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1.
Ulster Med J ; 88(3): 150-156, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31619848

ABSTRACT

Osteoporosis is a significant global health and economic burden associated with bone fracture, morbidity and mortality. Denosumab, a novel human monoclonal antibody second-line treatment, inhibits osteoclast-mediated bone resorption and increases bone mineral density (BMD). Treatment achieves reductions in vertebral, non-vertebral and hip fracture risk. We undertook a service evaluation to review clinical outcomes of patients treated with denosumab in an osteoporosis department that provides regional services. We identified 529 patients (95% female; mean age 72.8 years; 35-98 years), who had at least one dose of denosumab administered for the treatment of osteoporosis. The mean number of denosumab doses administered was 4.9 (range: 1 to 12). 330/529 patients had completed a baseline and post-treatment bone densitometry scan (DXA). The mean observed BMD change at around 18 months at the lumbar spine was +8.4% and at the hip was +3.5%. While the majority have transitioned to shared care administration of treatment within primary care (53%), 20% continue to attend hospital clinics to receive treatment. During follow-up, there were 66 deaths (12%). 15% switched to an alternative treatment or were discharged. This retrospective cohort study demonstrates the clinical effectiveness of denosumab in improving bone mineral density in a real life setting in an ageing, co-morbid population. There has been recent progress with adoption of shared care administration in primary care. As part of a quality improvement programme we have recently developed a dedicated denosumab database and day-case treatment clinic for those receiving treatment in secondary care.


Subject(s)
Bone Density Conservation Agents/administration & dosage , Denosumab/therapeutic use , Fractures, Spontaneous/prevention & control , Osteoporosis/diagnostic imaging , Osteoporosis/drug therapy , Absorptiometry, Photon/methods , Age Factors , Aged , Aged, 80 and over , Bone Density/drug effects , Cohort Studies , Databases, Factual , Female , Humans , Male , Middle Aged , Northern Ireland , Osteoporosis/diagnosis , Prognosis , Retrospective Studies , Risk Assessment , Severity of Illness Index , Sex Factors , Statistics, Nonparametric , Treatment Outcome
2.
Mol Microbiol ; 42(2): 495-502, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11703670

ABSTRACT

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) causes diseases ranging from mild, self-limiting pharyngitis to severe invasive infections. Regulation of the expression of GAS genes in response to specific environmental differences within the host is probably key in determining the course of the infectious process, however, little is known of global regulators of gene expression in GAS. Although secondary RNA polymerase sigma factors act as global regulators of gene expression in many other bacteria, none has yet been isolated from the GAS. The newly available GAS genome sequence indicates that the only candidate secondary sigma factor is encoded by two identical open reading frames (ORFS). These ORFS encode a protein that is 40% identical to the transcription factor ComX, believed to act as an RNA polymerase sigma factor in Streptococcus pneumoniae. To test whether the GAS ComX homologue functions as a sigma factor, we cloned and purified it from Escherichia coli. We found that in vitro, this GAS protein, which we call sigmaX, directed core RNA polymerase from Bacillus subtilis to transcribe from two GAS promoters that contain the cin-box region, required for transcription by S. pneumoniae ComX in vivo. On the other hand, GAS sigmaX did not promote transcription of a GAS promoter (hasA) expected to be dependent on sigmaA, the housekeeping or primary RNA polymerase sigma factor. Addition of monoclonal antibody that inhibited sigmaA-directed transcription had no effect on sigmaX-directed transcription, showing that the latter was not the result of contaminating sigmaA. Transcription of both cin-box-containing promoters initiated downstream of the cin-box and two different single basepair substitutions in the cin-box of the cinA promoter each caused a severe reduction of sigmaX-directed transcription in vitro. Thus, the cin-box is required for sigmaX-directed transcription.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial , Sigma Factor/metabolism , Streptococcus pyogenes/enzymology , Streptococcus pyogenes/genetics , Bacillus subtilis/enzymology , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/isolation & purification , Genes, Bacterial/genetics , Genes, Regulator/genetics , Mutation/genetics , Open Reading Frames/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Response Elements/genetics , Sigma Factor/genetics , Sigma Factor/isolation & purification , Transcription, Genetic
3.
J Bacteriol ; 183(14): 4183-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11418558

