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Journal of Applied Microbiology Oct 2022Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its... (Review)
Review
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.
Topics: Bacillus; Bacillus Phages; Bacillus anthracis; Bacillus subtilis; CRISPR-Cas Systems; Gene Editing
PubMed: 35797344
DOI: 10.1111/jam.15704 -
Microbiology Spectrum Jun 2022Bacillus anthracis is a spore-forming bacterium that causes life-threatening infections in animals and humans and has been used as a bioterror agent. Rapid and reliable...
Bacillus anthracis is a spore-forming bacterium that causes life-threatening infections in animals and humans and has been used as a bioterror agent. Rapid and reliable detection and identification of B. anthracis are of primary interest for both medical and biological threat-surveillance purposes. Few chromosomal sequences provide enough polymorphisms to clearly distinguish B. anthracis from closely related species. We analyzed 18 loci of the chromosome of B. anthracis and discovered eight novel single-nucleotide polymorphism (SNP) sites that can be used for the specific identification of B. anthracis. Using these SNP sites, we developed software-named AGILE V1.1 (nthracis enome-based dentification with high-fideity -probe)-for easy, user-friendly identification of B. anthracis from whole-genome sequences. We also developed a recombinase polymerase amplification-Cas12a-based method that uses nucleic acid extracts for the specific, rapid, in-the-field identification of B. anthracis based on these SNPs. Via this method and B. anthracis-specific CRISPR RNAs for the target CR5_2, CR5_1, and Ba813 SNPs, we clearly detected 5 aM genomic DNA. This study provides two simple and reliable methods suitable for use in local hospitals and public health programs for the detection of B. anthracis. Bacillus anthracis is the etiologic agent of anthrax, a fatal disease and a potential biothreat. A specific, accurate, and rapid method is urgently required for the identification of B. anthracis. We demonstrate the potential of using eight novel SNPs for the rapid and accurate detection of B. anthracis via and laboratory-based testing methods. Our findings have important implications for public health responses to disease outbreaks and bioterrorism threats.
Topics: Animals; Anthrax; Bacillus anthracis; Polymerase Chain Reaction; Polymorphism, Single Nucleotide
PubMed: 35575735
DOI: 10.1128/spectrum.02285-21 -
Frontiers in Cellular and Infection... 2021Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate... (Review)
Review
Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved pathway in bacteria that couples transport of sugars with phosphorylation to monitor host carbohydrate availability. A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Mga and AtxA. These paradigm PCVRs undergo phosphorylation, potentially the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from (Mga) and (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.
Topics: Bacillus anthracis; Bacterial Proteins; Gene Expression Regulation, Bacterial; Streptococcus pyogenes; Virulence
PubMed: 34737980
DOI: 10.3389/fcimb.2021.772874 -
Accounts of Chemical Research Dec 2021, present in two forms of vegetative cells and spores, is a pathogen that infects humans through contact with infected animals or contaminated animal products and is...
, present in two forms of vegetative cells and spores, is a pathogen that infects humans through contact with infected animals or contaminated animal products and is also maliciously used in terrorist acts. Therefore, a rapid and sensitive test for is necessary but challenging. The challenge comes from the following aspects: an accurate distinction of from other species due to their high genomic similarity and the horizontal gene transfer between members; direct detection of the spores without damaging them for component extraction to avoid the risk of spore atomization; and the rapid detections of in complex samples, such as soil and suspicious powders, without sample pretreatments and expensive large-scale equipment. Although culturing from samples is the conventional method for the detection of , it is time-consuming and the detection results would not be easy to interpret because many species share similar phenotypic features such as a lack of motility and hemolysis, resistance to gamma phages, and so on. Intensive and extensive effort has been expended to develop reliable detection technologies, among which biosensors exhibit comprehensive advantages in terms of sensitivity, specificity, and portability. Here, we briefly review the research progress, providing highlights of the latest achievements and our own practice and experience. The contents can be summarized in three aspects: the discovery of detection targets, including genes, toxins, and other components; the creation of molecular recognition elements, such as monoclonal antibodies, single-chain antibody fragments, specific peptides, and aptamers; and the design and construction of biosensing systems by the integration of appropriate molecular recognition elements and transducer devices. These sensor devices have their own characteristics and different principles. For example, the surface plasmon resonance biosensor and quartz crystal microbalance biosensor are very sensitive, while the multiplex PCR-on-a-chip can detect multitargets. Biosensors for direct spore detection are highly recommended because they are not only fast but also avoid contamination from aerosol-containing spores. The introduction of nanotechnology has significantly improved the performance of biosensors. Superparamagnetic nanoparticles and phage-displayed gold nanoparticle ligand peptides have made the results of spore detection visible to the naked eye. Because of space constraints, many advanced biosensors for are not described in detail but are cited as references. Although biosensors provide a variety of options for various application scenarios, the challenges have not been fully addressed, which leaves room for the development of more advanced and practical detection means.
