( 2003 )
In Bacillus subtilis W23, the duet sigmaXsigmaM, two sigma factors of the extracytoplasmic function subfamily, are required for septum and wall synthesis under batch culture conditions.
PMID : 12940998 : DOI : 10.1046/j.1365-2958.2003.03652.x
The synthesis of poly(RboP), the main Bacillus subtilis W23 teichoic acid, is encoded by tarDF-tarABIJKL operons, the latter being controlled by two promoters designated PtarA-int and PtarA-ext. Analysis by lacZ fusions reveals that PtarA-int activity exhibits sharp increases at the beginning and end of the transition between exponential and stationary growth phase. As confirmed by mRNA quantification, these increases are mediated by ECF sigma factors sigmaX and sigmaM respectively. In liquid media, strain W23 sigX sigM double mutants experience serious difficulties in the transition and stationary growth phases. Inactivation of sigmaX- and sigmaM-controlled regulons, which precludes transcription from PtarA-int, leads to (i) delays in chromosome segregation and septation and (ii) a transient loss of up to 30% of the culture OD or lysis. However, specific inactivation of PtarA-int, leading mainly to a shortage of poly(RboP), does not affect growth while, nevertheless, interfering with normal septation, as revealed by electron microscopy. The different sigM transcription in strains W23 and 168 is discussed. In W23, expression of tarA and sigM, which is shown to control divIC, is inversely correlated with growth rate, suggesting that the sigM regulon is involved in the control of cell division.
( 2002 )
Specific activation of the Bacillus quorum-sensing systems by isoprenylated pheromone variants.
PMID : 12067344 : DOI : 10.1046/j.1365-2958.2002.02977.x
Natural genetic competence in Bacillus subtilis is controlled by quorum-sensing (QS). The ComP- ComA two-component system detects the signalling molecule ComX, and this signal is transduced by a conserved phosphotransfer mechanism. ComX is synthesized as an inactive precursor and is then cleaved and modified by ComQ before export to the extracellular environment. The comQXP' loci of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum-sensing response. We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes. Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post-translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes. These results give new insights into peptidemediated quorum-sensing signalling in Gram-positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction.
( 2000 )
Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences.
PMID : 11204764 : DOI : 10.1023/a:1026555830014
Partial gyrA sequences were determined for twelve strains belonging to Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, B. mojavensis, B. subtilis subsp. subtilis, B. subtilis subsp. spizizenii and B. vallismortis. The average nucleotide and translated amino acid similarities for the seven type strains were 83.7 and 95.1%, respectively, whereas the corresponding value for the 16S rRNA sequences was 99.1%. All of the type strains were sharply separated; the closest relationship was found between B. atrophaeus and B. mojavensis which shared a nucleotide similarity of 95.8%. Phylogenetic trees were inferred from gyrA nucleotide sequences using the neighbor-joining, Fitch-Margoliash and maximum parsimony algorithms. The test strains were divided into four groups, which generally reflected results previously reported in restriction digest and DNA-DNA hybridization studies. It is concluded from the comparative sequence analysis that the gyrA sequences provide a firm framework for the rapid and accurate classification and identification of Bacillus subtilis and related taxa.
( 2001 )
Specificity and genetic polymorphism of the Bacillus competence quorum-sensing system.
PMID : 11133937 : DOI : 10.1128/JB.183.2.451-460.2001 PMC : PMC94899
A quorum-sensing mechanism involving the pheromone ComX and the ComP-ComA two-component system controls natural competence in Bacillus subtilis. ComX is expressed as a cytoplasmic inactive precursor that is released into the extracellular medium as a cleaved, modified decapeptide. This process requires the product of comQ. In the presence of ComX, the membrane-localized ComP histidine kinase activates the response regulator ComA. We compared the sequences of the quorum-sensing genes from four closely related bacilli, and we report extensive genetic polymorphism extending through comQ, comX, and the 5' two-thirds of comP. This part of ComP encodes the membrane-localized and linker domains of the sensor protein. We also determined the sequences of the comX genes of four additional wild-type bacilli and tested the in vivo activities of all eight pheromones on isogenic strains containing four different ComP receptor proteins. A striking pattern of specificity was discovered, providing strong evidence that the pheromone contacts ComP directly. Furthermore, we show that coexpression of comQ and comX in Escherichia coli leads to the production of active pheromone in the medium, demonstrating that comQ is the only dedicated protein required for the processing, modification, and release of active competence pheromone. Some of the implications of these findings for the evolution and the mechanism of the quorum-sensing system are discussed.
( 2007 )
Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group.
