Biomedical subjects
Publications and source records attributed to A D Grossman.
Despite the power of bacterial genetics, the prokaryotic cell cycle has remained poorly understood. But recent work with three different bacterial species has shed light on how chromosomes and plasmids are oriented and partitioned during the cell cycle, and on mechanisms regulating the initiation of DNA replication.
Many bacteria communicate by secreting and responding to extracellular peptides (pheromones). Some peptide pheromones act via receptors on the cell surface, which are often membrane-bound histidine protein kinases. Other peptide pheromones are transported into the cell by an oligopeptide permease and interact with intracellular receptors to modulate gene expression.
Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.
We examined the pattern of FtsZ localization in a Bacillus subtilis minCD mutant. When grown in minimal medium, the majority (approximately 89%) of the minCD mutant cells with an FtsZ ring had a single, medially positioned FtsZ ring. These results indicate that genes in addition to minCD function to restrict the number and position of FtsZ rings. When grown in rich medium, greater than 50% of the minCD mutant cells had multiple FtsZ rings, indicating significant differences in regulation of FtsZ ring formation based on growth medium.
The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport. Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon.
Competence development and sporulation in B. subtilis are partly controlled by peptides that accumulate in culture medium as cells grow to high density. We constructed two genes that encode mature forms of two different signaling molecules, the PhrA peptide that stimulates sporulation, and CSF, the competence- and sporulation-stimulating factor. Both pentapeptides are normally produced by secretion and processing of precursor molecules. The mature pentapeptides were functional when expressed inside the cell, indicating that they normally need to be imported to function. Furthermore, at physiological concentrations (10 nM), CSF was transported into the cell by the oligopeptide permease encoded by spo0K (opp). CSF was shown to have at least three different targets corresponding to its three activities: stimulating competence gene expression at low concentrations, and inhibiting competence gene expression and stimulating sporulation at high concentrations.
We have determined the subcellular localization of the chromosome partition protein Spo0J of Bacillus subtilis by immunofluorescence microscopy and visualizing fluorescence of a Spo0J-GFP fusion protein. Spo0J was associated with a region of the nucleoid proximal to the cell pole, both in growing cells dividing symmetrically and in sporulating cells dividing asymmetrically. Additional experiments indicated that Spo0J was bound to sites in the origin-proximal third of the chromosome. These results show that the replicating chromosomes are oriented in a specific manner during the division cycle, with the Spo0J binding region positioned toward the cell poles. Experiments characterizing cells at different stages of the cell cycle showed that chromosome orientation is established prior to the initiation of cell division. Our results indicate that there is a mechanism for orienting the chromosomes and that the chromosome partition protein Spo0J might be part of a bacterial mitotic-like apparatus.
To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.
Clp ATPase chaperone proteins are found in procaryotes and eucaryotes. Recently, ClpC of Bacillus subtilis was found to be part of a regulatory switch(1). ClpC, in combination with the MecA and ComS proteins, regulates the activity of a transcription factor, ComK, which is necessary for the development of genetic competence (the ability to bind and take up exogenous DNA). The complex of ClpC:MecA:ComK renders ComK inactive. Interaction between ComS and the ternary complex releases active ComK. This regulatory switch controls ComK activity in response to cell density signals that affect production of ComS. Regulated interaction between Clp ATPase and target proteins might prove to be widespread.
A Bacillus subtilis mutant with a deletion of citC, the gene encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, had a greatly reduced ability to sporulate. Analysis of expression of lacZ fusions to various sporulation gene promoters revealed that in the citC mutant development is probably blocked between stage 0 and stage II. That is, genes expressed very early in sporulation, under the direct control of the Spo0A transcription factor, were induced normally in the citC mutant. However, genes expressed after asymmetric septation (stage II) in wild-type cells were not induced in the citC mutant. Analysis of cell morphology by thin-section electron microscopy and immunofluorescence microscopy showed that the mutant formed axial chromosomal filaments and accumulated rings of FtsZ protein at potential polar division sites but failed to form asymmetric division septa, indicating that sporulation is blocked at stage I. The growth and sporulation defects of the B. subtilis citC mutant were fully overcome by introduction and expression of the Escherichia coli icd gene, encoding an isocitrate dehydrogenase similar to the enzyme from B. subtilis.
We have purified and characterized an extracellular peptide factor that serves as a cell density signal for both competence development and sporulation in Bacillus subtilis. This competence and sporulation stimulating factor (CSF) was purified from conditioned medium (culture supernatant) based on its ability to stimulate expression of srfA (comS) in cells at low cell density. CSF is a 5-amino-acid peptide, glu-arg-gly-met-thr (ERGMT), that is, the carboxy-terminal 5 amino acids of the 40-amino-acid peptide encoded by phrC. No detectable CSF was produced in a phrC null mutant. The activity of chemically synthesized CSF (ERGMT) was virtually indistinguishable from that of CSF that was purified from culture supernatants. At relatively low concentrations (1-10 nM), CSF stimulated expression of srfA, whereas high concentrations of CSF stimulated the ability of cells at low cell density to sporulate. Stimulation of srfA expression by CSF requires the oligopeptide permease encoded by spo0K, a member of the ATP-binding-cassette family of transporters, and the putative phosphatase encoded by rapC, the gene immediately upstream of phrC. RapC was found to be a negative regulator of srfA expression, suggesting that the target of RapC is the transcription factor encoded by comA. We propose that CSF is transported into the cell by the Spo0K oligopeptide permease and stimulates competence gene expression by inhibiting (either directly or indirectly) the RapC phosphatase.
