence analysis and liquid chromatography with mass tandem spectrometry analyses showed that these variations translated into discretely separated transcriptomic and proteomic profiles. When compared with historic strains, recent isolates showed increased expression of flagellar genes and genes involved in lipopolysaccharide biosynthesis and decreased expression of polysaccharide capsule genes. Compared with reference strain Tohama I, all strains had increased expression and production of the type III secretion system apparatus. We detected the potential link between observed effects and insertion sequence element–induced changes in gene context only for a few genes.

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the “flagellar switch” upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5’ leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

Two Tandem Mechanisms Control Bimodal Expression of the Flagellar Genes in Salmonella enterica.

Flagellar gene expression is bimodal in Salmonella enterica Under certain growth conditions, some cells express the flagellar genes whereas others do not. This results in mixed populations of motile and nonmotile cells. In the present study, we found that two independent mechanisms control bimodal expression of the flagellar genes. One was previously found to result from a double negative-feedback loop involving the flagellar regulators RflP and FliZ. This feedback loop governs bimodal expression of class 2 genes. In this work, a second mechanism was found to govern bimodal expression of class 3 genes. In particular, class 3 gene expression is still bimodal, even when class 2 gene expression is not. Using a combination of experimental and modeling approaches, we found that class 3 bimodality results from the σ28-FlgM developmental checkpoint.IMPORTANCE Many bacterial use flagella to swim in liquids and swarm over surface. In Salmonella enterica, over 50 genes are required to assemble flagella. The expression of these genes is tightly regulated. Previous studies have found that flagellar gene expression is bimodal in S. enterica, which means that only a fraction of cells express flagellar genes and are motile. In the present study, we found that two separate mechanisms induce this bimodal response. One mechanism, which was previously identified, tunes the fraction of motile cells in response to nutrients. The other results from a developmental checkpoint that couples flagellar gene expression to flagellar assembly. Collectively, these results further our understanding of how flagellar gene expression is regulated in S. enterica.

An ECF41 family σ factor controls motility and biogenesis of lateral flagella in Azospirillum brasilense Sp245.

ECF41 is a large family of bacterial extra-cytoplasmic function (ECF) σ factors. Their role in bacterial physiology or behavior, however, is not known. One of the 10 ECF σ factors encoded in the genome of Azospirillum brasilense Sp245, RpoE10, exhibits characteristic features of the typical ECF41-type σ factors. Inactivation of rpoE10 in A. brasilense Sp245 led to an increase in motility that could be complemented by the expression of rpoE10 By comparing the number of lateral flagella, transcriptome and proteome of A. brasilense Sp245 with its rpoE10::km mutant, we show here that this ECF41-type σ factor is involved in the negative regulation of swimming motility and biogenesis of lateral flagella of A. brasilense Sp245. The genome of A. brasilense Sp245 also encodes two OmpR-type regulators (LafR1 and LafR2), and three flagellins including Laf1, the major flagellin of lateral flagella. Elevated levels of laf1 transcripts and Laf1 protein in the rpoE10::km mutant indicated that RpoE10 negatively regulates the expression of Laf1. The elevated level of LafR1 in the rpoE10::km mutant indicated that LafR1 is also negatively regulated by RpoE10. The loss of motility and Laf1 in the lafR1::km mutant, complemented by lafR1 expression, showed that LafR1 is a positive regulator of Laf1 and motility in A. brasilense In addition, upregulation of laf1::lacZ and lafR1::lacZ fusions by RpoE10, and downregulation of the laf1::lacZ fusion by LafR1 suggests that RpoE10 negatively regulates swimming motility and the expression of LafR1 and Laf1. However, LafR1 positively regulates the swimming motility and Laf1 expression.Importance: Among extra-cytoplasmic function (ECF) σ factors, ECF41-type σ factors are unique due to the presence of a large C-terminal extension in place of a cognate anti- σ factor, which regulates their activity. Despite wide distribution and abundance in bacterial genomes, their physiological or behavioural roles are not known. We show here an indirect negative role of an ECF41-type of σ factor in the expression of lateral flagellar genes and motility in A.brasilense This study suggests that the motility of A. brasilense might be controlled by a regulatory cascade involving RpoE10, an unknown repressor, LafR1 and lateral flagellar genes including Laf1.

The extracellular matrix protein TasA is a developmental cue that maintains a motile subpopulation within Bacillus subtilis biofilms.