PMID : 17684269 : DOI : 10.1099/ijs.0.64685-0
The Bacillus subtilis group comprises eight closely related species that are indistinguishable from one another by 16S rRNA gene sequence analysis. Therefore, the gyrB gene, which encodes the subunit B protein of DNA gyrase, was selected as an alternative phylogenetic marker. To determine whether gyrB gene sequence analysis could be used for phylogenetic analysis and species identification of members of the B. subtilis group, the congruence of gyrB grouping with both 16S rRNA gene sequencing and DNA-DNA hybridization data was evaluated. Ranges of gyrB nucleotide and translated amino acid sequence similarities among the eight type strains were 75.4-95.0 % and 88.5-99.2 %, respectively, whereas 16S rRNA gene sequence similarities were 98.1-99.8 %. Results showed that gyrB gene sequences provide higher resolution than 16S rRNA gene sequences. The classification achieved by gyrB sequence analysis was in agreement with results obtained with DNA-DNA hybridization. It is concluded that the gyrB gene may be an efficient alternative target for identification and taxonomic analysis of members of the B. subtilis group.
( 2007 )
Bacillus subtilis genome diversity.
PMID : 17114265 : DOI : 10.1128/JB.01343-06 PMC : PMC1797320
Microarray-based comparative genomic hybridization (M-CGH) is a powerful method for rapidly identifying regions of genome diversity among closely related organisms. We used M-CGH to examine the genome diversity of 17 strains belonging to the nonpathogenic species Bacillus subtilis. Our M-CGH results indicate that there is considerable genetic heterogeneity among members of this species; nearly one-third of Bsu168-specific genes exhibited variability, as measured by the microarray hybridization intensities. The variable loci include those encoding proteins involved in antibiotic production, cell wall synthesis, sporulation, and germination. The diversity in these genes may reflect this organism's ability to survive in diverse natural settings.
( 2007 )
In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir.
PMID : 17172458 : DOI : 10.1128/AEM.02264-06 PMC : PMC1828672
Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO(2) and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 +/- 0.01 h(-1)), carbon balances (107% +/- 34%), biosurfactant production rates (0.02 +/- 0.001 h(-1)), and biosurfactant yields (0.015 +/- 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.
( 2006 )
Effects of streptomycin resistance mutations on posttranslational modification of ribosomal protein S12.
PMID : 16484214 : DOI : 10.1128/JB.188.5.2020-2023.2006 PMC : PMC1426572
Ribosomal protein S12 contains a highly conserved aspartic acid residue that is posttranslationally beta-methylthiolated. Using mass spectrometry, we have determined the modification states of several S12 mutants of Thermus thermophilus and conclude that beta-methylthiolation is not a determinant of the streptomycin phenotype.
( 2005 )
Structure of the Bacillus subtilis quorum-sensing peptide pheromone ComX.
PMID : 16407988 : DOI : 10.1038/nchembio709
The ComX pheromone is an extracellular signaling molecule that stimulates natural competence in response to crowding in the gram-positive bacterium Bacillus subtilis. The pheromone is formed by isoprenylation of an inactive precursor peptide, but its precise structure is not known. Here we report the structure of the ComX pheromone, showing that addition of a geranyl group to a tryptophan residue results in the formation of an unusual ring structure.
( 2004 )
Diversifying selection at the Bacillus quorum-sensing locus and determinants of modification specificity during synthesis of the ComX pheromone.
PMID : 14679219 : DOI : 10.1128/jb.186.1.15-21.2004 PMC : PMC303460
The competence quorum-sensing system of Bacillus subtilis consists of two-component regulatory proteins, ComP (histidine kinase) and the response regulator, ComA, an extracellular pheromone (ComX), and a protein that is needed for the proteolytic cleavage and modification of pre-ComX (ComQ). ComQ and pre-ComX are both necessary and sufficient for the production of active pheromone, which is released as an isoprenylated peptide. Laboratory strain 168 and a number of natural isolates of bacilli differ in the primary sequences of their pheromones as well as in the masses of their isoprenyl adducts. We have shown that ComX, ComQ, and the membrane-localized sensor domain of ComP are highly polymorphic in natural isolates of bacilli all closely related to the laboratory strain of B. subtilis. In this study, we used two statistical tests (the ratio of synonymous and nonsynonymous substitution rates and the Tajima D test) to demonstrate that these polymorphic sequences evolved by diversifying selection rather than by neutral drift. We show that the choice of isoprenyl derivative is determined by the C-terminal (mature) sequence of pre-ComX rather than by the ComQ protein. The implications of these findings for the evolution of the quorum-sensing system and for the protein-protein interactions involved in determining specificity are discussed.
( 2011 )
Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity.
PMID : 21239550 : DOI : 10.1128/AEM.01962-10 PMC : PMC3067280
Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.