ABSTRACT

Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins. Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins. GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family. Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif. Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding. Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA. We examined the effects of single base pair substitutions in the high-affinity GerE binding site on the sigK promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on the sigK promoter are important for a high-affinity interaction with GerE. We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites. We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites. These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Helix-Turn-Helix Motifs , Promoter Regions, Genetic , Sigma Factor , Transcription Factors/genetics , Amino Acid Sequence , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Binding Sites , DNA-Binding Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid
4.
J Bacteriol ; 183(10): 2995-3003, 2001 May.
Article in English | MEDLINE | ID: mdl-11325926

ABSTRACT

The Bacillus subtilis genome encodes two members of the Lon family of prokaryotic ATP-dependent proteases. One, LonA, is produced in response to temperature, osmotic, and oxidative stress and has also been implicated in preventing sigma(G) activity under nonsporulation conditions. The second is encoded by the lonB gene, which resides immediately upstream from lonA. Here we report that transcription of lonB occurs during sporulation under sigma(F) control and thus is restricted to the prespore compartment of sporulating cells. First, expression of a lonB-lacZ transcriptional fusion was abolished in strains unable to produce sigma(F) but remained unaffected upon disruption of the genes encoding the early and late mother cell regulators sigma(E) and sigma(K) or the late forespore regulator sigma(G). Second, the fluorescence of strains harboring a lonB-gfp fusion was confined to the prespore compartment and depended on sigma(F) production. Last, primer extension analysis of the lonB transcript revealed -10 and -35 sequences resembling the consensus sequence recognized by sigma(F)-containing RNA polymerase. We further show that the lonB message accumulated as a single monocistronic transcript during sporulation, synthesis of which required sigma(F) activity. Disruption of the lonB gene did not confer any discernible sporulation phenotype to otherwise wild-type cells, nor did expression of lonB from a multicopy plasmid. In contrast, expression of a fusion of the lonB promoter to the lonA gene severely reduced expression of the sigma(G)-dependent sspE gene and the frequency of sporulation. In confirmation of earlier observations, we found elevated levels of sigma(F)-dependent activity in a spoIIIE47 mutant, in which the lonB region of the chromosome is not translocated into the prespore. Expression of either lonB or the P(lonB)-lonA fusion from a plasmid in the spoIIIE47 mutant reduced sigma(F) -dependent activity to wild-type levels. The results suggest that both LonA and LonB can prevent abnormally high sigma(F) activity but that only LonA can negatively regulate sigma(G).


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Sigma Factor/genetics , Transcription, Genetic , ATP-Dependent Proteases , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Base Sequence , Blotting, Northern , Gene Expression Regulation, Bacterial , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/metabolism , Sigma Factor/metabolism , Spores, Bacterial/physiology
5.
J Bacteriol ; 183(10): 3041-9, 2001 May.
Article in English | MEDLINE | ID: mdl-11325931

ABSTRACT

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.


Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Peptide Synthases/metabolism , Spores, Bacterial/metabolism , Bacillus subtilis/genetics , Escherichia coli/metabolism , Microscopy, Fluorescence , Mutation , Saccharomyces cerevisiae/metabolism , Two-Hybrid System Techniques
6.
J Bacteriol ; 183(6): 2032-40, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11222602

