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At least 73 records · Page 4Linked to original sources

PubMed1999

Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis.

Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

Bacillus subtilis↗

PubMed1995

Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis.

Bacillus subtilis contains at least three classes of heat-shock genes regulated by different mechanisms. We are studying class I heat-shock genes encoded by the operons dnaK and groE. These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat-shock element designated CIRCE. Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis. To answer the question of whether dnaK is involved in the deregulation of the heat-shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39-grpE-dnaK-dnaJ). In one mutant a cat cassette was inserted at the beginning of orf39. Transcriptional analysis revealed that this mutation abolished expression of the whole operon. In contrast, the basal level of groE mRNA was significantly increased at 37 degrees C, followed by a prolonged delay in the shut off after temperature upshift. These data point to a crucial role for the orf39 gene in the regulation of class I heat-shock genes. In the other mutant an internal 0.8 kb Bg/II fragment of dnaK was replaced by the cat cassette. In contrast to E. coli dnaK null mutants, the two B. subtilis dnaK operon mutants could grow within a temperature range from 16-52 degrees C. At temperatures above 52 degrees C, they failed to form colonies on agar plates, started to filament, and lost motility. Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗

PubMed1991

[Subcloning and study of the GTP-cyclohydrolase gene of Bacillus subtilis].

Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells. The position of the gene within the riboflavine operon was defined. The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity.

Bacillus subtilis↗

PubMed2000

Expression of the ftsY gene, encoding a homologue of the alpha subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis.

Bacillus subtilis FtsY (Srb) is a homologue of the alpha subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor sigma(A). During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of sigma(K) and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a sigma(K)- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.

Bacillus subtilis↗

PubMed2004

FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis.

Bacillus subtilis is an aerobic endospore forming bacterium widely spread in different environments. Because it represents a biological agent of some health relevance, its rapid detection and identification is highly desirable. By using FT-IR spectroscopy for this purpose slightly different characteristics were obtained from cell mass grown in differently composed cultural media, and harvested in different phases of bacterial growth. If cultivated uniformly, i.e., 24h at 30 degrees C in a minimum-strength nutrient broth, cell mass of B. subtilis delivered a well differentiated spectrum with major absorption bands of nucleic acid structures at 3300cm(-1), cell wall constituents at 3000-2800cm(-1), proteinaceous structures at 1660, 1544 and 1235cm(-1), and some aliphatic structural units at 1080cm(-1). Attenuated total reflectance, and absorption/transmission scanning techniques, delivered structurally identical spectra but those obtained by the former technique were more expressed.

Bacillus subtilis↗

PubMed2006

The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis.

Bacillus species are valuable producers of industrial enzymes and biopharmaceuticals, because they can secrete large quantities of high-quality proteins directly into the growth medium. This requires the concerted action of quality control factors, such as folding catalysts and 'cleaning proteases'. The expression of two important cleaning proteases, HtrA and HtrB, of Bacillus subtilis is controlled by the CssRS two-component regulatory system. The induced CssRS-dependent expression of htrA and htrB has been defined as a protein secretion stress response, because it can be triggered by high-level production of secreted alpha-amylases. It was not known whether translocation of these alpha-amylases across the membrane is required to trigger a secretion stress response or whether other secretory proteins can also activate this response. These studies show for the first time that the CssRS-dependent response is a general secretion stress response which can be triggered by both homologous and heterologous secretory proteins. As demonstrated by high-level production of a nontranslocated variant of the alpha-amylase, AmyQ, membrane translocation of secretory proteins is required to elicit this general protein secretion stress response. Studies with two other secretory reporter proteins, lipase A of B. subtilis and human interleukin-3, show that the intensity of the protein secretion stress response only partly reflects the production levels of the respective proteins. Importantly, degradation of human interleukin-3 by extracellular proteases has a major impact on the production level, but only a minor effect on the intensity of the secretion stress response.

Bacillus subtilis↗

PubMed2001

Influence of culture conditions on lipopeptide production by Bacillus subtilis.

