Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.     

Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis.     

Synthesis of Bacterial Flagella II. PBSl Transduction of Flagella-specific Markers in Bacillus subtilis

The linkage relationship of mutants involved in the synthesis of flagella was determined by PBSl transduction. Mutants that affect the structure of flagellin (hag) and temperature-sensitive mutants (flaTS) that produce flagella when grown at 37 C but not when grown at 46 C were examined. All of the mutants were found to be linked to the hisA1 marker. The flaTS mutants fell into three clusters. Group A contained the majority of mutants which were loosely grouped around the hag locus. Group B mutants were segregated from the hag locus and appeared closely linked to the phage adsorption site gene (gtaA), and group C was only loosely linked to hisA1 and thus far contains only one mutant. A flagella locus (ifm) affecting both the degree of motility and level of flagellation was shown to map near group A. Mutants affecting motility (mot) were not linked to hisA1 by PBSl transduction. Several markers previously shown to link to hisA1 were ordered with respect to hisA1 and the flagellar genes.     

Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter

The Agrobacterium VirG protein is normally expressed from two promoters in response to multiple stimuli, including plant-released phenolics (at promoter P1) and acidic growth media (at promoter P2). To simplify the analysis of vir gene induction, we sought to create Agrobacterium strains in which virG could be expressed in a controllable fashion. To study the possibility of using the lac promoter and repressor, we constructed a plasmid containing the lac promoter fused to the lacZ structural gene. A derivative of this plasmid containing the lacIq gene was also constructed. The plasmid not containing lacIq expressed high levels of beta-galactosidase. The plasmid containing lacIq expressed beta-galactosidase at very low levels in the absence of o-nitrophenyl-beta-D-galactoside (IPTG) and at moderate levels in the presence of IPTG. We also fused the lac promoter to a virG::lacZ translational fusion and found that IPTG elevated expression of this translational fusion to moderate levels, though not to levels as high as from the stronger of the two native virG promoters. Finally, the lac promoter was used to express the native virG gene in strains containing a virB::lacZ translational fusion. virB expression in this strain depended on addition of IPTG as well as the vir gene inducer acetosyringone. In a similar strain lacking lacIq, virB expression was greater than in a strain in which virG was expressed from its native promoters. Expression of virG from the lac promoter did not alter the acidic pH optimum for vir gene induction, indicating that the previously observed requirement for acidic media was not due solely to the need to induce P2.     

Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella

The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.     

A novel gene involved in regulating the flagellar gene cascade in Proteus mirabilis

In this study, we identified a transposon insertion in a novel gene, designated disA, that restored swarming motility to a putrescine-deficient speA mutant of Proteus mirabilis. A null allele in disA also increased swarming in a wild-type background. The DisA gene product was homologous to amino acid decarboxylases, and its role in regulating swarming was investigated by examining the expression of genes in the flagellar cascade. In a disA mutant background, we observed a 1.4-fold increase in the expression of flhDC, which encodes FlhD(2)C(2), the master regulator of the flagellar gene cascade. However, the expressions of class 2 (fliA, flgM) and class 3 (flaA) genes were at least 16-fold higher in the disA background during swarmer cell differentiation. Overexpression of DisA on a high-copy-number plasmid did not significantly decrease flhDC mRNA accumulation but resulted in a complete block in mRNA accumulation for both fliA and flaA. DisA overexpression also blocked swarmer cell differentiation. The disA gene was regulated during the swarming cycle, and a single-copy disA::lacZ fusion exhibited a threefold increase in expression in swarmer cells. Given that DisA was similar to amino acid decarboxylases, a panel of decarboxylated amino acids was tested for effects similar to DisA overexpression, and phenethylamine, the product of phenylalanine decarboxylation, was capable of inhibiting both swarming and the expression of class 2 and class 3 genes in the flagellar regulon. A DisA-dependent decarboxylated amino acid may inhibit the formation of active FlhD(2)C(2) heterotetramers or inhibit FlhD(2)C(2) binding to DNA.     

Isolation of specialized lambda transducing bacteriophages for flagellar genes (fla) of Escherichia coli K-12.

Specialized transducing lambda phages carrying the region III flagellar genes (fla) of Escherichia coli K-12 were isolated by a new method. A strain carrying both a cryptic lambda prophage near the his genes and a deletion of the attlambda gene was used as a starting strain. The lysogen of lambdacI857pga18-bio69 was isolated in which the prophage was integrated within the lambda cryptic genes by means of recombination with the residual lambda DNA. The strains with deletions starting within the prophage and ending in these fla genes were selected from among the heat-resistant survivors of the lysogen. They were then infected with heat-inducible and lysis-defective lambda phages and, thus, specialized transducing phage lines for hag and fla were obtained. High-frequency transfer lines of rare phages carrying the fla genes were isolated by inducing a strain carrying a heat-inducible lambda prophage near the his genes and selecting by transduction of a fla deletion strain. Preliminary characterization of these transducing phages is also reported.     

Gene expression profiling of flagellar disassembly in Chlamydomonas reinhardtii

Flagella are sensory organelles that interact with the environment through signal transduction and gene expression networks. We used microarray profiling to examine gene regulation associated with flagellar length change in the green alga Chlamydomonas reinhardtii. Microarrays were probed with fluorescently labeled cDNAs synthesized from RNA extracted from cells before and during flagellar assembly or disassembly. Evaluation of the gene expression profiles identified >100 clones showing at least a twofold change in expression during flagellar length changes. Products of these genes are associated not only with flagellar structure and motility but also with other cellular responses, including signal transduction and metabolism. Expression of specific genes from each category was further characterized at higher resolution by using quantitative real-time PCR (qRT-PCR). Analysis and comparison of the gene expression profiles coupled to flagellar assembly and disassembly revealed that each process involves a new and uncharacterized whole-cell response to flagellar length changes. This analysis lays the groundwork for a more comprehensive understanding of the cellular and molecular networks regulating flagellar length changes.