Role and Regulation of Bacillus subtilis Glutamate Dehydrogenase Genes

The complete Bacillus subtilis genome contains two genes with the potential to encode glutamate dehydrogenase (GlutDH) enzymes. Mutations in these genes were constructed and characterized. The rocG gene proved to encode a major GlutDH whose synthesis was induced in media containing arginine or ornithine or, to a lesser degree, proline and was repressed by glucose. A rocG null mutant was impaired in utilization of arginine, ornithine, and proline as nitrogen or carbon sources. The gudB gene was expressed under all growth conditions tested but codes for a GlutDH that seemed to be intrinsically inactive. Spontaneous mutations in gudB that removed a 9-bp direct repeat within the wild-type gudB sequence activated the GudB protein and allowed more-efficient utilization of amino acids of the glutamate family.     

Differential Regulation of Horizontally Acquired and Core Genome Genes by the Bacterial Modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.     

Genes controlling xylan utilization by Bacillus subtilis.

Eight mutants of Bacillus subtilis deficient in xylan utilization were isolated and characterized genetically and biochemically. Each mutant was obtained independently after nitrosoguanidine mutagenesis. All of the analyzed mutations were shown to be linked. Reciprocal transformation crosses revealed the existence of two genes controlling xylan utilization which have been designated xynA and xynB. Available data have indicated that these two genes code for two xylan-degrading enzymes existing in the wild-type strains, an extracellular beta-xylanase (xynA) and a cell-associated beta-xylosidase (xynB).     

Lysis Genes of the Bacillus subtilis Defective Prophage PBSX

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are xepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show that xepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression of xhlB (encoding the putative holin) together with xlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.     

Bacterial Sporulation and Regulation of Dihydrodipicolinate Synthase in Ribonucleic Acid Polymerase Mutants of Bacillus subtilis

The activity of dihydrodipicolinate synthase increased late in sporulation in Bacillus subtilis. Mutants blocked at several stages of sporulation due to having an altered ribonucleic acid polymerase failed to exhibit this increase.     

Genes involved in formation of structured multicellular communities by Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.     

Regulation of polyglutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular gamma-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.     

Regulation of Polyglutamic Acid Synthesis by Glutamate in Bacillus licheniformis and Bacillus subtilis

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular γ-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.     

Regulation of Bacillus subtilis macrofiber twist development by D-alanine.

Twist states of Bacillus subtilis macrofibers were found to vary as a function of the concentration of D-alanine in the medium during growth. L-Alanine in the same concentration range had no effect. Increasing concentrations of D-alanine resulted in structures progressively more right-handed (or less left-handed). All strains examined in this study, including mutants fixed in the left-hand domain as a function of temperature, responded to D-alanine in the same way. All twist states from tight left- to tight right-handedness could be achieved solely by varying the D-alanine concentration. The D-alanine-requiring macrofiber strain 2C8, which carries a genetic defect (dal-1) in the alanine racemase, behaved in a similar fashion. The combined effects of D-alanine and ammonium sulfate (a factor known to influence macrofiber twist development in the leftward direction) were examined by using both strains able to undergo temperature-induced helix hand inversion and others incapable of doing so. In all cases, the effects of D-alanine predominated. A synergism was found in which increasing the concentration of ammonium sulfate in the presence of D-alanine enhanced the right-factor activity of the latter. A D-alanine pulse protocol provided evidence that structures undergo a transient inversion indicative of "memory." Chloramphenicol treatment inhibited the establishment of memory in the D-alanine-induced right to left inversion, supporting the existence of a "left twist protein(s)" that is required for the attainment of left-handed twist states. Chemical analysis of cell walls obtained from right- and left-handed macrofibers produced in the presence and absence of D-alanine, respectively, failed to reveal twist state-specific differences in the overall composition of either peptidoglycan or wall teichoic acids.     

Regulation of Bacillus subtilis macrofiber twist development by D-cycloserine.

The effect of D-cycloserine on the establishment of twist states in Bacillus subtilis macrofibers was examined. Macrofibers produced in the presence of the drug differed in twist compared with those produced in its absence. The degree of twist alteration was dependent on the concentration of D-cycloserine in the growth medium. Macrofibers of different twist states representative of the entire twist spectrum from tight left-handedness to tight right-handedness were produced in strains FJ7 and C6D in four different ways: by control of the concentration of D-alanine, magnesium sulfate, or ammonium sulfate in the growth medium or by control of the growth temperature. The structures so produced were used to determine the effect of D-cycloserine on twist establishment starting from different twist states throughout the twist spectrum. In all but one case, twist resulting from growth in the presence of D-cycloserine was further towards the left-hand end of the twist spectrum than that produced in its absence, the exception being the unusual left-handed twist states produced in strains C6D and the closely related RHX 11S at high D-alanine concentrations described here. Studies of the interaction between D-cycloserine and D-alanine both used alone and used independently with the other twist-modifying systems (temperature, magnesium sulfate, and ammonium sulfate) revealed that changes in twist resulting from D-cycloserine were always in the opposite direction from those resulting from D-alanine. This antagonism suggests that the biochemical mechanism of twist regulation involves the metabolism of peptidoglycan, particularly reactions involving D-alanine or the dipeptide D-alanyl-D-alanine. This antagonism suggests that the biochemical mechanism of twist regulation involves the metabolism of peptidoglycan, particularly reactions involving D-alanine or the dipeptide D-alanyl-D-alanine. The possibility that peptidoglycan cross-linking is involved is discussed.