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Journal of Bacteriology Jun 2015D-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-D-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative...
UNLABELLED
D-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-D-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2, are each essential for growth of B. anthracis, supplying D-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1(+) pXO2(-)), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1(+) pXO2(+)), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous D-glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis, similar to Bacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesize D-glutamic acid for peptidoglycan synthesis.
IMPORTANCE
Glutamate racemases, enzymes that convert L-glutamate to D-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis, requires d-glutamate for the synthesis of peptidoglycan and poly-γ-D-glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic efficacy.
Topics: Amino Acid Isomerases; Bacillus anthracis; Bacterial Proteins; Gene Deletion; Glutamic Acid; Polyglutamic Acid; Sequence Deletion
PubMed: 25777674
DOI: 10.1128/JB.00070-15 -
MBio Apr 2017In 1998, it was claimed that an 80-year-old glass tube intentionally filled with and embedded in a sugar lump as a WWI biological weapon still contained viable spores....
In 1998, it was claimed that an 80-year-old glass tube intentionally filled with and embedded in a sugar lump as a WWI biological weapon still contained viable spores. Today, genome sequencing of three colonies isolated in 1998 and subjected to phylogenetic analysis surprisingly identified a well-known reference strain isolated in the United States in 1981, pointing to accidental laboratory contamination. Next-generation sequencing and subsequent phylogenetic analyses are useful and reliable tools for the classification of recent and historical samples. The reliability of sequences obtained and bioinformatic algorithms has increased in recent years, and research has uncovered the identity of a presumed bioweapon agent as a contaminant.
Topics: Bacillus anthracis; Biological Warfare Agents; Phylogeny; Sequence Analysis, DNA; United Kingdom; United States
PubMed: 28442608
DOI: 10.1128/mBio.00440-17 -
Bioengineered Jan 2018Surrogate microorganisms, in short surrogates, are an essential part of pathogen research. Compared to surrogates used in controlled laboratory environments, surrogates... (Review)
Review
Surrogate microorganisms, in short surrogates, are an essential part of pathogen research. Compared to surrogates used in controlled laboratory environments, surrogates for field release are restricted by concerns about human and environmental safety. For field research of food-borne pathogens, strains of an attenuated pathogen or strains of genetically close non-pathogenic species have been used as surrogates. Genetic modification is usually performed to attenuate virulence, through for examples deletion of genes of virulence and transcriptional regulators and removal of virulence plasmids, and to facilitate detection and monitoring through observing antibiotic resistance, fluorescence, and bioluminescence. For field research of a biological warfare agent Bacillus anthracis, strains of genetically close non-pathogenic species or strains of genetically distant non-pathogenic species have been used, mostly without any genetic modification. Recently, we constructed strains of Bacillus thuringiensis as surrogates for B. anthracis, demonstrating that strain engineering could significantly enhance the utility of surrogates, and that the application of a simple genetic circuit could significantly impact surrogate safety. Thus far, enormous potential of biotechnology has not been exploited enough due to safety concerns regarding the field release of genetically engineered microorganisms. However, synthetic biology is rapidly developing, providing new concepts for biocontainment as well as ingenious genetic circuits and devices, which should be applied in future research of field-use surrogates.
Topics: Bacillus anthracis; Bacillus thuringiensis; Biotechnology; Containment of Biohazards; Genetic Engineering; Humans; Plasmids; Synthetic Biology; Virulence
PubMed: 28692329
DOI: 10.1080/21655979.2017.1349044 -
Microbial Genomics Aug 2021the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. While analyses of genetic data from across the globe have...
the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. While analyses of genetic data from across the globe have increased our understanding of this bacterium’s population genomic structure, the influence of selective pressures on this successful pathogen is not well understood. In this study, we investigate the effects of antimicrobial resistance, phage diversity, geography and isolation source in shaping population genomic structure. We also identify a suite of candidate genes potentially under selection, driving patterns of diversity across 356 globally extant genomes. We report ten antimicrobial resistance genes and 11 different prophage sequences, resulting in the first large-scale documentation of these genetic anomalies for this pathogen. Results of random forest classification suggest genomic structure may be driven by a combination of antimicrobial resistance, geography and isolation source, specific to the population cluster examined. We found strong evidence that a recombination event linked to a gene involved in protein synthesis may be responsible for phenotypic differences between comparatively disparate populations. We also offer a list of genes for further examination of evolution, based on high-impact single nucleotide polymorphisms (SNPs) and clustered mutations. The information presented here sheds new light on the factors driving genomic structure in this notorious pathogen and may act as a road map for future studies aimed at understanding functional differences in terms of biogeography, virulence and evolution.
