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PubMed1986

Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene.

The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits.

Amino Acids↗

PubMed1986

Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus".

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP. Genes coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.

Amino Acid Sequence↗

PubMed1986

Regulatory elements common to the Bacillus pumilus and Bacillus subtilis trp operons.

The trp operon regulatory region of Bacillus pumilus was cloned and sequenced. The cloned B. pumilus trp promoter-leader region functioned in Bacillus subtilis to express the adjacent leukocyte interferon A gene on a multicopy transcriptional fusion plasmid, pBpIFI. In strains carrying this plasmid, anthranilate synthetase levels were elevated, possible due to titration of a B. subtilis trp regulatory factor by multiple copies of the transcript of the plasmid-borne B. pumilus trp leader region. The B. pumilus trp promoter was recognized efficiently in vitro by B. subtilis sigma 43 RNA polymerase. Approximately 12% of the transcripts produced in vitro terminated in the leader region immediately following synthesis of a transcript structure resembling rho-independent terminators of enteric bacteria. An analogous terminator exists in the B. subtilis trp leader transcript. Nucleotide sequence comparison of the B. pumilus and B. subtilis trp leader regions revealed conservation of these and other sequences that could form transcript secondary structures postulated to regulate transcription termination in B. subtilis (H. Shimotsu, M.I. Kuroda, C. Yanofsky, and D.J. Henner, J. Bacteriol. 166:461-471, 1986). We propose that two elements implicated in B. subtilis trp operon regulation are conserved in the related organism B. pumilus: alternative transcription antiterminator and terminator structures in the leader transcript, and a trans-acting factor present in limiting amounts that is required for transcription termination in the leader region.

Anthranilate Synthase↗

PubMed1987

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis.

The sacQ gene from Bacillus licheniformis was cloned and expressed in Bacillus subtilis. Deletion analysis shows that it encodes a 46-amino-acid polypeptide homologous to the B. subtilis sacQ gene product. The polypeptide, when it is overexpressed, activates the expression of a number of target genes in B. subtilis, all encoding secreted enzymes: alkaline protease, levansucrase, beta-glucanase(s), xylanase, and alpha-amylase. The maximum stimulations measured for alkaline protease and levansucrase were by a factor of 70 and 50, respectively, when the sacQ gene from B. licheniformis was present on a multicopy plasmid in B. subtilis. The sacQ genes from B. subtilis and B. licheniformis, cloned in the same multicopy plasmid, were compared under the same conditions. The sacQ gene from B. licheniformis was more efficient than the sacQ gene from B. subtilis in producing the hypersecretion phenotype. The sacQ structural genes from B. subtilis and B. licheniformis were placed under the control of the same inducible promoter. Hypersecretion was specifically obtained under conditions of full induction of the promoter. The target site of levansucrase regulation by sacQ was identified as a 440-base-pair fragment located in the 5' noncoding region of sacB, suggesting transcriptional control.

Amino Acid Sequence↗

PubMed1987

Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis.

Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis. We identified the promoter for the B. brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B. brevis and Bacillus subtilis. Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B. brevis cells growing in sporulation medium. To study the regulation of tycA in B. subtilis, we constructed a derivative of the B. subtilis bacteriophage SP beta containing a transcriptional fusion of the tycA promoter to the lacZ gene of Escherichia coli and introduced the tycA-lacZ operon fusion by means of specialized transduction into sporulation mutants known to be blocked in sporulation-associated antibiotic production. Our principal finding was that tycA-directed lacZ expression was impaired in the stage-0 mutants with mutations spo0A, spo0B, and spo0E but not in spo0C, spo0F, spo0H, or spo+ bacteria. The dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation, which caused tycA-lacZ to be transcribed constitutively at all stages of growth. A simple model is proposed for the mechanism of tycA induction based on the Spo0A-dependent inactivation of Ab-B protein, which is proposed to be a negative regulator of tycA transcription.

Bacillus↗

PubMed1989

In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47.

Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall. Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B. brevis 47. Biochemical analysis indicated that most of the MWP synthesized by B. subtilis was localized in the cytoplasmic fraction. This was further confirmed by using immunogold electron microscopy. The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B. subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein. The precursor of the MWP possessed the ability to reassemble in vitro on the B. brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B. brevis 47, judging from images averaged at a resolution of about 2.5 nm. Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein.

Amino Acid Sequence↗

PubMed1989

Bacillus licheniformis alpha-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis.

Expression of the Bacillus licheniformis alpha-amylase gene, amyL, was temporally activated and subject to catabolite repression both in its natural host and when cloned on a 3.55-kilobase fragment in Bacillus subtilis. A subclone from which the promoter region of amyL and sequences upstream from the promoter were deleted had a low level of amylase activity. Expression of the promoterless gene was still subject to repression by glucose when the gene was present either on a multicopy plasmid or integrated into the B. subtilis chromosome. Catabolite repression occurred independently of the amylase promoter and irrespective of the distance of the promoterless amyL gene from the promoter which transcribed it. The transcriptional start sites of amyL activated by its own promoter and by a vector sequence promoter were determined by S1 mapping. alpha-Amylase-specific mRNA levels were measured in repressing and nonrepressing media, and catabolite repression was found to act at the level of transcription.

Bacillus↗

PubMed1989

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.

Bacillus↗

PubMed1991

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.

Amidohydrolases↗

PubMed1993

The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked.

Determination by amino acid analyses of the percentage of diaminopimelic acid in the spore cortex of Bacillus megaterium and Bacillus subtilis which is involved in interpeptide cross-links gave values of 31 to 37%. This finding supports the idea that the cortex volume could undergo significant changes in response to changes in pH or ionic strength and could thus play an active role in reducing the water content of the spore protoplast during sporulation.

Amino Acids↗

PubMed1995

The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis.

The mtrB gene from Bacillus pumilus encodes a 76-amino-acid polypeptide with 77% identity to the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis. B. pumilus TRAP binds trp leader RNA from either B. subtilis or B. pumilus in a tryptophan-dependent manner. Altering threonine 52 to alanine eliminated RNA-binding activity of B. pumilus TRAP.

Bacillus↗

PubMed2003

A bacitracin-resistant Bacillus subtilis gene encodes a homologue of the membrane-spanning subunit of the Bacillus licheniformis ABC transporter.

Bacitracin is a peptide antibiotic nonribosomally produced by Bacillus licheniformis. The bcrABC genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells. Bacillus subtilis 168, which has no bacitracin synthesizing operon, has several genes homologous to bcrABC. It was found that the disruption of ywoA, a gene homologous to bcrC, resulted in hypersensitivity to bacitracin. Resistance to other drugs such as surfactin, iturin A, vancomycin, tunicamycin, gramicidin D, valinomycin and several cationic dyes were not changed in the ywoA disruptant. Spontaneous bacitracin-resistant mutants (Bcr-1 and -2) isolated in the presence of bacitracin have a single base substitution from A to G in the ribosome binding region. Northern hybridization analysis and determination of the expression of ywoA-LacZ transcriptional fusion gene revealed that the transcription of the ywoA gene was dependent on extracytoplasmic function (ECF) sigma factors sigma(M) and sigma(X). Preincubation of wild-type cells in the presence of a low concentration of bacitracin induced increased resistance to bacitracin about two- to threefold, although the mechanism of this induction has not yet been elucidated. It has been reported that a commercially available bacitracin is a mixture of several components and also contains impurity. Bacitracin A was purified by reverse phase high-performance liquid chromatography (HPLC). Similar results were obtained with bacitracin A as those with crude bacitracin, indicating that contaminating substances were not responsible for the results obtained in this study.

ATP-Binding Cassette Transporters↗

PubMed2003

The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state.

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.

Amino Acid Sequence↗

PubMed2003

Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis.

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

Bacillus anthracis↗

PubMed2004

Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus.

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

Bacillus anthracis↗

PubMed2005

GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements.

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.

Bacillus cereus↗

PubMed1966

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus.

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.

Acids↗

PubMed1967

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis.

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.

Adenine↗