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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SummaryThe exosporium is the outermost layer of the Bacillus anthracis spore. The predominant protein on the exosporium surface is BclA, a collagen-like glycoprotein. BclA is incorporated on the spore surface late in the B. anthracis sporulation pathway. A second collagen-like protein, BclB, has been shown to be surface-exposed on B. anthracis spores. We have identified sequences near the N-terminus of the BclA and BclB glycoproteins responsible for the incorporation of these proteins into the exosporium layer of the spore and used these targeting domains to incorporate reporter fluorescent proteins onto the spore surface. The BclA and BclB proteins are expressed in the mother cell cytoplasm and become spore-associated in a two-step process involving first association of the protein with the spore surface followed by attachment of the protein in a process that involves a proteolytic cleavage event. Protein domains associated with each of these events have been identified. This novel targeting system can be exploited to incorporate foreign proteins into the exosporium of inactivated, spores resulting in the surface display of recombinant immunogens for use as a potential vaccine delivery system.

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [15N]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [15N]glycine incorporation ranged from 26 to 45 % of 15NH4+ incorporation and was 2- to 3-fold greater than 15NO3- incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [15N]glycine and 15NO3- in similar amounts, 18–26 % of 15NH4+ incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of 15N from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of 15N- label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of 15N from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.

S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (σS), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that σS is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that σS is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a σS mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that σS is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network.