ABSTRACT

During endospore formation in Bacillus subtilis, over two dozen polypeptides are localized to the developing spore and coordinately assembled into a thick multilayered structure called the spore coat. Assembly of the coat is initiated by the expression of morphogenetic proteins SpoIVA, CotE, and SpoVID. These morphogenetic proteins appear to guide the assembly of other proteins into the spore coat. For example, SpoVID forms a complex with the SafA protein, which is incorporated into the coat during the early stages of development. At least two forms of SafA are found in the mature spore coat: a full-length form and a shorter form (SafA-C(30)) that begins with a methionine encoded by codon 164 of safA. In this study, we present evidence that the expression of SafA-C(30) arises from translation initiation at codon 164. We found only a single transcript driving expression of SafA. A stop codon engineered just upstream of a predicted ribosome-binding site near codon M164 abolished formation of full-length SafA, but not SafA-C(30). The same effect was observed with an alanine substitution at codon 1 of SafA. Accumulation of SafA-C(30) was blocked by substitution of an alanine codon at codon 164, but not by a substitution at a nearby methionine at codon 161. We found that overproduction of SafA-C(30) interfered with the activation of late mother cell-specific transcription and caused a strong sporulation block.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Protein Biosynthesis , Sigma Factor , Transcription Factors , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Codon/genetics , Mutation , Promoter Regions, Genetic/genetics , Spores, Bacterial/genetics , Spores, Bacterial/metabolism , Transcription, Genetic
7.
J Bacteriol ; 182(7): 1828-33, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10714986

ABSTRACT

During endospore formation in Bacillus subtilis, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination. In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore. A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat. Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly. This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as yrbA. We confirmed the results of a previous study that showed safA mutant spores have defective coats which are missing several proteins. We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation. Therefore, SafA may associate with SpoVID during the early stage of coat assembly. We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores. SafA appears to have a modular design. The C-terminal region of SafA is similar to those of several inner spore coat proteins. The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall. This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Sigma Factor , Transcription Factors , Amino Acid Motifs , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Consensus Sequence/genetics , Conserved Sequence/genetics , Microscopy, Immunoelectron , Mutation/genetics , Peptide Library , Phenotype , Precipitin Tests , Protein Binding , Spores, Bacterial/genetics , Spores, Bacterial/growth & development , Spores, Bacterial/metabolism , Spores, Bacterial/ultrastructure , Time Factors
8.
Methods ; 20(1): 95-110, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10610808

ABSTRACT

Many biological processes are mediated through the action of multiprotein complexes, often assembled at specific cellular locations. Bacterial endospores for example, are encased in a proteinaceous coat, which confers resistance to lysozyme and harsh chemicals and influences the spore response to germinants. In Bacillus subtilis, the coat is composed of more than 20 polypeptides, organized into three main layers: an amorphous undercoat; a lamellar, lightly staining inner structure; and closely apposed to it, a striated electron-dense outer coat. Synthesis of the coat proteins is temporally and spatially governed by a cascade of four mother cell-specific transcription factors. However, the order of assembly and final destination of the coat structural components may rely mainly on specific protein-protein interactions, as well as on the action of accessory morphogenetic proteins. Proteolytic events, protein-protein crosslinking, and protein glycosylation also play a role in the assembly process. These modifications are carried out by enzymes that may themselves be targeted to the coat layers. Coat genes have been identified by reverse genetics or, more recently, by screens for mother cell-specific promoters or for peptide sequences able to interact with certain bait proteins. A role for a given locus in coat assembly is established by a combination of regulatory, functional, morphological, and topological criteria. Because of the amenability of B. subtilis to genetic analysis (now facilitated by the knowledge of its genome sequence), coat formation has become an attractive model for the assembly of complex macromolecular structures during development.


Subject(s)
Spores, Bacterial/chemistry , Bacterial Proteins/analysis , Bacterial Proteins/metabolism , Gene Expression Regulation , Glycosylation , Protein Processing, Post-Translational , Spores, Bacterial/genetics , Spores, Bacterial/physiology
9.
J Bacteriol ; 181(14): 4365-73, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10400595

