Biomedical subjects

P J Piggot

Publications and source records attributed to P J Piggot.

At least 19 recordsLinked to original sources

PubMed2001

Development of a two-part transcription probe to determine the completeness of temporal and spatial compartmentalization of gene expression during bacterial development.

We have developed a two-part test, using the Bacillus subtilis sacB/SacY transcription antitermination system, to evaluate the completeness of temporal and spatial compartmentalization of gene expression during bacterial cell development. Transcription of sacY(1-55) (encoding a constitutively active form of the antiterminator, SacY) is directed by one promoter, whereas transcription of sacB'-'lacZ (the target of SacY action) is directed by the same or another promoter. To obtain beta-galactosidase activity, SacY(1-55) needs to be present when sacB'-'lacZ is being transcribed. We tested the system by analyzing the spatial compartmentalization of the activities of RNA polymerase final sigma factors, which are tightly regulated during sporulation of B. subtilis: final sigma(F) and then final sigma(G) in the prespore, final sigma(E) and then final sigma(K) in the mother cell. We have confirmed that the activities of final sigma(F) and final sigma(E) are spatially compartmentalized. We have demonstrated that there is also sharp temporal compartmentalization, with little or no overlap in the activities of final sigma(F) and final sigma(G) or of final sigma(E) and final sigma(K). In contrast, we found no compartmentalization of the activity of the main vegetative factor, final sigma(A), which continued to be active alongside all of the sporulation-specific final sigma factors. We also found no temporal compartmentalization of expression of loci that are activated during the development of competent cells of B. subtilis, a developmental program distinct from spore formation. A possible mechanism to explain the temporal compartmentalization of final sigma(F) and final sigma(G) activities is that the anti-sigma factor SpoIIAB transfers from final sigma(G) to final sigma(F).

Bacillus subtilis↗

PubMed2001

Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides.

Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma factors. Of these, sigma(F) is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma(F) (E-sigma(F)), and only two genes of known function are exclusively under the control of E-sigma(F), spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma(F), we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma(F) in vivo. The results indicate a weak sigma(F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma factors, as well as to sigma(A). There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma factors. The looseness of the sigma(F) promoter requirement contrasts with the strict requirement for sigma(A)-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma(F) in vivo.

Amino Acid Sequence↗

PubMed2001

Identification and characterization of the dif Site from Bacillus subtilis.

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6 degrees counterclockwise from the B. subtilis terminus of replication (172 degrees ), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPbeta-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains.

Bacillus subtilis↗

PubMed2001

Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant.

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.

Bacillus subtilis↗

PubMed2000

A tale of two genomes: resolution of dimeric chromosomes in Escherichia coli and Bacillus subtilis.

Dimeric chromosomes can be formed during replication of circular bacterial chromosomes by an odd number of homologous recombination events between sister chromosomes. In the absence of a compensating recombination reaction such dimers cannot be segregated from each other as the cell divides. This review highlights the shared and divergent mechanisms employed by Escherichia coli and Bacillus subtilis in their effort to resolve and partition dimeric chromosomes safely. In particular, we discuss the Xer-type recombinases, RecA, FtsK/SpoIIIE, and dif.

Bacillus subtilis↗

PubMed2000

The putative DNA translocase SpoIIIE is required for sporulation of the symmetrically dividing coccal species Sporosarcina ureae.

The spoIIIE gene of Sporosarcina ureae encodes a 780-residue protein, showing 58% identity to the SpoIIIE protein of Bacillus subtilis, which is thought to be a DNA translocase. Expression of the S. ureae spoIIIE gene is able to restore sporulation in a B. subtilis spoIIIE mutant. Inactivation of the S. ureae spoIIIE gene blocks sporulation of S. ureae at stage III. Within the limits of detection, the sporulation division in S. ureae shows the same symmetry, or near symmetry, as the vegetative division (in contrast to the highly asymmetric location of the sporulation division for B. subtilis), and so it is inferred that SpoIIIE facilitates chromosome partitioning during sporulation, even when the division is not grossly asymmetric. It is suggested that chromosome partitioning lags behind division during sporulation but not during vegetative growth.

Bacillus subtilis↗

PubMed2000

The chromosomal location of the Bacillus subtilis sporulation gene spoIIR is important for its function.

Formation of the asymmetrically located septum during sporulation of Bacillus subtilis results in enclosure of the origin-proximal 30% of the chromosome in the prespore compartment. The rest of the chromosome is then translocated into the prespore from the mother cell. Transcription of spoIIR is initiated in the prespore by RNA polymerase containing sigma(F) soon after the septum is formed. The SpoIIR protein is required for the activation of the transcription program directed by sigma(E) in the mother cell. The spoIIR locus is located at 324 degrees, near the origin of replication (0/360 degrees ). We show here that movement of spoIIR to 28 degrees had little effect on sporulation. However, movement to regions not in the origin-proximal part of the chromosome substantially reduced sporulation efficiency. At 283 degrees sporulation was reduced to less than 20% of the level obtained when spoIIR was at its natural location, and movement to 190 degrees reduced sporulation to about 6% of that level. These positional effects were also seen in the transcription of a spoIIR-lacZ fusion. In contrast, movement of other spo-lacZ fusions from 28 degrees to 190 degrees had little effect on their expression. These results suggest that spoIIR is the subject of "positional regulation," in the sense that the chromosomal position of spoIIR is important for its expression and function.

Bacillus subtilis↗

PubMed1999

Physical and genetic map of Streptococcus mutans GS-5 and localization of five rRNA operons.

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

Blotting, Southern↗

PubMed1999

Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon.

In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.

5' Untranslated Regions↗

PubMed1999

The ripX locus of Bacillus subtilis encodes a site-specific recombinase involved in proper chromosome partitioning.

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.

Bacillus subtilis↗

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↗