PubMed Health⌕ Search

Biomedical subjects

P J Piggot

Publications and source records attributed to P J Piggot.

At least 19 recordsLinked to original sources
PubMed1998

Control of cell shape and elongation by the rodA gene in Bacillus subtilis.

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli, the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA, encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG-inducible Pspac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.

Artificial Gene Fusion↗
PubMed1998

Suppression of TGA mutations in the Bacillus subtilis spoIIR gene by prfB mutations.

An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2. These are the first mutations shown to map to the B. subtilis prfB locus. The sequence of the prfB gene was completed, and two revisions of the published sequence were made. The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86(+), compared to the readthrough level of 6% in the prfB+ strain. N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.

Amino Acid Sequence↗
PubMed1998

The spoIIE locus is involved in the Spo0A-dependent switch in the location of FtsZ rings in Bacillus subtilis.

A switch in the location of FtsZ ring structures from medial to polar is one of the earliest morphological indicators of sporulation in Bacillus subtilis. This switch can be artificially caused during vegetative growth by induction of an active form, Sad67, of the transcription regulator, Spo0A (P. A. Levin and R. Losick, Genes Dev. 10:478-488, 1996). We have used immunofluorescence microscopy to show that the switch in FtsZ ring location during vegetative growth caused by Sad67 induction is blocked by a spoIIE deletion mutation. The spoIIE mutation also impaired polar FtsZ ring formation during sporulation. These results suggest that SpoIIE mediates the Spo0A-directed formation of polar FtsZ rings.

Bacillus subtilis↗
PubMed1997

Neomycin- and spectinomycin-resistance replacement vectors for Bacillus subtilis.

A plasmid is described for Bacillus subtilis that facilitates replacement of the widely used neomycin resistance gene (neo) with a spectinomycin resistance (spcE) gene. A second plasmid is described that facilitates replacement of spcS, associated with mini-Tn10 mutagenesis in B. subtilis, with neo. These plasmids can also function as integrative vectors for B. subtilis. They expand the scope of strain construction and gene analysis in B. subtilis.

Anti-Bacterial Agents↗
PubMed1997

The division during bacterial sporulation is symmetrically located in Sporosarcina ureae.

Immunofluorescence microscopy was used to visualize the FtsZ band that marks the site of septation in Sporosarcina ureae. Image analysis indicated that the vegetative division was symmetrically located with respect to the ends of the cells. Fusions of lacZ to the sporulation loci, spollA and cotE, of Bacillus subtilis were introduced into S. ureae by mobilization of plasmids containing the fusions from Escherichia coli. The fusions showed similar patterns of sporulation-associated expression in S. ureae to those observed in B. subtilis. Formation of beta-galactosidase encoded by the spollA-lacZ fusion made it possible to identify early sporulating cells by immunofluorescence microscopy. Analysis of the position of FtsZ bands in cells expressing spollA-lacZ indicated that the location of sporulation division was symmetrical with respect to the ends of the cells, in sharp contrast to the asymmetrical location of septation in sporulating Bacilli. It is inferred that asymmetry of location of the sporulation division is not essential for the compartmentalization of gene expression that follows the division.

Bacillus subtilis↗
PubMed1996

Spore development in Bacillus subtilis.

Cell-cell and starvation signals are funneled through the phosphorelay to initiate sporulation by activating the transcription regulator SpoOA. Activation of SpoOA leads to synthesis of the transcription factors sigmaF and sigmaE. Substantial advances have been made in our understanding of the signal circuitry of the phosphorelay and of the cell-type-specific activation of the sigma factors.

Bacillus subtilis↗
PubMed1996

Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis.

A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric division. The development programs in these two cells involve the compartmentalized activities of sigma E in the larger mother cell and of sigma F in the smaller prespore. Activation of sigma E requires expression of the sigma F-directed gene spoIIR. By immunofluorescence microscopy of a strain containing a spoIIR-lacZ fusion, we have shown that spoIIR is transcribed exclusively in the prespore. By placing spoIIR under the control of PspoIIE, it was possible to express spoIIR before the spore septum was formed. Strains containing the PspoIIE-spoIIR construct activated sigma E only in the mother cell in organisms that underwent the asymmetric sporulation division. Thus, compartmentalization of sigma E activity did not require the compartmentalization of spoIIR expression. Nor did the compartmentalization of sigma E require SpoIIAA, SpoIIAB, sigma F, or sigma F-dependent transcription, all of which are required for prespore-specific gene expression. It is inferred that although sigma F and sigma E direct compartmentalized gene expression, neither of these sigma factors, nor the genes under their control, directs the process of compartmentalization.