ABSTRACT

During endospore formation in Bacillus subtilis, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing sigmaK. GerE binds to a site on one of these promoters, cotX, that overlaps its -35 region. We tested the model that GerE interacts with sigmaK at the cotX promoter by seeking amino acid substitutions in sigmaK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the sigmaK-dependent, GerE-independent promoter gerE. We identified two amino acid substitutions in sigmaK, E216K and H225Y, that decrease cotX promoter utilization but do not affect gerE promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in sigmaK on transcription in vitro. We found that these substitutions specifically reduced utilization of the cotX promoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent cotX promoter activity, that the histidine at position 225 of sigmaK may interact with GerE at the cotX promoter, and that this interaction may facilitate the initial binding of sigmaK RNA polymerase to the cotX promoter. We also found that the alanine substitutions at positions 216 and 225 of sigmaK had no effect on utilization of the GerE-dependent promoter cotD, which contains GerE binding sites that do not overlap with its -35 region.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Sigma Factor , Transcription Factors/genetics , Bacillus subtilis/physiology , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Mutagenesis , Plasmids/genetics , Polymerase Chain Reaction , Recombinant Fusion Proteins , Spores, Bacterial/physiology , Transcription Factors/metabolism , Transcription, Genetic
10.
J Bacteriol ; 181(12): 3632-43, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10368135

ABSTRACT

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of sigmaH. When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Sigma Factor , Transcription Factors , Bacillus subtilis/genetics , Bacillus subtilis/ultrastructure , Gene Deletion , Genotype , Kinetics , Models, Biological , Operon , Phenotype , Polymerase Chain Reaction , Protein Sorting Signals/genetics , Recombinant Proteins/metabolism , Spores, Bacterial/genetics , Spores, Bacterial/physiology , Spores, Bacterial/ultrastructure
11.
J Bacteriol ; 181(8): 2631-3, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10198031

ABSTRACT

We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Sigma Factor , Transcription Factors , Bacillus subtilis/ultrastructure , Bacterial Proteins/analysis , Cell Wall/chemistry , Spores, Bacterial/physiology , Spores, Bacterial/ultrastructure
12.
J Bacteriol ; 181(2): 426-33, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9882655

ABSTRACT

We have identified a locus essential for galacturonate utilization in Bacillus subtilis. Genes homologous to Escherichia coli and Erwinia chrysanthemi glucuronate and galacturonate metabolic genes were found in a cluster consisting of 10 open reading frames (ORFs) in the B. subtilis chromosome. A mutant of B. subtilis containing a replacement of the second and third ORFs was unable to grow with galacturonate as its primary carbon source. Galacturonate induced expression from a sigmaA-dependent promoter, exuP1, located upstream from the first ORF. The eighth ORF in this cluster (the exu locus) encodes a LacI and GalR homolog that negatively regulated expression from exuP1. A 26-bp inverted repeat sequence centered 15 bp downstream from the exuP1 start point of transcription acted in cis to negatively regulate expression from exuP1 under noninducing conditions. Expression from the exuP1 promoter was repressed by high levels of glucose, which is probably mediated by CcpA (catabolite control protein A). A sigmaE-dependent promoter, exuP2, was localized between the second and third ORFs and was active during sporulation.


Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Genes, Bacterial , Glucuronates/metabolism , Hexuronic Acids/metabolism , Aldose-Ketose Isomerases/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Bacterial , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Dickeya chrysanthemi/genetics , Escherichia coli/genetics , Genotype , Glucuronic Acid , Molecular Sequence Data , Multigene Family , Open Reading Frames , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid
13.
J Bacteriol ; 180(18): 4987-90, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9733708

ABSTRACT

Spo0A activates transcription in Bacillus subtilis from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, sigmaA, and RNA polymerase containing a secondary sigma factor, known as sigmaH. The region of sigmaA near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of sigmaA at these promoters. To determine if the amino acids in the corresponding region of sigmaH are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in sigmaH (201 to 210). Two alanine substitutions in sigmaH, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, sigmaH-dependent promoter spoIIA but did not affect expression from the sigmaH-dependent, Spo0A-independent promoters citGp2 and spoVG. Therefore, promoter activation by Spo0A requires homologous regions in sigmaA and sigmaH. A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in sigmaA did not suppress the sporulation defects caused by the Q201A and R205A substitutions in sigmaH. This result and others indicate that different surfaces of Spo0A probably interact with sigmaA and sigmaH RNA polymerases.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/physiology , Promoter Regions, Genetic , Sigma Factor/physiology , Transcription Factors/physiology , Amino Acid Sequence , Molecular Sequence Data , Sigma Factor/chemistry , Structure-Activity Relationship
14.
J Bacteriol ; 180(14): 3578-83, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9658000