In nature, bacteria form biofilms-differentiated multicellular communities attached to surfaces. Within these generally sessile biofilms, a subset of cells continues to express motility genes. We found that this subpopulation enabled Bacillus subtilis biofilms to expand on high-friction surfaces. The extracellular matrix (ECM) protein TasA was required for the expression of flagellar genes. In addition to its structural role as an adhesive fiber for cell attachment, TasA acted as a developmental signal stimulating a subset of biofilm cells to revert to a motile phenotype. Transcriptomic analysis revealed that TasA stimulated the expression of a specific subset of genes whose products promote motility and repress ECM production. Spontaneous suppressor mutations that restored motility in the absence of TasA revealed that activation of the biofilm-motility switch by the two-component system CssR/CssS antagonized the TasA-mediated reversion to motility in biofilm cells. Our results suggest that although mostly sessile, biofilms retain a degree of motility by actively maintaining a motile subpopulation.

Characterization of the extracellular matrix of biofilms formed by Xanthomonas citri subsp. citri strains with different host ranges

Xanthomonas citri subsp. citri (Xcc) causes citrus bacterial canker (CBC) on several Citrus species. Xcc requires bacterial aggregation and biofilm formation to colonize the host plant. Those biofilms are dependent on the environmental condition and their structures differ according to the host range of the Xcc strain. Herein, we have visualized and evaluated the extracellular structures produced by Xcc strains with different host range at early stages of biofilm formation, in mature biofilms and during planktonic growth. Moreover, the presence of these structures was related to the transcription of quorum sensing, flagellar and fimbrial genes. Our results demonstrate that the variation in biofilm formation between Xcc strains is associated with expression of fimbrial and flagellar genes which in some cases is dependent on a quorum sensing system.

Small RNAs OmrA and OmrB promote class III flagellar gene expression by inhibiting the synthesis of anti-Sigma factor FlgM

ABSTRACT Bacteria can move by a variety of mechanisms, the best understood being flagella-mediated motility. Flagellar genes are organized in a three-tiered cascade allowing for temporally regulated expression that involves both transcriptional and post-transcriptional control. The class I operon encodes the master regulator FlhDC that drives class II gene transcription. Class II genes include fliA and flgM, which encode the Sigma factor σ28, required for class III transcription, and the anti-Sigma factor FlgM, which inhibits σ28 activity, respectively. The flhDC mRNA is regulated by several small regulatory RNAs (sRNAs). Two of these, the sequence-related OmrA and OmrB RNAs, inhibit FlhD synthesis. Here, we report on a second layer of sRNA-mediated control downstream of FhlDC in the flagella pathway. By mutational analysis, we confirm that a predicted interaction between the conserved 5ʹ seed sequences of OmrA/B and the early coding sequence in flgM mRNA reduces FlgM expression. Regulation is dependent on the global RNA-binding protein Hfq. In vitro experiments support a canonical mechanism: binding of OmrA/B prevents ribosome loading and decreases FlgM protein synthesis. Simultaneous inhibition of both FlhD and FlgM synthesis by OmrA/B complicated an assessment of how regulation of FlgM alone impacts class III gene transcription. Using a combinatorial mutation strategy, we were able to uncouple these two targets and demonstrate that OmrA/B-dependent inhibition of FlgM synthesis liberates σ28 to ultimately promote higher expression of the class III flagellin gene fliC.

Articles of Significant Interest in This Issue

Nieto et al. (e00578-19) revisit YcgR::c-di-GMP action at the flagellar motor in Escherichia coli, a controversial subject due to differing results from several laboratories. Using a high-resolution single motor assay, the authors show that YcgR acts sequentially, altering motor bias first and then decreasing motor speed, thus demonstrating clearly that YcgR affects both rotor and stator components of the motor. While investigating why motility in soft agar does not return to normal in the absence of YcgR, the authors discovered that it is because the σS regulator protein RssB contributes to negative regulation of flagellar gene expression under elevated c-di-GMP.

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB.

In Escherichia coli and Salmonella, the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of ycgR restores wild-type motor behavior in E. coli, indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In Salmonella, c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in E. coli Instead, we found through reversion analysis that deletion of rssB, which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σS for degradation, restored wild-type motility in the ycgR mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression.IMPORTANCE Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli, we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σS regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.

Detecting Salmonella Type II flagella production by transmission electron microscopy and immunocytochemistry.

The bacterial flagellum is an appendage structure that provides a means for motility to promote survival in fluctuating environments. For the intracellular pathogen Salmonella enterica serovar Typhimurium to survive within macrophages, flagellar gene expression must be tightly regulated, and thus, is controlled at multiple levels, including DNA recombination, transcription, post-transcription, protein synthesis, and assembly within host cells. To understand the contribution of flagella to Salmonella pathogenesis within the host, it is critical to detect flagella production within macrophages via microscopy. In this paper, we describe two methods for detecting bacterial flagella by microscopy both in vitro and in vivo infection models.

A GntR Family Transcription Factor (VPA1701) for Swarming Motility and Colonization of Vibrio parahaemolyticus.