Topics: Animals; Bacillus anthracis; Biosensing Techniques; Gold; Humans; Metal Nanoparticles; Quartz Crystal Microbalance Techniques
PubMed: 34846836
DOI: 10.1021/acs.accounts.1c00407 -
Journal of Bacteriology Sep 2022The Bacillus anthracis gene, encoding the protective antigen component of anthrax toxin, is part of a bicistronic operon on pXO1 that codes for its own repressor,...
The Bacillus anthracis gene, encoding the protective antigen component of anthrax toxin, is part of a bicistronic operon on pXO1 that codes for its own repressor, PagR1. In addition to the operon, PagR1 regulates and , two chromosome genes encoding components of the surface layer, a mounting structure for surface proteins involved in virulence. Genomic studies have revealed a PagR1 paralog, PagR2, encoded by a gene on pXO2. The amino acid sequences of the paralogues are 71% identical and show similarity to the ArsR family of transcription regulators. We determined that the expression of either rPagR1 or rPagR2 in a Δ pXO1/pXO2 (PagR1-PagR2) background repressed the expression of , , , and a newly discovered target, encoding virulence activator AtxA. Despite the redundancy in PagR1 and PagR2 function, we determined that purified rPagR1 bound DNA corresponding to the control regions of all four target genes and existed as a dimer in cell lysates, whereas rPagR2 exhibited weak binding to the DNA of the and promoters, did not bind or promoter DNA, and did not appear as a dimer in cell lysates. A single amino acid change in PagR2, S81Y, designed to match the native Y81 of PagR1, allowed for DNA-binding to the and promoters. Moreover, the S81Y mutation allowed for the detection of PagR2 homomultimers in coaffinity purification experiments. Our results expand our knowledge of the roles of the paralogues in B. anthracis gene expression and provide a potential mechanistic basis for differences in the functions of these repressors. The protective antigen component of the anthrax toxin is essential for the delivery of the enzymatic components of the toxin into host target cells. The toxin genes and other virulence genes of B. anthracis are regulated by multiple -acting regulators that respond to a variety of host-related signals. PagR1, one such -acting regulator, connects the regulation of plasmid-encoded and chromosome-encoded virulence genes by controlling both protective antigen and surface layer protein expression. Whether PagR2, a paralog of PagR1, also functions as a -acting regulator was unknown. This work advances our knowledge of the complex model of virulence regulation in B. anthracis and furthers our understanding of the intriguing evolution of this pathogen.
Topics: Amino Acids; Antigens, Bacterial; Bacillus anthracis; Bacterial Proteins; DNA; Gene Expression Regulation, Bacterial; Membrane Proteins; Plasmids
PubMed: 36005808
DOI: 10.1128/jb.00208-22 -
Scientific Reports Jul 2021Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity....
Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity. Despite the occurrence of zoonotic pathogens in wildlife, their prevalence in bushmeat remains unknown. To assess the risk of exposure to major pathogens in bushmeat, a total of 3784 samples, both fresh and processed, were collected from three major regions in Tanzania during both rainy and dry seasons, and were screened by real-time PCR for the presence of DNA signatures of Bacillus anthracis (B. anthracis), Brucella spp. (Brucella) and Coxiella burnetii (Coxiella). The analysis identified DNA signatures of B. anthracis (0.48%), Brucella (0.9%), and Coxiella (0.66%) in a total of 77 samples. Highest prevalence rates of B. anthracis, Brucella, and Coxiella were observed in wildebeest (56%), dik-dik (50%), and impala (24%), respectively. Fresh samples, those collected during the rainy season, and samples from Selous or Serengeti had a greater relative risk of being positive. Microbiome characterization identified Firmicutes and Proteobacteria as the most abundant phyla. The results highlight and define potential risks of exposure to endemic wildlife diseases from bushmeat and the need for future investigations to address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption.