Topics: Anti-Bacterial Agents; Bacillus anthracis; Bacteriophages; Biodiversity; Drug Resistance, Bacterial; Genome, Bacterial; Genomics; Polymorphism, Single Nucleotide; Virulence
PubMed: 34402777
DOI: 10.1099/mgen.0.000616 -
Journal of Bacteriology Jan 2017The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic...
UNLABELLED
The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan.
IMPORTANCE
The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→) with α-Gal and β-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.
Topics: Amino Acid Sequence; Bacillus anthracis; Bacterial Proteins; Cell Wall; Gene Deletion; Gene Expression Regulation, Bacterial; Polysaccharides, Bacterial
PubMed: 27795328
DOI: 10.1128/JB.00613-16 -
Journal of Applied Microbiology Aug 1999Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S-layer). Their corresponding genes have been cloned, shown to be...
Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S-layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three 'S-layer homology' motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of both the non-capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S-layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a gram-positive bacterium.
Topics: Bacillus anthracis; Bacterial Capsules; Bacterial Outer Membrane Proteins; Cloning, Molecular; Genes, Bacterial
PubMed: 10475960
DOI: 10.1046/j.1365-2672.1999.00882.x -
PloS One 2020A study was conducted to assess the efficacy of ozone gas in inactivating spores of both Bacillus anthracis and Bacillus subtilis inoculated onto six building materials...
A study was conducted to assess the efficacy of ozone gas in inactivating spores of both Bacillus anthracis and Bacillus subtilis inoculated onto six building materials (glass, wood, carpet, laminate, galvanized metal, and wallboard paper). Testing conditions consisted of ozone gas concentrations ranging from 7,000-12,000 parts per million (ppm), contact times from 4 to 12 h, and two relative humidity (RH) levels of 75 and 85%. Results showed that increasing the ozone concentration, contact time, and RH generally increased decontamination efficacy. The materials in which the highest decontamination efficacy was achieved for B. anthracis spores were wallboard paper, carpet, and wood with ≥ 6 log10 reduction (LR) occurring with 9,800 ppm ozone, 85% RH, for 6 h. The laminate and galvanized metal materials were generally more difficult to decontaminate, requiring 12,000 ppm ozone, 85% RH, and 9-12 h contact time to achieve ≥6 LR of B. anthracis. Lastly, overall, there were no significant differences in decontamination efficacy between the two species.
Topics: Bacillus anthracis; Bacillus subtilis; Construction Materials; Decontamination; Disinfectants; Disinfection; Fumigation; Humans; Ozone; Species Specificity; Spores, Bacterial; Virulence
PubMed: 32437373
DOI: 10.1371/journal.pone.0233291 -
Journal of Bacteriology Oct 1965Avakyan, A. A. (Academy of Medical Sciences, Moscow, USSR), L. N. Katz, K. N. Levina, and I. B. Pavlova. Structure and composition of the Bacillus anthracis capsule. J....
Avakyan, A. A. (Academy of Medical Sciences, Moscow, USSR), L. N. Katz, K. N. Levina, and I. B. Pavlova. Structure and composition of the Bacillus anthracis capsule. J. Bacteriol. 90:1082-1095. 1965.-Observations by various methods of light microscopy (phase contrast, dark-field, and fluorescence) revealed the complex structure of the Bacillus anthracis capsule, which changes regularly during the growth cycle of the culture. Special cytological methods of staining the capsule made it possible to study its fine structure, which is not revealed by negative staining with India ink. For example, the capsule shows a membranelike outline, fine transverse lines, and interruptions and transverse septa traversing the entire capsule. By using cytochemical methods, it was found that the capsule has a stratified structure and that the various layers of the capsule differ as to the value of the isoelectric point, metachromatic ability, sensitivity to various enzymes, and, consequently, chemical composition. It was thus shown that the membranelike outline of the capsule consists of peptides and neutral mucopolysaccharides. The middle part of the capsule consists of a complex of substances of both polysaccharide and protein nature, and the inner part consists of acid mucopolysaccharides. Observation of the capsular forms of B. anthracis by means of an electron microscope revealed differences in the osmiophilia and submicroscopic structure of the membranelike outline and the middle and inner parts of the capsule. Immunochemical studies conducted by the fluorescent-antibody method revealed localization of antigens in different parts of the capsule, and made it possible to differentiate the capsular antigens according to their serum-staining ability and according of their relations to enzymes, i.e., their chemical composition. This paper concerns the possibility of studying the fine structure of bacterial capsules in fixed preparations, and the differences and similarities of the antigens of the capsule and cell wall of B. anthracis and of the related species, B. megaterium.