Motility is important for virulence, biofilm formation, and the environmental adaptation of many bacteria. Vibrio parahaemolyticus (V. parahaemolyticus) contains two flagellar systems that are responsible for motility, and are tightly regulated by transcription regulators and sigma factors. In this study, we identified a novel transcription factor, VPA1701, which regulates the swarming motility of V. parahaemolyticus. The VPA1701 deletion mutant (ΔVPA1701) eliminated the swarming motility on the surface of BHI agar plates and reduced colonization in infant rabbits. RNA-seq assays, confirmed by qRT-PCR, indicated that VPA1701 regulated the expression of lateral flagellar cluster genes. Further analyses revealed that VPA1701 directly binds to the promoter region of the flgBCDEFGHIJKL cluster to regulate the expression of lateral flagellar genes. CalR was originally identified as a repressor for the swarming motility of V. parahaemolyticus, and it was inhibited by calcium. In this study, we found that VPA1701 could inhibit the expression of the calR gene to increase the swarming motility of V. parahaemolyticus. Calcium downregulated the expression of calR, indicating that calcium could increase swarming motility of ΔVPA1701 by inhibiting calR. Thus, this study illustrates how the transcription factor VPA1701 regulates the expression of lateral flagellar genes and calR to control the swarming motility of V. parahaemolyticus.

The flagella of 'Candidatus Liberibacter asiaticus' and its movement in planta.

SummaryCitrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin‐encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin‐encoding genes. Only flgJAtu but not flgJLas restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull‐down assays demonstrated that FlgJLas interacts with FlgB but not with FliE. Ectopic expression of flaALas in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin‐specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.

Multidrug Resistance Regulators MarA, SoxS, Rob, and RamA Repress Flagellar Gene Expression and Motility in Salmonella enterica Serovar Typhimurium.

Production of flagella is costly and subject to global multilayered regulation, which is reflected in the hierarchical control of flagellar production in many bacterial species. For Salmonella enterica serovar Typhimurium and its relatives, global regulation of flagellar production primarily occurs through the control of flhDC transcription and mRNA translation. In this study, the roles of the homologous multidrug resistance regulators MarA, SoxS, Rob, and RamA (constituting the mar-sox-rob regulon in S Typhimurium) in regulating flagellar gene expression were explored. Each of these regulators was found to inhibit flagellar gene expression, production of flagella, and motility. To different degrees, repression via these transcription factors occurred through direct interactions with the flhDC promoter, particularly for MarA and Rob. Additionally, SoxS repressed flagellar gene expression via a posttranscriptional pathway, reducing flhDC translation. The roles of these transcription factors in reducing motility in the presence of salicylic acid were also elucidated, adding a genetic regulatory element to the response of S Typhimurium to this well-characterized chemorepellent. Integration of flagellar gene expression into the mar-sox-rob regulon in S Typhimurium contrasts with findings for closely related species such as Escherichia coli, providing an example of plasticity in the mar-sox-rob regulon throughout the Enterobacteriaceae family.IMPORTANCE The mar-sox-rob regulon is a large and highly conserved stress response network in the Enterobacteriaceae family. Although it is well characterized in E. coli, the extent of this regulon in related species is unclear. Here, the control of costly flagellar gene expression is connected to the mar-sox-rob regulon of S Typhimurium, contrasting with the E. coli regulon model. These findings demonstrate the flexibility of the mar-sox-rob regulon to accommodate novel regulatory targets, and they provide evidence for its broader regulatory role within this family of diverse bacteria.

Synergistic action of SPI-1 gene expression in Salmonella enterica serovar typhimurium through transcriptional crosstalk with the flagellar system

BackgroundSalmonella enterica serovar Typhimurium is a common food-borne pathogen. S. enterica uses a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1) to invade intestinal epithelial cells. A complex network of interacting transcription factors regulates SPI-1 gene expression. In addition, SPI-1 gene expression is coupled to flagellar gene expression. Both SPI-1 and flagellar gene expression are bistable, with co-existing populations of cells expressing and not expressing these genes. Previous work demonstrated that nutrients could be used to tune the fraction of cells expressing the flagellar genes. In the present study, we tested whether nutrients could also tune the fraction of cells expressing the SPI-1 genes through transcriptional crosstalk with the flagellar genes.ResultsNutrients alone were not found to induce SPI-1 gene expression. However, when the cells were also grown in the presence of acetate, the concentration of nutrients in the growth medium was able to tune the fraction of cells expressing the SPI-1 genes. During growth in nutrient-poor medium, acetate alone was unable to induce SPI-1 gene expression. These results demonstrate that acetate and nutrients synergistically activate SPI-1 gene expression. The response to acetate was governed by the BarA/SirA two-component system and the response to nutrients was governed by transcriptional crosstalk with the flagella system, specifically through the action of the flagellar regulator FliZ.ConclusionsAcetate and nutrients are capable of synergistically activating SPI-1 gene expression. In addition, these signals were found to tune the fraction of cells expressing the SPI-1 genes. The governing mechanism involves transcriptional crosstalk with the flagellar gene network. Collectively, these results further our understanding of SPI-1 gene regulation and provide the basis for future studies investigating this complex regulatory mechanism.