Topics: Animals; Animals, Wild; Bacillus anthracis; Bacterial Zoonoses; Brucella; Coxiella burnetii; DNA, Bacterial; Food Microbiology; Meat; Proteobacteria; Real-Time Polymerase Chain Reaction; Risk; Seasons; Tanzania
PubMed: 34290271
DOI: 10.1038/s41598-021-94112-9 -
Journal of Bacteriology Dec 2022BxpB (also known as ExsFA) and ExsFB are an exosporium basal layer structural protein and a putative interspace protein of Bacillus anthracis that are known to be...
BxpB (also known as ExsFA) and ExsFB are an exosporium basal layer structural protein and a putative interspace protein of Bacillus anthracis that are known to be required for proper incorporation of the BclA collagen-like glycoprotein on the spore surface. Despite extensive similarity of the two proteins, their distribution in the spore is markedly different. We utilized a fluorescent fusion approach to examine features of the two genes that affect spore localization. The timing of expression of the and genes and their distinct N-terminal sequences were both found to be important for proper assembly into the exosporium basal layer. Results of this study provided evidence that the BclA nap glycoprotein is not covalently attached to BxpB protein despite the key role that the latter plays in BclA incorporation. Assembly of the BxpB- and ExsFB-containing outer basal layer appears not to be completely abolished in mutants lacking the ExsY and CotY basal layer structural proteins despite these spores lacking a visible exosporium. The BxpB and, to a lesser extent, the ExsFB proteins, were found to be capable of self-assembly into higher-molecular-weight forms that are stable to boiling in SDS under reducing conditions. The genus consists of spore-forming bacteria. Some species of this genus, especially those that are pathogens of animals or insects, contain an outermost spore layer called the exosporium. The zoonotic pathogen B. anthracis is an example of this group. The exosporium likely contributes to virulence and environmental persistence of these pathogens. This work provides important new insights into the exosporium assembly process and the interplay between BclA and BxpB in this process.
Topics: Animals; Bacillus anthracis; Membrane Glycoproteins; Spores, Bacterial; Bacterial Proteins; Glycoproteins
PubMed: 36394311
DOI: 10.1128/jb.00290-22 -
Viruses Jul 2014Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to... (Review)
Review
Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.
Topics: Bacillus anthracis; Bacillus cereus; Bacillus thuringiensis; Bacteriophages; Chromosomes, Bacterial; Genome, Viral; Lysogeny; Prophages; Transduction, Genetic; Virulence; Virus Integration
PubMed: 25010767
DOI: 10.3390/v6072623 -
Revista Chilena de Infectologia :... Aug 2014
Topics: Bacillus anthracis
PubMed: 25327200
DOI: 10.4067/S0716-10182014000400012 -
Microbiology Spectrum Oct 2015In some Bacillus species, including Bacillus subtilis, the coat is the outermost layer of the spore. In others, such as the Bacillus cereus family, there is an... (Review)
Review
In some Bacillus species, including Bacillus subtilis, the coat is the outermost layer of the spore. In others, such as the Bacillus cereus family, there is an additional layer that envelops the coat, called the exosporium. In the case of Bacillus anthracis, a series of fine hair-like projections, also referred to as a "hairy" nap, extends from the exosporium basal layer. The exact role of the exosporium in B. anthracis, or for any of the Bacillus species possessing this structure, remains unclear. However, it has been assumed that the exosporium would play some role in infection for B. anthracis, because it is the outermost structure of the spore and would make initial contact with host and immune cells during infection. Therefore, the exosporium has been a topic of great interest, and over the past decade much progress has been made to understand its composition, biosynthesis, and potential roles. Several key aspects of this spore structure, however, are still debated and remain undetermined. Although insights have been gained on the interaction of exosporium with the host during infection, the exact role and significance of this complex structure remain to be determined. Furthermore, because the exosporium is a highly antigenic structure, future strategies for the next-generation anthrax vaccine should pursue its inclusion as a component to provide protection against the spore itself during the initial stages of anthrax.
Topics: Bacillus anthracis; Bacterial Proteins; Host-Pathogen Interactions; Microscopy, Electron, Transmission; Spores, Bacterial
PubMed: 26542035
DOI: 10.1128/microbiolspec.TBS-0021-2015