Brucella and Its Hidden Flagellar System

Brucella, a Gram-negative bacterium with a high infective capacity and a wide spectrum of hosts in the animal world, is found in terrestrial and marine mammals, as well as amphibians. This broad spectrum of hosts is closely related to the non-classical virulence factors that allow this pathogen to establish its replicative niche, colonizing epithelial and immune system cells, evading the host’s defenses and defensive response. While motility is the primary role of the flagellum in most bacteria, in Brucella, the flagellum is involved in virulence, infectivity, cell growth, and biofilm formation, all of which are very important facts in a bacterium that to date has been described as a non-motile organism. Evidence of the expression of these flagellar proteins that are present in Brucella makes it possible to hypothesize certain evolutionary aspects as to where a free-living bacterium eventually acquired genetic material from environmental microorganisms, including flagellar genes, conferring on it the ability to reach other hosts (mammals), and, under selective pressure from the environment, can express these genes, helping it to evade the immune response. This review summarizes relevant aspects of the presence of flagellar proteins and puts into context their relevance in certain functions associated with the infective process. The study of these flagellar genes gives the genus Brucella a very high infectious versatility, placing it among the main organisms in urgent need of study, as it is linked to human health by direct contact with farm animals and by eventual transmission to the general population, where flagellar genes and proteins are of great relevance.

Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1

Background: Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role on biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids’ modulation of those group behaviors. Results: Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the non-motile phenotype of the rhlA-minus strain. Swarming motility was restored with the addition of exogenous rhamnolipids obtained from the wild-type strain. Conclusions: Our results show significant evidence that rhamnolipids and/or their precursors, 3-(3-hydroxyalkanoyloxy) alkanoic acids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa. Swarming motility assays support this hypothesis, since the non-motile rhlA-mutant strain had its swarming ability restored by the addition of exogenous rhamnolipids.

An ECF41 Family σ Factor Controls Motility and Biogenesis of Lateral Flagella in Azospirillum brasilense Sp245

ABSTRACT ECF41 is a large family of bacterial extracytoplasmic 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 features characteristic 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 those of 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 suggest 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 extracytoplasmic 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 their wide distribution and abundance in bacterial genomes, their physiological or behavioral 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 that encoding Laf1.

Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

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

ABSTRACT 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.

Flagellum-Mediated Mechanosensing and RflP Control Motility State of Pathogenic Escherichia coli

ABSTRACT Bacterial flagellar motility plays an important role in many processes that occur at surfaces or in hydrogels, including adhesion, biofilm formation, and bacterium-host interactions. Consequently, expression of flagellar genes, as well as genes involved in biofilm formation and virulence, can be regulated by the surface contact. In a few bacterial species, flagella themselves are known to serve as mechanosensors, where an increased load on flagella experienced during surface contact or swimming in viscous media controls gene expression. In this study, we show that gene regulation by motility-dependent mechanosensing is common among pathogenic Escherichia coli strains. This regulatory mechanism requires flagellar rotation, and it enables pathogenic E. coli to repress flagellar genes at low loads in liquid culture, while activating motility in porous medium (soft agar) or upon surface contact. It also controls several other cellular functions, including metabolism and signaling. The mechanosensing response in pathogenic E. coli depends on the negative regulator of motility, RflP (YdiV), which inhibits basal expression of flagellar genes in liquid. While no conditional inhibition of flagellar gene expression in liquid and therefore no upregulation in porous medium was observed in the wild-type commensal or laboratory strains of E. coli, mechanosensitive regulation could be recovered by overexpression of RflP in the laboratory strain. We hypothesize that this conditional activation of flagellar genes in pathogenic E. coli reflects adaptation to the dual role played by flagella and motility during infection. IMPORTANCE Flagella and motility are widespread virulence factors among pathogenic bacteria. Motility enhances the initial host colonization, but the flagellum is a major antigen targeted by the host immune system. Here, we demonstrate that pathogenic E. coli strains employ a mechanosensory function of the flagellar motor to activate flagellar expression under high loads, while repressing it in liquid culture. We hypothesize that this mechanism allows pathogenic E. coli to regulate its motility dependent on the stage of infection, activating flagellar expression upon initial contact with the host epithelium, when motility is beneficial, but reducing it within the host to delay the immune response.

Two tandem mechanisms control bimodal expression of the flagellar genes in Salmonella enterica

ABSTRACTFlagellar 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 non-motile 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 YdiV 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 bimodalilty results from the σ28-FlgM developmental checkpoint.IMPORTANCEMany bacterial use flagella to swim in liquids and swarm over surface. In Salmonella enterica, over fifty genes are required to assemble flagella. The expression of these genes is tightly regulated. Previous studies have found that flagella 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.

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.