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.
Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3 end of the operon. Two of these promoters, fla/che P A and P D-3 , lie upstream of the >26-kb fla/che operon. The third promoter, P sigD , lies within the operon, immediately upstream of sigD. fla/che P A , transcribed by E A , lies >24 kb upstream of sigD and appears to be largely responsible for sigD expression. P D-3 , transcribed by E D , has been proposed to participate in an autoregulatory positive feedback loop. P sigD , a minor A -dependent promoter, has been implicated as essential for normal expression of the fla/che operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P A , P D-3 , or P sigD . Our analysis of these strains indicates that fla/che P A is absolutely essential for motility, that P D-3 does not function in positive feedback regulation of sigD expression, and that P sigD is not essential for normal fla/che expression. Further, our results suggest that an additional promoter(s) contributes to sigD expression.Motility and chemotaxis functions in Bacillus subtilis are encoded within the fla/che operon. This large (Ͼ26-kb) operon includes both structural and regulatory components required for motility (6,19,32). The proximal region of the operon includes genes that encode the hook and basal body (HBB) complex, a structure that is required for tethering the flagellar filament to the cell. The distal-most region of the fla/che operon encodes the flagellum-specific sigma factor, D (19). D activity is required for transcription of the genes encoding flagellin (hag) and for the motA and motB genes, which encode the motor proteins that drive flagellar rotation (21,22 (11,15). It is believed that FlgM activity is also controlled by export through the HBB in B. subtilis (5,21,24). Expression of the fla/che operon thus controls motility in a complex manner. First, HBB components are expressed concurrently with D . Subsequent assembly of the HBB structure allows export of FlgM. This activates D to promote transcription of D -dependent motility genes. Recent studies have implicated three promoters in the expression of the fla/che operon and the sigD gene (1, 6) (see schematic, Fig. 1A). One of these promoters, P sigD , is located within the fla/che operon, immediately upstream of the sigD gene. Transcription from P sigD is dependent upon A , the major, vegetative sigma factor in B. subtilis. Previous studies indicated that P sigD contributed only weakly to overall expression of the sigD gene (1). However, genetic data suggested that this slight level of expression might be required to control temporal regulation of the entire fla/che operon (1). This requirement would presumably be indirect, since the location of P sigD precludes it from directly promoting transcription of the fla/che operon (see schematic in Fig. 1A).Two additional promoters, fla/ch...
The fla/che region contains more than 30 genes required for flagellar synthesis and chemotaxis in Bacillus subtilis, including the gene for the flagellum-specific ςD factor, sigD. Sequence and primer extension data demonstrate that a PA promoter immediately upstream of flgB, henceforth referred to as thefla/che PA, and the PD-3 promoter are active in vivo. Transcription from the PD-3 element is dependent on ςD activity and is regulated by the flagellum-specific negative regulator, FlgM. In a strain containing a deletion of fla/che PA(PAΔ), ςD protein was not detected, demonstrating that the fla/che PA is necessary for wild-type expression of the sigD gene. Thus,sigD is part of the >26-kb fla/che operon. Consistent with a lack of detectable ςD protein, the PAΔ strain grows as long filaments and does not express a ςD-dependent hag::lacZreporter construct. These phenotypes are indicative of a lack of sigD expression or complete inhibition of ςD activity by FlgM. However, ςD activity is found in a double mutant containing the PAΔ and a null mutation in flgM. The double mutant no longer grows as long filaments, and expression ofhag::lacZ is partially restored. These data demonstrate that a low level of ςDactivity does exist in the PAΔ mutant but can be detected only in the presence of a null mutation inflgM. Therefore, normal expression ofsigD may also involve another promoter(s) within thefla/che operon.
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