枯草芽孢杆菌近缘种群鉴定方法研究进展

枯草芽孢杆菌近缘种群(简称枯草群)是一群表型相似的革兰氏阳性、产芽孢的杆菌,目前该群已包括10个有效发表种。该群细菌广泛应用于生物技术、工业和农业等领域,因此实现菌株的快速准确鉴别与鉴定,确保其使用的安全性和有效性是首要条件。本文在本研究室进行的枯草群鉴定研究基础上,总结提出了基于形态和生化特性测定的传统经典方法和以分子生物学技术为基础的基因序列分析技术、特异PCR技术,并在实践上进行了大量的验证,对指导该种群的准确鉴定与快速鉴别具有重要应用价值。     

Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species.     

Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences

Partial gyrA sequences were determined for twelve strains belonging to Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, B. mojavensis,B. subtilis subsp. subtilis, B. subtilissubsp. 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.     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

Background

Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.

Results

We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.

Conclusions

Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.     

A PCR test to identify bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment

A PCR test based on the 16S rRNA gene was set up that could identify any of the five species of the 'Bacillus subtilis group' (B. subtilis, B. pumilus, B. atrophaeus, B. lichenijormis and B. amyloliquefaciens). The test was directly applicable to single colonies and showed excellent specificity. In the mixed population context of wastewater analysis, direct detection of the target Bacillus species by PCR on either crude or purified DNA extracts had poor sensitivity. When assayed on cell suspensions derived from enriched wastewater samples, sensitivity was increased. Using a simple calibration method, it was possible to estimate the proportion of the target organisms. This method was found suitable for easy monitoring of a wastewater bioaugmentation experiment carried out with a mixture of sporulated Bacillus strains.     

Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.     

Nucleotide sequence of a cellulase gene of Bacillus subtilis

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.     

Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.     

Nucleotide sequence of the sucrase gene of Bacillus subtilis

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.     

Nucleotide sequence of the Bacillus subtilis developmental gene spoVE

We have determined the nucleotide sequence of a 1159 bp DNA fragment containing the spoVE locus of Bacillus subtilis. The locus contained a single open reading frame of 293 codons. On the basis of the predicted amino acid sequence, the product of the spoVE gene is believed to be a protein with an Mr of 31,539. The amino-terminal portion of the spoVE gene was used to construct a translational fusion with the lacZ' gene. The hybrid spoVE-lacZ' gene was shown to be expressed in Escherichia coli and, therefore, it seems reasonable to conclude that the proposed open reading frame for the spoVE gene does indeed function in vivo.     

Nucleotide sequence of the Bacillus subtilis phoR gene.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

Genealogical inference of closely related species based on microsatellites

Despite their unmatched popularity in many research areas, microsatellites have not yet become a major tool for the inference of genealogical relationships of closely related species. Recent studies have successfully extended the repertoire of microsatellite analysis beyond population genetics and demonstrate that phylogenetic relationships of closely related species can be inferred accurately with fewer loci than previously assumed.