Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis

Abstract

We have used Tn9171acZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsl. An additional transposon insert that maps in the hag locus was obtained. The pattern of P-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtllis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response.The mechanism of bacterial chemotaxis has been the focus of an intense study over the last decade, and recently a model has been proposed for Escherichia coli that appears to be consistent with most genetic, biochemical, and physiological data (2). Over this same period, we have shown that the mechanism of chemotaxis in Bacillus subtilis appears to differ from that in E. coli in several respects (23-25).Methanol does not appear to be produced from a direct hydrolysis of methyl groups from the methyl-accepting chemotaxis proteins (MCPs) (24). We also observe that methanol is released in response to the addition or removal of attractants or repellents (24). In E. coli, negative stimuli (removal of attractants or addition of repellents) cause methanol formation but positive stimuli (addition of attractants or removal of repellents) prevent even basal levels of methanol formation (6,22,26). Methanol release in response to all stimuli has also been reported for Halobacterium halobium (1). B. subtilis motile cells move toward all amino acids (16), whereas cells of E. coli have a neutral response to some amino acids and are repelled by others (12,27). This more elaborate methylation mechanism and the greater variety of chemoeffectors detected should be reflected in the existence of additional gene products that are not found in E. coli.There are only six proteins that are known to be required for chemotaxis in E. coli and Salmonella typhimurium. These are encoded by the cheA, cheB, cheR, cheW, cheY, and cheZ genes (8). Other proteins are also involved, but mutants lacking these proteins have specific defects in flagellum biosynthesis, flagellum structure, motility (rotation of the flagellum), or switching (flagellum reversal) or are unable to respond to specific chemoeffectors.

Characterization of the sigD transcription unit of Bacillus subtilis

“…Interestingly, a cluster of genes required for chemotaxis (che) was also found to map to this region of DNA (42). This cluster of genes has now been shown to contain primarily genes renamed for flagellar synthesis (fla) (1,6,16,37,(39)(40)(41) and has therefore been renamed the fla/che region (33). To understand the nature of the transcription unit encoding sigD, as well as to determine its relationship to the fla/che region, we cloned and analyzed DNA upstream of sigD.…”

“…Transposon insertions within this region, however, gave rise to a lack of flagellin expression (41). Since the aU factor is required for transcription of the flagellin gene (30), we hypothesized that the effect generated by the transposon insertions was due to a complete block to transcription of the sigD gene from upstream promoter elements, whereas the plasmid insertions in the first study resulted in leaky expression of sigD.…”

“…The plasmid insertions used to argue for the existence of a single operon also did not result in the lack of flagellin expression, since those strains were motile but nonchemotactic (42). Both the transposon insertions and the plasmid insertions are located throughout the >24-kb region upstream of sigD (19,28,41,42) (for the approximate location of those used in this study, see Fig. 1), but whereas the transposon insertions disrupt the genes into which they have inserted, the plasmid insertions regenerate the genes into which they have inserted by Campbell-like recombination.…”

“…The che locus has therefore been renamed the fla/che region to more accurately represent the function of the proteins encoded therein (33). Strains containing transposon insertions within the fla/che region of DNA do not express flagellin (41), as is the case with strains lacking the CD factor (19,28). In fact the structural gene for aD, sigD, maps to the same area of the bacterial chromosome as the fla/che region does (28).…”

Motility and chemotaxis in Bacillus sphaericus dependence upon stage of growth

“…Thus, such regulation seems to be universal in the eubacterial world. However, at least one sigh-independent the locus has been identified in B. subtilis [16].…”