( 2010 )
Ecology of speciation in the genus Bacillus.
PMID : 20048064 : DOI : 10.1128/AEM.01988-09 PMC : PMC2832372
Microbial ecologists and systematists are challenged to discover the early ecological changes that drive the splitting of one bacterial population into two ecologically distinct populations. We have aimed to identify newly divergent lineages ("ecotypes") bearing the dynamic properties attributed to species, with the rationale that discovering their ecological differences would reveal the ecological dimensions of speciation. To this end, we have sampled bacteria from the Bacillus subtilis-Bacillus licheniformis clade from sites differing in solar exposure and soil texture within a Death Valley canyon. Within this clade, we hypothesized ecotype demarcations based on DNA sequence diversity, through analysis of the clade's evolutionary history by Ecotype Simulation (ES) and AdaptML. Ecotypes so demarcated were found to be significantly different in their associations with solar exposure and soil texture, suggesting that these and covarying environmental parameters are among the dimensions of ecological divergence for newly divergent Bacillus ecotypes. Fatty acid composition appeared to contribute to ecotype differences in temperature adaptation, since those ecotypes with more warm-adapting fatty acids were isolated more frequently from sites with greater solar exposure. The recognized species and subspecies of the B. subtilis-B. licheniformis clade were found to be nearly identical to the ecotypes demarcated by ES, with a few exceptions where a recognized taxon is split at most into three putative ecotypes. Nevertheless, the taxa recognized do not appear to encompass the full ecological diversity of the B. subtilis-B. licheniformis clade: ES and AdaptML identified several newly discovered clades as ecotypes that are distinct from any recognized taxon.
( 2009 )
Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov.
PMID : 19622642 : DOI : 10.1099/ijs.0.009126-0
The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).
( 2008 )
The origins of 168, W23, and other Bacillus subtilis legacy strains.
PMID : 18723616 : DOI : 10.1128/JB.00722-08 PMC : PMC2580678
Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.
( 2008 )
CSF, a species-specific extracellular signaling peptide for communication among strains of Bacillus subtilis and Bacillus mojavensis.
PMID : 18375560 : DOI : 10.1128/JB.00187-08 PMC : PMC2395031
ComX and CSF are Bacillus subtilis extracellular signaling peptides. Many different strains of B. subtilis do not communicate due to strain-specific variation of ComX. We demonstrate that CSF is a species-specific signaling molecule that partially compensates for the lack of ComX-mediated communication between different strains of B. subtilis.
( 2008 )
Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics.
PMID : 18272490 : DOI : 10.1073/pnas.0712205105 PMC : PMC2268166
The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach ("ecotype simulation") to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the "Evolution Canyons" of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.
( 2007 )
Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from fermented soybean foods manufactured in Asian countries.
PMID : 18187886 :
Natto-like fermented soybean products are manufactured and consumed in many Asian countries. In this study, we isolated thirty-four Bacillus strains capable of producing gamma-polyglutamic acid (PGA) from natto in mountainous areas of South Asia and Southeast Asia and from soils in Japan. To elucidate the phylogeny of these PGA-producing strains, phylogenetic trees based on sequences of 16S rDNA, housekeeping genes of rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) were constructed. A phylogenetic tree based on 16S rDNA sequences showed that twenty-one isolates were clustered in the same group of B. subtilis. The other thirteen isolates were located in the cluster of B. amyloliquefaciens. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. The results of the present study indicate that PGA-producing strains isolated from local natto in Asian countries and soil in Japan can be divided into two species, B. subtilis and B. amyloliquefaciens.
( 2016 )
Establishment and application of an analytical in-house database (IHDB) for rapid discrimination of Bacillus subtilis group (BSG) using whole-cell MALDI-TOF MS technology.
PMID : 27507023 : DOI : 10.1016/j.mcp.2016.08.002
Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.
( 2012 )
The quorum sensing diversity within and between ecotypes of Bacillus subtilis.
PMID : 22390407 : DOI : 10.1111/j.1462-2920.2012.02717.x
Ecological sociobiology is an emerging field that aims to frame social evolution in terms of ecological adaptation. Here we explore the ecological context for evolution of quorum sensing diversity in bacteria, where social communication is limited to members of the same quorum sensing type (pherotype). We sampled isolates of Bacillus subtilis from soil on a microgeographical scale and identified three ecologically distinct phylogenetic groups (ecotypes) and three pherotypes. Each pherotype was strongly associated with a different ecotype, suggesting that it is usually not adaptive for one ecotype to 'listen' to the signalling of another. Each ecotype, however, contained one or more minority pherotypes shared with the other B. subtilis ecotypes and with more distantly related species taxa. The pherotype diversity within ecotypes is consistent with two models: first, a pherotype cycling model, whereby minority pherotypes enter a population through horizontal genetic transfer and increase in frequency through cheating the social interaction; and second, an occasional advantage model, such that when two ecotypes are each below their quorum densities, they may benefit from listening to one another. This is the first survey of pherotype diversity in relation to ecotypes and it will be interesting to further test the hypotheses raised and supported here, and to explore other bacterial systems for the role of ecological divergence in fostering pherotype diversity.
