The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB ؊ mutant. The DivIVA ؊ mutants, but not the DivIVB ؊ mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.Bacillus subtilis is a gram-positive rod-shaped endosporeforming soil bacterium. Like other rod-shaped bacteria, this bacterium replicates by a process in which the cell doubles its mass by doubling its length and then divides into two daughter cells. The division process is highly regulated both temporally and spatially. Temporal regulation ensures that chromosomal replication and segregation precede cytokinesis and spatial regulation results in the division septum forming at the central site of the cell, equidistant from the cell poles. The cellular architecture which defines a septation site remains one of the most significant unanswered questions left in procaryotic biology. It is known that bacterial cell division can occur at sites other than the cell midpoint. Minicell-producing divisions have been described for mutant cells of both Escherichia coli and Bacillus subtilis (1,21,27,32). Minicells are small, generally spherical, anucleate cells produced by a septation event occurring near one pole of the bacterial cell, rather than at the midpoint. Studies utilizing minicell mutants have indicated that there are three potential septation sites in a rod-shaped cell. One is the normal division site at the midpoint of the cell, and two polar sites occur near the ends of the cell. The latter sites are utilized in the minicell divisions. Pioneering work by Rothfield and coworkers has elucidated the mechanism of the regulation of septum site selection by the minB operon of E. coli (12). The minB operon encodes three genes, designated minCDE. The MinC and MinD proteins function as an inhibitor of septation which is capable of blocking division at all three potential division sites. The former is the actual inhibitor, whereas MinD is the regul...

The bacterial phosphotransferase system (PTS) catalyzes the transport and phosphorylation of its sugar substrates. The protein‐kinase‐catalyzed phosphorylation of serine 46 in the phosphocarrier protein, HPr, inhibits PTS activity, but neither the mechanism of this inhibition nor its physiological significance is known. Site‐specific HPr mutants were constructed in which serine 46 was replaced by alanine (S46A), threonine (S46T), tyrosine (S46Y) or aspartate (S46D). The purified S46D protein exhibited markedly lower Vmax and higher Km values than the wild‐type, S46T or S46A protein for the phosphoryl transfer reactions involving HPr(His approximately P). Interactions of HPr with the enzymes catalyzing phosphoryl transfer to and from HPr regulated the kinase‐catalyzed reaction. These results establish the inhibitory effect of a negative charge at position 46 on PTS‐mediated phosphoryl transfer and suggest that HPr is phosphorylated on both histidyl and seryl residues by enzymes that recognize its tertiary rather than its primary structure. In vivo studies showed that a negative charge on residue 46 of HPr strongly inhibits PTS‐mediated sugar uptake, but that competition of two PTS permeases for HPr(His approximately P) is quantitatively more important to the regulation of PTS function than serine 46 phosphorylation.

SUMMARYMuch of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacteriumBacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present onB. subtilisspores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of theBacillus cereusfamily, principallyBacillus anthracisandBacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol61:555–588, 2007,http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

Mutation of the divIVB locus in BaciUlus subtilis causes frequent misplacement of the division septum, resulting in circular minicells, short rods, and filaments of various sizes. The divIVBI mutant allele maps to a region of the chromosome also known to encode sporulation (spoOB, spoIVF, spoIIB) and cell shape (rodB) determinants. This study reports the cloning and sequence analysis of 4.4 kb of the B. subtilis chromosome encompassing the divIVB locus. This region contains five open reading frames (ORFs) arranged in two functionally distinct gene clusters (mre and min) and transcribed colinearly with the direction of replication.Although sequence analysis reveals potential promoters preceding each gene cluster, studies with integrational plasmids suggest that all five ORFs are part of a single transcription unit. The first gene cluster contains three ORFs (mreBCD) homologous to the mre genes of Escherichia coli. We show that rodBI is allelic to mreD and identify the rodB) mutation. The second gene cluster contains two ORFs (minCD) homologous to minC and minD ofE. coli but lacks a minE homolog. We show that divIVBI is allelic to minD and identify two mutations in the divIVlBI allele. Insertional inactivation of either minC or minD or the presence of the divIVB region on plasmids produces a severe minicell phenotype in wild-type cells. Moreover, E. coli cells carrying the divIVB region on a low-copy-number plasmid produce minicells, suggesting that a product of this locus may retain some function across species boundaries.

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