Bacillus subtilis↗
PubMed1995

Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation.

A series of gusA transcriptional fusion vectors is described for Bacillus subtilis (Bs). The series includes a vector for use with the amyE system of Shimotsu and Henner [Gene 43 (1986) 85-94], an integrative vector and vectors that provide gusA or gusA neo cassettes. The gusA fusions are compatible with lacZ fusion vectors that are widely used with Bs, and gusA and lacZ fusions are expressed at similar levels. beta-Glucuronidase (beta Glu) and beta-galactosidase (beta Gal) do not exhibit any cross-reactivity, there is very little endogenous beta Glu activity in Bs, and there is no indication of mutation to high-level expression. We have use strains containing both gusA and lacZ fusions to compare the times of expression of different genes during sporulation.

Bacillus subtilis↗
PubMed1995

Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis.

Sporulation of Bacillus subtilis requires the coordinated expression of two separate developmental programs in the mother cell and forespore compartments by sigma E and sigma F, respectively. This coordination is maintained through the action of cross-regulatory factors that control the activities of the various sporulation-specific sigma factors. We present here the isolation and characterization of one such cross-regulatory factor, the spoIIR gene. Using a genetic screen, we have isolated four mutant alleles of spoIIR. These mutants were isolated as expressing sigma F-directed genes but not sigma E-directed genes. The block in sigma E-directed gene expression in spoIIR mutants was caused by an inability to process pro-sigma E to its active form. Cloning and characterization of the spoIIR gene determined that its transcription is directed by sigma F. Thus, SpoIIR is required for linking the activation of sigma E to the activation of sigma F and coordinating the initiation of the two developmental programs required to form a spore.

Amino Acid Sequence↗
PubMed1995

The cytochrome bc complex (menaquinone:cytochrome c reductase) in Bacillus subtilis has a nontraditional subunit organization.

We have identified an operon in Bacillus subtilis, designated qcr, that is thought to encode a quinone: cytochrome c reductase. Northern (RNA blot) analysis suggests a tricistronic operon. The operon is located at about 200 degrees on the B. subtilis map. Disruption of the operon leads to loss of a 22-kDa cytochrome c from membrane preparations. The structure of the putative protein products of the qcr operon suggests a protein complex that is closely related to but distinct from known cytochrome bc1 and b6f complexes, which catalyze electron transfer from a quinol to a c-type cytochrome or to plastocyanin. QcrA is similar to Rieske-type iron-sulfur proteins; QcrB is similar in size and sequence to b-type cytochromes from b6f complexes; and QcrC has a novel structure that resembles a fusion of a subunit IV (found in b6f complexes) to a cytochrome c. Transcription of the operon is induced at the end of exponential growth from a sigma A-like promoter. This transition state induction appears to be dependent on the downregulation of abrB expression, which is mediated by Spo0A activation. As bacteria move from the transition state into sporulation, transcription of the operon is reduced in a sigma F-dependent manner.

Amino Acid Sequence↗
PubMed1994

A pho regulon promoter induced under sporulation conditions.

Sporulation-induced alkaline phosphatases (APases) of Bacillus subtilis require the products of the sporulation stage-0 genes and certain stage-II genes, including the spoIIA operon, for induction. Mutations in either sapA or sapB bypass this requirement [Piggot and Taylor, J. Gen. Microbiol. 10 (1977) 69-80], resulting in APase production in a spoIIA sapA or spoIIA sapB strain, under sporulation conditions. B. subtilis has multiple structural genes encoding APases, which are induced either by phosphate starvation or during sporulation, or under both conditions. We report studies designed to determine which APase(s) were being expressed in the sap mutants, and from which promoters. phoB (formerly phoAIII), one of the structural genes encoding an APase in B. subtilis, is expressed under both sporulation and phosphate starvation conditions, but from separate promoters [Chesnut et al., Mol. Microbiol. 5 (1991) 2181-2190]. The spoIIA sapA and spoIIA sapB strains express phoB under sporulation conditions. Interestingly, the expression of phoB during sporulation was from Pv, the phosphate starvation-inducible promoter of phoB, rather than from Ps, the sporulation-specific promoter. Since the induction of phosphate starvation-inducible promoters during phosphate limitation requires the phoPR operon [Miki et al., Genetics 52 (1965) 1093-1100], we asked if the phoPR products were involved in regulating Pv expression under sporulation conditions. The phoPR genes are transcribed under sporulation conditions, regulated by sapA and sapB under sporulation conditions, and required for expression from Pv under sporulation conditions.