Topics: Bacillus anthracis; Chemical Phenomena; Chemistry; Histocytochemistry; Immunochemistry; In Vitro Techniques; Microscopy; Microscopy, Electron
PubMed: 4954516
DOI: 10.1128/jb.90.4.1082-1095.1965 -
Journal of Clinical Microbiology Jan 1984Bacillus anthracis was agglutinated by several lectins, including those from Griffonia simplicifolia, Glycine max, Abrus precatorius, and Ricinus communis. Some strains... (Comparative Study)
Comparative Study
Bacillus anthracis was agglutinated by several lectins, including those from Griffonia simplicifolia, Glycine max, Abrus precatorius, and Ricinus communis. Some strains of Bacillus cereus var. mycoides (B. mycoides) were strongly reactive with the lectin from Helix pomatia and weakly reactive with the G. max lectin. The differential interactions between Bacillus species and lectins afforded a means of distinguishing B. anthracis from other bacilli. B. cereus strains exhibited heterogeneity with respect to agglutination patterns by lectins but could readily be differentiated from B. anthracis and the related B. mycoides. Spores of B. anthracis and B. mycoides retained lectin receptors, although the heating of spores or vegetative cells at 100 degrees C resulted in a decrease in their ability to be specifically agglutinated. Fluorescein-conjugated lectin of G. max stained vegetative cells of B. anthracis uniformly, suggesting that the distribution of lectin receptors was continuous over the entire cellular surface. B. anthracis cells grown under conditions to promote the production of capsular poly(D-glutamyl peptide) were also readily agglutinated by the lectins, suggesting that the lectin reactive sites penetrate the polypeptide layer. Trypsin, subtilisin, lysozyme, and mutanolysin did not modify the reactivity of B. anthracis with the G. max agglutinin, although the same enzymes markedly diminished the interaction between the lectin and B. mycoides. Because the lectins which interact with B. anthracis are specific for alpha-D-galactose or 2-acetamido-2-deoxy-alpha-D-galactose residues, it is likely that the bacteria possess cell surface polymers which contain these sugars. Lectins may prove useful in the laboratory identification of B. anthracis and possibly other pathogenic Bacillus species, such as B. cereus.
Topics: Agglutination Tests; Bacillus; Bacillus anthracis; Bacillus cereus; Lectins; Peptide Hydrolases; Receptors, Mitogen; Spores, Bacterial
PubMed: 6418761
DOI: 10.1128/jcm.19.1.48-53.1984 -
Virulence 2010Quorum-sensing (QS), the regulation of bacterial gene expression in response to changes in cell density, involves pathways that synthesize signaling molecules...
Quorum-sensing (QS), the regulation of bacterial gene expression in response to changes in cell density, involves pathways that synthesize signaling molecules (auto-inducers). The luxS/AI-2-mediated QS system has been identified in both gram-positive and gram-negative bacteria. Bacillus anthracis, the etiological agent of anthrax, possesses genes involved in luxS/AI-2-mediated QS, and deletion of luxS in B. anthracis Sterne strain 34F2 results in inhibition of AI-2 synthesis and a growth defect. In the present study, we created a ΔluxS B. anthracis strain complemented in trans by insertion of a cassette, including luxS and a gene encoding erythromycin resistance, into the truncated plcR regulator locus. The complemented ΔluxS strain has restored AI-2 synthesis and wild-type growth. A B. anthracis microarray study revealed consistent differential gene expression between the wild-type and ΔluxS strain, including downregulation of the B. anthracis S-layer protein gene EA1 and pXO1 virulence genes. These data indicate that B. anthracis may use luxS/AI-2-mediated QS to regulate growth, density-dependent gene expression and virulence factor expression.
Topics: Bacillus anthracis; Bacterial Proteins; Carbon-Sulfur Lyases; Gene Expression Regulation, Bacterial; Homoserine; Lactones; Membrane Glycoproteins; Molecular Sequence Data; Quorum Sensing; Virulence Factors
PubMed: 21178420
DOI: 10.4161/viru.1.2.10752