ABSTRACT

Spo0A is a DNA binding protein in Bacillus subtilis required for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the sigmaA subunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in sigmaA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in sigmaA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the sigmaA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIA promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, sigmaH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in sigmaA-dependent promoter activation.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/physiology , Promoter Regions, Genetic/genetics , Sigma Factor/genetics , Transcription Factors/genetics , Transcription Factors/physiology , Alanine/metabolism , Alleles , Bacterial Proteins/chemistry , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation , Gene Expression Regulation, Bacterial , Mutation/genetics , Phenotype , Sigma Factor/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic
15.
Mol Microbiol ; 28(2): 235-47, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9622350

ABSTRACT

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.


Subject(s)
Bacillus subtilis/cytology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Escherichia coli Proteins , Genes, Bacterial/physiology , Membrane Proteins , Artificial Gene Fusion , Bacillus subtilis/growth & development , Bacillus subtilis/ultrastructure , Escherichia coli/genetics , Genes, Bacterial/genetics , Microscopy, Electron , Microscopy, Fluorescence , Molecular Sequence Data , Soil Microbiology , Time Factors
16.
J Bacteriol ; 180(9): 2285-91, 1998 May.
Article in English | MEDLINE | ID: mdl-9573176

ABSTRACT

Endospores of Bacillus subtilis are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the sod4 locus. sod4 is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from B. subtilis. Disruption of the sod4 locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The sodA mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of sodA mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the cotG gene in sporulating cells of the sodA mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a sodA mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in a cotG mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer.


Subject(s)
Bacillus subtilis/physiology , Superoxide Dismutase/metabolism , Bacillus subtilis/ultrastructure , Bacterial Proteins/genetics , Cross-Linking Reagents , Manganese/metabolism , Models, Biological , Mutation , Oxidation-Reduction , Spores, Bacterial/physiology , Spores, Bacterial/ultrastructure , Superoxide Dismutase/deficiency , Superoxide Dismutase/genetics
17.
Mol Microbiol ; 25(5): 955-66, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9364920

ABSTRACT

During Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon (cotJA and cotJC) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA. Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co-immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two-hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.


Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Sigma Factor , Spores, Bacterial/chemistry , Transcription Factors , Bacterial Proteins/analysis , Fluorescent Antibody Technique , Gene Expression/genetics , Gene Expression/physiology , Genes, Bacterial/genetics , Genes, Bacterial/physiology , Operon/genetics , Operon/physiology
18.
J Bacteriol ; 179(17): 5605-8, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9287022

ABSTRACT

Bacillus subtilis Spo0A activates transcription from both sigmaA- and sigmaH-dependent promoters. Baldus et al. (2) identified two amino acid substitutions in the carboxyl terminus of sigmaA, K356E and H359R, that specifically impaired Spo0A-activated transcription in vivo. To test the model in which the K356E and H359R substitutions in sigmaA interfere with the interaction of Spo0A and sigmaA, we examined the effects of alanine substitutions at these positions in sigmaA on sigmaA's ability to direct transcription in vivo and in vitro. We found that alanine substitutions at these positions specifically reduced expression from the sigmaA-dependent, Spo0A-dependent promoters, spoIIG and spoIIE, in vivo. Furthermore, we found that stimulation of spoIIG promoter activity by Spo0A in vitro was reduced by the single substitutions H359A and H359R in sigmaA.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Sigma Factor/genetics , Sigma Factor/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/physiology , Amino Acid Sequence , DNA-Directed RNA Polymerases/genetics , Histidine/physiology , Lysine/physiology , Molecular Sequence Data , Mutation , Promoter Regions, Genetic/genetics
19.
J Bacteriol ; 179(6): 1887-97, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9068633