Bacillus subtilis produces various families of lipopeptides with different homologous compounds. To produce "new molecules" with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family. This study focused on two B. subtilis strains cultivated in flasks. Optimized medium for lipopeptide production and Landy medium modified by replacing glutamic acid with other alpha-amino acids were used. We found that the intensity of production of homologous compounds depends on the strain and the culture medium. Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B. subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium. Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L c15.

Amino Acids↗

PubMed2005

Metal ion homeostasis in Bacillus subtilis.

Bacillus subtilis, a Gram-positive soil bacterium, provides a model system for the study of metal ion homeostasis. Metalloregulatory proteins serve as the arbiters of metal ion sufficiency and regulate the expression of metal homeostasis pathways. In B. subtilis, uptake systems are regulated by the highly selective metal-sensing repressors Fur (iron), Zur (zinc), and MntR (manganese). Metal efflux systems are regulated by MerR and ArsR family homologs which, by contrast, can be rather non-specific with regard to metal selectivity. A Fur homolog, PerR, functions as an Fe(II)-dependent peroxide stress sensor and regulates putative metal transport and storage functions.

Bacillus subtilis↗

PubMed1999

Spore peptidoglycan structure in a cwlD dacB double mutant of Bacillus subtilis.

Bacillus subtilis cwlD and dacB mutants produce spore peptidoglycan (PG) with increased cross-linking but with little change in spore core hydration compared to the wild type. A cwlD dacB double mutant produced spores with a two- to fourfold greater increase in PG cross-linking and novel muropeptides containing glycine residues but no significant changes in spore resistance or core hydration.

Amino Acids↗

PubMed2006

Preparation, crystallization and preliminary X-ray analysis of protein YtlP from Bacillus subtilis.

Bacillus subtilis YtlP is a protein that is predicted to belong to the bacterial and archael 2'-5' RNA-ligase family. It contains 183 residues and two copies of the HXTX sequence motif conserved among proteins belonging to this family. In order to determine the structure of YtlP and to compare it with the paralogue YjcG and identified 2'-5' RNA ligases, the gene ytlP was amplified from B. subtilis genomic DNA and cloned into expression vector pET-21a. The soluble protein was produced in Escherichia coli, purified to homogeneity and crystals suitable for X-ray analysis were obtained. The crystal diffracted to 2.0 A and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.16, b = 48.54, c = 105.75 A.

Amino Acid Sequence↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed2005

Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis.

Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2'-5' RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 A and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 A, beta = 113.56 degrees.

Amino Acid Sequence↗

PubMed2006

Phosphate starvation induces the sporulation killing factor of Bacillus subtilis.

Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.

Bacillus subtilis↗

PubMed2003

Expression of a Bacillus thuringiensisdelta-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum, Mills).

AIMS: To introduce a cry gene into microorganisms that naturally colonize the phylloplane of tomato plants to improve the persistence of the Cry proteins for controlling a South American tomato moth (Tuta absoluta, Meyrick, 1917). METHODS AND RESULTS: A cry1Ab gene isolated from a native Bacillus thuringiensis strain (LM-466), showing a relevant activity against T. absoluta larvae, was cloned into the shuttle vector pHT315 (Arantes and Lereclus 1991). The construct was introduced by electroporation into native Bacillus subtilis and Bacillus licheniformis strains, both natural inhabitants of the tomato phylloplane. Western analysis and toxicity assays against the target larvae proved that the successful expression of the gene was accomplished in host bacteria. Recombinant toxin displayed a similar LC50 value in comparison to native donor strain LM-466. Both transformed Bacillus survived for at least 45 days on the tomato leaf surface. CONCLUSIONS: Plant-associated microorganisms that naturally colonize the phylloplane could be useful as recombinant microbial delivery systems of toxin genes of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Modified microorganisms capable of surviving on leaf surfaces for several weeks with insecticidal activity should allow for a reduction in pesticide application.

Animals↗

PubMed1994

Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis.