( 2012 )
Bacteriocin formation by dominant aerobic sporeformers isolated from traditional maari.
PMID : 22240061 : DOI : 10.1016/j.ijfoodmicro.2011.12.003
The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursor peptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.
( 2011 )
Biosurfactant-producing Bacillus are present in produced brines from Oklahoma oil reservoirs with a wide range of salinities.
PMID : 21562978 : DOI : 10.1007/s00253-011-3326-z
Nine wells producing from six different reservoirs with salinities ranging from 2.1% to 15.9% were surveyed for presence of surface-active compounds and biosurfactant-producing microbes. Degenerate primers were designed to detect the presence of the surfactin/lichenysin (srfA3/licA3) gene involved in lipopeptide biosurfactant production in members of Bacillus subtilis/licheniformis group and the rhlR gene involved in regulation of rhamnolipid production in pseudomonads. Polymerase chain reaction amplification, cloning, and sequencing confirmed the presence of the srfA3/licA3 genes in brines collected from all nine wells. The presence of B. subtilis/licheniformis strains was confirmed by sequencing two other genes commonly used for taxonomic identification of bacteria, gyrA (gyrase A) and the 16S rRNA gene. Neither rhlR nor 16S rRNA gene related to pseudomonads was detected in any of the brines. Intrinsic levels of surface-active compounds in brines were low or not detected, but biosurfactant production could be stimulated by nutrient addition. Supplementation with a known biosurfactant-producing Bacillus strain together with nutrients increased biosurfactant production. The genetic potential to produce lipopeptide biosurfactants (e.g., srfA3/licA3 gene) is prevalent, and nutrient addition stimulated biosurfactant production in brines from diverse reservoirs, suggesting that a biostimulation approach for biosurfactant-mediated oil recovery may be technically feasible.
( 2019 )
Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the Phosphono-oligopeptide Antibiotic Rhizocticin.
PMID : 30830751 : DOI : 10.1021/acschembio.9b00030
Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal (Z) -l -2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC (BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.
( 2017 )
Production of a bioactive unnatural ginsenoside by metabolically engineered yeasts based on a new UDP-glycosyltransferase from Bacillus subtilis.
PMID : 28778764 : DOI : 10.1016/j.ymben.2017.07.008
Ginsenosides are the main bioactive constituents of Panax species, which are biosynthesized by glycosylation at C3-OH and/or C20-OH of protopanaxadiol (PPD), C6-OH and/or C20-OH of protopanaxatriol (PPT). The C12-glycosylated ginsenosides have scarcely been identified from Panax species. The C12-glycosylated ginsenosides produced from PPD by chemical semi-synthesis have been reported to exhibit higher cytotoxicity than the natural ginsenosides. However, the chemical semi-synthesis approach is not practical due to its complexity and high cost. In our study, a new UDP-glycosyltransferase UGT109A1 was identified from Bacillus subtilis. This enzyme transferred a glucose moiety to C3-OH and C20-OH of dammarenediol-II (DM), C3-OH and C12-OH of PPD and PPT respectively to produce the unnatural ginsenosides 3�]-O-Glc-DM, 3�],20S-Di-O-Glc-DM, 3�],12�]-Di-O-Glc-PPD and 3�],12�]-Di-O-Glc-PPT. Among these unnatural ginsenosides, 3�],12�]-Di-O-Glc-PPT is a new compound which has never been reported before. The anti-cancer activities of these unnatural ginsenosides were evaluated in vitro and in vivo. 3�],12�]-Di-O-Glc-PPD exhibited higher anti-lung cancer activity than Rg3, which is the most active natural ginsenoside against lung cancer. Finally, we constructed metabolically engineered yeasts to produce 3�],12�]-Di-O-Glc-PPD by introducing the genes encoding B. subtilis UGT109A1, Panax ginseng dammarenediol-II synthase (DS), P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS) together with Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1) into Saccharomyces cerevisiae INVSc1. The yield of 3�],12�]-Di-O-Glc-PPD was increased from 6.17mg/L to 9.05mg/L by overexpressing tHMG1. Thus, this study has established an alternative route to produce the unnatural ginsenoside 3�],12�]-Di-O-Glc-PPD by synthetic biology strategies, which provides a promising candidate for anti-cancer drug discovery.