ABSTRACT

We cloned and characterized a gene, cotM, that resides in the 173 degrees region of the Bacillus subtilis chromosome and is involved in spore outer coat assembly. We found that expression of the cotM gene is induced during development under sigma K control and is negatively regulated by the GerE transcription factor. Disruption of the cotM gene resulted in spores with an abnormal pattern of coat proteins. Electron microscopy revealed that the outer coat in cotM mutant spores had lost its multilayered type of organization, presenting a diffuse appearance. In particular, significant amounts of material were absent from the outer coat layers, which in some areas had a lamellar structure more typical of the inner coat. Occasionally, a pattern of closely spaced ridges protruding from its surface was observed. No deficiency associated with the inner coat or any other spore structure was found. CotM is related to the alpha-crystallin family of low-molecular-weight heat shock proteins, members of which can be substrates for transglutaminase-mediated protein cross-linking. CotM was not detected among the extractable spore coat proteins. These observations are consistent with a model according to which CotM is part of a cross-linked insoluble skeleton that surrounds the spore, serves as a matrix for the assembly of additional outer coat material, and confers structural stability to the final structure.


Subject(s)
Bacillus subtilis/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/physiology , Sigma Factor , Spores, Bacterial/ultrastructure , Amino Acid Sequence , Bacillus subtilis/physiology , Bacterial Proteins/analysis , Base Sequence , Chromosome Mapping , Cloning, Molecular , Crystallins/chemistry , Genes, Bacterial , Heat-Shock Proteins/chemistry , Microscopy, Electron , Molecular Sequence Data , Mutagenesis, Insertional , RNA, Bacterial/genetics , RNA, Messenger/genetics , Spores, Bacterial/chemistry , Transcription Factors/metabolism , Transcription, Genetic
20.
J Bacteriol ; 179(2): 389-98, 1997 Jan.
Article in English | MEDLINE | ID: mdl-8990290

ABSTRACT

We report on the characterization of three new transcription units expressed during sporulation in Bacillus subtilis. Two of the units, cse15 and cse60, were mapped at about 123 degrees and 62 degrees on the genetic map, respectively. Their transcription commenced around h 2 of sporulation and showed an absolute requirement for sigmaE. Maximal expression of both cse15 and cse60 further depended on the DNA-binding protein SpoIIID. Primer extension results revealed -10 and -35 sequences upstream of the cse15 and cse60 coding sequences very similar to those utilized by sigmaE-containing RNA polymerase. Alignment of these and other regulatory regions led to a revised consensus sequence for sigmaE-dependent promoters. A third transcriptional unit, designated csk22, was localized at approximately 173 degrees on the chromosome. Transcription of csk22 was activated at h 4 of sporulation, required the late mother-cell regulator sigmaK, and was repressed by the GerE protein. Sequences in the csk22 promoter region were similar to those of other sigmaK-dependent promoters. The cse60 locus was deduced to encode an acidic product of only 60 residues. A 37.6-kDa protein apparently encoded by cse15 was weakly related to the heavy chain of myosins, as well as to other myosin-like proteins, and is predicted to contain a central, 100 residue-long coiled-coil domain. Finally, csk22 is inferred to encode a 18.2-kDa hydrophobic product with five possible membrane-spanning helices, which could function as a transporter.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Regulon , Amino Acid Sequence , Bacillus subtilis/physiology , Base Sequence , Chromosome Mapping , Chromosomes, Bacterial , Cloning, Molecular , DNA, Bacterial , Molecular Sequence Data , Mutagenesis, Insertional , Promoter Regions, Genetic , Sigma Factor/genetics , Spores, Bacterial , Transcription Factors/genetics , Transcription, Genetic
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