Bacillus subtilis has an alkaline phosphatase (APase) gene family composed of at least four genes. All members of this gene family are expressed postexponentially, either in response to phosphate starvation or sporulation induction or, in some cases, in response to both. The phoA gene (formerly called phoAIV) and the phoB gene (formerly called phoAIII) products have both been isolated from phosphate-starved cells, and a mutation in either gene decreased the total APase expressed under phosphate starvation conditions. Data presented here show that a phoA phoB double mutant reduced APase production during phosphate starvation by 98%, indicating that these two genes are responsible for most of the APase activity during phosphate-limited growth. The promoter for phoA was cloned and used, with the phoB promoter, to examine phosphate regulation in B. subtilis. phoA-lacZ reporter gene assays showed that the expression of the phoA gene commences as the culture enters stationary phase as a result of limiting phosphate concentrations in the growth medium, thereby mimicking the pattern of total APase expression. Induction persists for approximately 2 h and is then turned off. phoA is transcribed from a single promoter which initiates transcription 19 bp before the translation initiation codon. PhoP and PhoR are members of the two-component signal transduction system believed to regulate gene expression in response to limiting phosphate. The expression of phoA or phoB in response to phosphate starvation was equally dependent on PhoP and PhoR for induction. lacZ-promoter fusions showed that both phoA and phoB were hyperinduced, or failed to turn off induction after 2 h, in a spo0A strain of B. subtilis. Mutations in genes which are required for phosphorylation of Spo0A, spo0B and spo0F, also resulted in phoA and phoB hyperinduction, suggesting that phosphorylation of Spo0A is required for the repression of both APases in wild-type strains. The hyperinduction of either APase gene in a spo0A strain was dependent on PhoP and PhoR. Analysis of a phoP-lacZ promoter fusion showed that the phoPR operon is hyperinduced in a spo0A mutant strain, suggesting that Spo0A approximately P represses APases by repressing phoPR transcription. We propose a model for PHO regulation in B. subtilis whereby the phoPR operon is transcribed in response to limiting phosphate concentration, resulting in activation of the PHO regulon transcription, including transcription of phoA and phoB. When the phosphate response fails to overcome the nutrient deficiency, signals for phosphorylation of Spo0A result in production of Spo0A approximately P, which represses transcription of phoPR, thereby repressing synthesis of the PHO regulon.

Alkaline Phosphatase↗

PubMed1978

Two thymidylate synthetases in Bacillus subtilis.

Bacillus subtilis grown at temperatures below 37 degrees contains two thymidylate synthetases, TSaseA and TSaseB. Their presence is dependent on functional thyA and thyB genes, respectively. When cells are grown at 46 degrees they only contain TSaseA activity. This allous an easy positive selection for thyA and thyA, THYB mutants. The two TSases have been physically separated, and they show similar overall requirements for activity. However, they differ significantly in both their kinetic and their physicochemical properties.

Bacillus subtilis↗

PubMed1975

Ultraviolet sensitivity and photoproducts in spore-like bodies of an excision-repair-deficient and dipicolinic-acid-less strain of Bacillus subtilis.

Bacillus subtilis strain UVS-42DPA is defective in both excision-repair capability and dipicolonic acid(DPA)accumulation. In sporulation medium, it forms spore-like bodies, which are sensitive to ultraviolet light (UV) as the vegetative cells and produce mostly cyclobutane dimers instead of "spore photoproduct" upon UV irradiation. The results suggest that the drastic change in the photochemical reactivity of DNA during sporulation might be induced and(or) maintained by the accumulation of DPA.

Bacillus subtilis↗

PubMed2003

Expression, purification, crystallization and preliminary crystallographic analysis of a putative GTP-binding protein, YsxC, from Bacillus subtilis.

Bacillus subtilis YsxC has been putatively identified as a member of the GTP-binding protein family. Gene-knockout/deletion analysis has suggested that this protein is essential for survival of the microorganism and hence may represent a target for the development of a novel anti-infective agent. The B. subtilis ysxC gene was cloned and the protein was overexpressed in Escherichia coli and subsequently purified. Using hanging-drop vapour-diffusion crystallization techniques, two different crystal forms of YsxC were obtained in the presence and absence of GDP and which have one and two copies of YsxC in the asymmetric unit, respectively. Both crystal forms diffract to beyond 2.0 A resolution and are suitable for structure determination.

Bacillus subtilis↗