Multiple copies of flhDC in Paraburkholderia unamae regulate flagellar gene expression, motility, and biofilm formation

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia and two additional flhC copies also present in P. unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1 , plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter- lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia . In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2 . The varying impact each copy of flhDC has on downstream processes indicates that regulation of FlhDC in P. unamae, and likely other Paraburkholderia, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well-studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC . In P. unamae , the flhDC1 copy showed higher expression and greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in E. coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

SoxS represses flagellar gene expression through RflP in Salmonella enterica Serovar Typhimurium

ABSTRACTFlagellar gene expression is subject to regulation by many global transcription factors in response to environmental and nutritional signals. One of the primary ways this occurs in Salmonella enterica serovar Typhimurium, and its close relatives, is through controlling levels of FlhD4C2 (the flagellar master regulator) via transcriptional, post-transcriptional, and post-translational mechanisms. Recently, we found the homologous transcription factors MarA, Rob, and SoxS repress flhDC expression by distinct mechanisms. MarA and Rob, regulators involved in inducible multidrug resistance, repressed flhDC transcription by interacting directly with the flhDC promoter. Alternatively, SoxS, the oxidative stress response regulator, altered FlhD4C2 levels independent of flhDC transcription by post-transcriptional or post-translational mechanism. Here, using a forward genetic screen, we discovered that SoxS-dependent repression of flagellar gene expression occurs through RflP, an anti-FlhD4C2 factor that targets FlhD4C2 for proteolytic degradation. Elevated soxS expression resulted in concomitant increases in rflP expression, indicating SoxS may work through RflP at the level of rflP transcription. Mapping of the rflP promoter and a bioinformatic search yielded a putative SoxS binding site proximal to the rflP transcription start site. Comparison of the rflP promoter region in S. Typhimurium and Escherichia coli indicate substantial differences, providing a possible mechanism for differential expression of rflP between these species.IMPORTANCESalmonella enterica is a major cause of foodborne illness. Understanding environmental and intracellular signals used by Salmonella to control expression of virulence-associated traits is critical to advancing treatment and prevention of Salmonella-related disease. Reduced expression of flagella at key points during Salmonella infection aids in evasion of the host innate immune system. Within macrophages Salmonella is non-flagellated and exposed to oxidative stress. SoxS-dependent repression of flagellar genes may provide a potential link between oxidative stress and reductions in flagellar expression.

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.

A mathematical model of flagellar gene regulation and construction in Salmonella enterica

Millions of people worldwide develop foodborne illnesses caused by Salmonella enterica (S. enterica) every year. The pathogenesis of S. enterica depends on flagella, which are appendages that the bacteria use to move through the environment. Interestingly, populations of genetically identical bacteria exhibit heterogeneity in the number of flagella. To understand this heterogeneity and the regulation of flagella quantity, we propose a mathematical model that connects the flagellar gene regulatory network to flagellar construction. A regulatory network involving more than 60 genes controls flagellar assembly. The most important member of the network is the master operon, flhDC, which encodes the FlhD4C2 protein. FlhD4C2 controls the construction of flagella by initiating the production of hook basal bodies (HBBs), protein structures that anchor the flagella to the bacterium. By connecting a model of FlhD4C2 regulation to a model of HBB construction, we investigate the roles of various feedback mechanisms. Analysis of our model suggests that a combination of regulatory mechanisms at the protein and transcriptional levels induce bistable FlhD4C2 levels and heterogeneous numbers of flagella. Also, the balance of regulatory mechanisms that become active following HBB construction is sufficient to provide a counting mechanism for controlling the total number of flagella produced.Author summarySalmonella causes foodborne illnesses in millions of people worldwide each year. Flagella, which are appendages that the bacteria use to move through the environment, are a key factor in the infection process. Populations of genetically identical bacteria have been observed to contain both motile cells, generally with 6–10 flagella, and nonmotile cells, with no flagella. In this paper, we use mathematical models of the gene network that regulates flagellar construction to explore how the bacteria controls the number of flagella produced. We suggest that a bacterium must accumulate a threshold amount of a master regulator protein to initiate flagella production and failure to reach the threshold results in no flagella. Downstream mechanisms that impact the amount of master regulator protein are sufficient to determine how many flagella are produced.

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.

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

ABSTRACT 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.