Natural genetic competence, the ability of cells to bind to and to take up exogenous DNA, is widespread among bacteria and might be an important mechanism for the horizontal transfer of genes. Competent cells express specialized proteins that assemble into a DNA-uptake complex. In many organisms, the development of competence and expression of the uptake machinery is regulated in response to cell-cell signaling and/or nutritional conditions. Exciting new progress has been made in characterizing the signals and pathways that regulate the development of competence.
In Bacillus subtilis, the major sigma factor, sigma-A (rpoD), and the minor sigma factor, sigma-H (spo0H), are present during growth and are required for the initiation of sporulation. Our experiments indicate that sigma-A and sigma-H compete for binding to core RNA polymerase. We used a fusion of rpoD to the LacI-repressible IPTG-inducible promoter, Pspac, to vary the levels of sigma-A in the cell. Increasing the amount of sigma-A caused a decrease in expression of genes controlled by sigma-H, and a delay in the production of heat-resistant spores. Decreasing the amount of sigma-A, in a strain deleted for the chromosomal rpoD, caused an increase in expression of genes controlled by sigma-H. As rpoD itself is controlled by at least two promoters recognized by RNA polymerase that contains sigma-H, the effect of sigma-A levels on expression of sigma-H-controlled promoters represents a feedback mechanism that might contribute to maintaining appropriate levels of sigma-A. While the level of sigma-A was important for efficient sporulation, our results indicate that the normal transcriptional control of rpoD, in the context of the rpoD operon and the numerous promoters in that operon, is not required for efficient sporulation or germination, provided that the sigma-A level from a heterologous promoter is comparable to that in wild-type cells.
Expression of genes early during sporulation in Bacillus subtilis requires the activity of the transcription factor encoded by spo0A. The active, phosphorylated form of Spo0A is produced through the action of a multicomponent pathway, the phosphorelay. A mutant defective in the first three enzymes of the Krebs citric acid cycle was unable to express early sporulation genes, apparently because of a failure to activate the phosphorelay. Cells that produce an altered Spo0A protein that can be phosphorylated by an alternative pathway were not dependent on Krebs cycle function for early sporulation gene expression. These findings suggest that Krebs cycle enzymes transmit a signal to activate the phosphorelay and that B. subtilis monitors its metabolic potential before committing itself to spore formation.
Development of genetic competence in Bacillus subtilis is regulated by extracellular signaling molecules, including the ComX pheromone, a modified 9- or 10-amino-acid peptide. Here, we present characterization of a second extracellular competence stimulating factor (CSF). CSF appears to be, at least in part, a small peptide of between 520 and 720 daltons. Production of CSF requires several genes that are needed both for initiation of sporulation and development of competence (spo0H, spo0A, spo0B, and spo0F). Although both peptide factors regulate competence, two different sensing pathways mediate the response to the ComX pheromone and CSF. Analysis of double mutants indicated that ComX pheromone is on the same genetic pathway as the membrane-bound histidine protein kinase encoded by comP and that CSF is on the same genetic pathway as the oligopeptide permease encoded by spo0K. Furthermore, the cellular response to partly purified ComX pheromone requires the ComP histidine protein kinase, whereas the response to partly purified CSF requires the Spo0K oligopeptide permease. These two sensing pathways converge to activate competence genes. Both factors and their convergent sensing pathways are required for normal development of competence and might function to integrate different physiological signals.
Phosphorylation of the transcription factor encoded by spo0A is required for the initiation of sporulation in Bacillus subtilis. Production and accumulation of Spo0A-P is controlled by histidine protein kinases and the spo0 gene products. To identify additional genes that might be involved in the initiation of sporulation and production of Spo0A-P, we isolated genes which when present on a multicopy plasmid could suppress the sporulation defect of a spo0K mutant. kinC was one gene isolated in this way. A multicopy plasmid containing kinC completely or partially suppressed the sporulation defect caused by mutations in spo0K, kinA, spo0F, and spo0B, indicating that at least when overexpressed, KinC is capable of stimulating phosphorylation of Spo0A independently of the normal phosphorylation pathway. The predicted product of kinC is 428 amino acids long and is most similar to KinA and KinB, the histidine protein kinases involved in the initiation of sporulation. In otherwise wild-type strains, kinC null mutations caused little or no defect in sporulation under the conditions tested. However, in the absence of a functional phosphorelay (spo0F or spo0B), KinC appears to be the kinase responsible for phosphorylation of the sof-1 and rvtA11 forms of Spo0A.
We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter Pspac. Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0A approximately P. These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.
spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.