The Periplasmic Chaperone SurA Affects Motility and Biofilm Formation via the RcsCDB Pathway in Salmonella enterica Serovar Typhi.

SurA, a periplasmic chaperone, is a key factor in the biogenesis of β-barrel outer membrane proteins (OMPs). It is also associated with virulence, invasion and biofilm formation in Escherichia and Salmonella species. To investigate whether SurA affects bacterial motility and biofilm formation in Salmonella enterica serovar Typhi, we prepared a surA deleted mutant. Motility assay and quantitative real-time PCR (qRT-PCR) indicated that surA deletion reduced swimming motility and decreased flagellar gene expression, respectively. β-galactosidase assay and qRT-PCR further showed that surA deletion also activated the RcsCDB pathway, which we verified affected motility. We also examined the effects of the surA deletion on biofilm formation and established that the surA mutant exhibited significantly reduced ability to form biofilms compared with the wild-type. From our findings, we proposed that the periplasmic chaperone, SurA, affects flagella expression via the RcsCDB pathway thereby influencing biofilm formation in Salmonella enterica serovar Typhi. Collectively, these studies confirmed a new physiological role for SurA in Salmonella enterica serovar Typhi.

Sensor I Regulated ATPase Activity of FleQ Is Essential for Motility to Biofilm Transition in Pseudomonas aeruginosa.

Members of the AAA+ (ATPase associated with various cellular activities) family of ATPases couple chemical energy derived from ATP hydrolysis for generation of mechanical force, resulting in conformational changes. The hydrolysis is brought about by highly conserved domains and motifs. The sensor I motif is critical for sensing and hydrolysis of the nucleotide. Pseudomonas aeruginosa FleQ is an ATPase that is a positive regulator of flagellar gene expression. We have determined the crystal structures of the ATPase domain of wild-type FleQ and sensor I mutants H287N and H287A in complex with ATPγS and Mg2+ to 2.4, 1.95, and 2.25 Å resolution, respectively. The structural data highlight the role of sensor I in regulating the ATPase activity. The in vitro and in vivo data demonstrate that the moderate ATPase activity of FleQ due to the presence of histidine in sensor I is essential for maintaining the monotrichous phenotype and for the rapid motility to biofilm transition.

Bordetella pertussis Can Be Motile and Express Flagellum-Like Structures.

Bordetella bronchiseptica encodes and expresses a flagellar apparatus. In contrast, Bordetella pertussis, the causative agent of whooping cough, has historically been described as a nonmotile and nonflagellated organism. The previous statements that B. pertussis was a nonmotile organism were consistent with a stop codon located in the flagellar biosynthesis gene, flhA, discovered when the B. pertussis Tohama I genome was sequenced and analyzed by Parkhill et al. in 2003 (J. Parkhill, M. Sebaihia, A. Preston, L. D. Murphy, et al., Nat Genet, 35:32-40, 2003, https://doi.org/10.1038/ng1227). The stop codon has subsequently been found in all annotated genomes. Parkhill et al. also showed, however, that B. pertussis contains all genetic material required for flagellar synthesis and function. We and others have determined by various transcriptomic analyses that these flagellar genes are differentially regulated under a variety of B. pertussis growth conditions. In light of these data, we tested for B. pertussis motility and found that both laboratory-adapted strains and clinical isolates can be motile. Upon isolation of motile B. pertussis, we discovered flagellum-like structures on the surface of the bacteria. B. pertussis motility appears to occur primarily in the Bvg(-) phase, consistent with regulation present in B. bronchiseptica Motility can also be induced by the presence of fetal bovine serum. These observations demonstrate that B. pertussis can express flagellum-like structures, and although it remains to be determined if B. pertussis expresses flagella during infection or if motility and/or flagella play roles during the cycle of infection and transmission, it is clear that these data warrant further investigation.IMPORTANCE This report provides evidence for motility and expression of flagella by B. pertussis, a bacterium that has been reported as nonmotile since it was first isolated and studied. As with B. bronchiseptica, B. pertussis cells can express and assemble a flagellum-like structure on their surface, which in other organisms has been implicated in several important processes that occur in vivo The discovery that B. pertussis is motile raises many questions, including those regarding the mechanisms of regulation for flagellar gene and protein expression and, importantly, the role of flagella during infection. This novel observation provides a foundation for further study of Bordetella flagella and motility in the contexts of infection and transmission.