Alkaline Phosphatase↗
PubMed1994

Identification of a control region for expression of the forespore-specific Bacillus subtilis locus spoVA.

The role of a 20 bp conserved region located 45-64 nucleotides 5' of the spoVA transcription start point in Bacillus subtilis and Bacillus licheniformis was investigated by deletion analysis and by mobility shift assay. Deletions 5' of this conserved sequence had little effect on expression of a spoVA-lacZ fusion, whereas deletions extending into the sequence reduced expression of the spoVA-lacZ fusion by 85%. The timing of expression of spoVA was not affected by deletion of the sequence. The region was shown by mobility shift assays to bind specifically to a protein. Binding activity was detected in protein extracts prepared from bacteria 1 h or more after they had started to sporulate, but not in extracts prepared from vegetative bacteria. Mutations in all known spoO loci were screened but none prevented appearance of the binding activity; nor did mutations in any of the stage II and III loci tested. It is concluded that the 20 bp conserved region is the binding site of an activator that is subject to temporal regulation independent of known spo loci.

Bacillus subtilis↗
PubMed1994

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.

Adhesins, Bacterial↗
PubMed1994

The dacF-spoIIA operon of Bacillus subtilis, encoding sigma F, is autoregulated.

The spoIIA operon of Bacillus subtilis encodes sigma F and two proteins that may regulate sigma factor activity. High level induction of the tricistronic spoIIA operon occurs early during spore formation. At later times, the locus is cotranscribed with the upstream gene dacF, which encodes a putative DD-carboxypeptidase. In this study, the regulation of dacF-spoIIA transcription has been analyzed. Expression of a dacF-lacZ transcriptional fusion during sporulation required sigma F but not the later-expressed sporulation-associated sigma factors. Induction of sigma F synthesis during vegetative growth caused expression of dacF-lacZ fusions. The dacF-spoIIA promoter sequence is similar to sequences of previously identified sigma F promoters. It is concluded that dacF-spoIIA is transcribed by E sigma F. We present evidence that dacF-spoIIA is also transcribed by E sigma G, as is the case for the three other promoters known to be transcribed by E sigma F.

Bacillus subtilis↗
PubMed1993

Chromosome organization of Streptococcus mutans GS-5.

Twenty-eight genetic loci have been physically mapped to specific large restriction fragments of the Streptococcus mutans GS-5 chromosome by hybridization with probes of cloned genes or, for transposon-generated amino acid auxotrophs, with probes for Tn916. In addition, restriction fragments generated by one low-frequency-cleavage enzyme were used as probes to identify overlapping fragments generated by other restriction enzymes. The approach allowed construction of a low resolution physical map of the S. mutans GS-5 genome using restriction enzymes ApaI (5'-GGGCC/C), SmaI (5'-CCC/GGG), and NotI (5'-GC/GGCCGC).

Blotting, Southern↗
PubMed1993

Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase.

When chloramphenicol was added to a culture of Bacillus subtilis in early exponential growth, microscopic observation of cells stained by 4',6-diamidino-2-phenylindole showed nucleoids that had changed in appearance from irregular spheres and dumbbells to large, brightly stained spheres and ovals. In contrast, the addition of chloramphenicol to cultures in mid- and late exponential growth showed cells with elongated nucleoids whose frequency and length increased as the culture approached stationary phase. The kinetics of nucleoid elongation after the addition of chloramphenicol to exponential-phase cultures was complex. Immediately after treatment, the rate of nucleoid elongation was very rapid. The nucleoid then elongated steadily for about 4 min, after which the rate of elongation decreased considerably. Nucleoids of cells treated with 6-(p-hydroxyphenylazo)-uracil (an inhibitor of DNA synthesis) exhibited the immediate rapid elongation upon chloramphenicol treatment but not the subsequent changes. These observations suggest that axial filament formation during stationary phase (stage I of sporulation) in the absence of chloramphenicol results from changes in nucleoid structure that are initiated earlier, during exponential growth.

Bacillus subtilis↗
PubMed1992

Septal membrane fusion--a pivotal event in bacterial spore formation?

Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro-sigma E to sigma E in the cytoplasm by exposing it to a membrane-bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why sigma E is associated with transcription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, sigma F, to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.

Bacillus subtilis↗