Conjugation in bacilli

The review considers experimental data on the conjugal transfer of plasmids in the Bacillus cereus and Bacillus subtilis groups (the transfer of large self-transmissible plasmids and the mobilization of small plasmids). Conjugation in bacilli is compared with conjugation in E. coli dependent on the F factor. Conjugation of bacilli in their natural habitats is also discussed.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.     

Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

Self-transfer and mobilisation capabilities of the pXO2-like plasmid pBT9727 from Bacillus thuringiensis subsp. konkukian 97-27

Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp. kurstaki HD73 and the heretofore cryptic plasmid pBT9727 from the clinical strain B. thuringiensis subsp. konkukian 97-27. Comparative sequence analysis of these three plasmids suggested that they were derived from an ancestral conjugative plasmid, with pAW63 retaining its self-transfer capabilities, and pXO2 having lost them through genetic drift. Such properties had not been investigated in pBT9727, but sequence homologies led us to predict that it may possess self-transfer capabilities. Here, we report that pBT9727 is indeed conjugative, and is able to promote its own transfer as well as that of small mobilisable plasmids.     

Isolation and expression of a Bacillus cereus gene encoding benzil reductase.

Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01. To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes. The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin. Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase. The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase.     

Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

To facilitate the analysis of genetic determinants carried by large resident plasmids of Bacillus anthracis, a mating system was developed which promotes plasmid transfer among strains of B. anthracis, B. cereus, and B. thuringiensis. Transfer of the selectable tetracycline resistance plasmid pBC16 and other plasmids from B. thuringiensis to B. anthracis and B. cereus recipients occurred during mixed incubation in broth. Two plasmids, pXO11 and pXO12, found in B. thuringiensis were responsible for plasmid mobilization. B. anthracis and B. cereus transcipients inheriting either pXO11 or pXO12 were, in turn, effective donors. Transcipients harboring pXO12 were more efficient donors than those harboring pXO11; transfer frequencies ranged from 10(-4) to 10(-1) and from 10(-8) to 10(-5), respectively. Cell-to-cell contact was necessary for plasmid transfer, and the addition of DNase had no effect. The high frequencies of transfer, along with the fact that cell-free filtrates of donor cultures were ineffective, suggested that transfer was not phage mediated. B. anthracis and B. cereus transcipients which inherited pXO12 also acquired the ability to produce parasporal crystals (Cry+) resembling those produced by B. thuringiensis, indicating that pXO12 carries a gene(s) involved in crystal formation. Transcipients which inherited pXO11 were Cry-. This mating system provides an efficient method for interspecies transfer of a large range of Bacillus plasmids by a conjugation-like process.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.     

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.     

Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

Insights into the genetic organization of the Bacillus mycoides cryptic plasmids pDx14.2 and pSin9.7 deduced from their complete nucleotide sequence

Bacillus mycoides, a member of the Bacillus cereus group of bacteria, can be easily distinguished from close species because of colony shape, made by filaments of cells, resembling fungal hyphae, curving clock- or counterclockwise depending on the strain. Two plasmids, one from a strain curving to the right (pDx14.2), the other from a strain curving to the left (pSin9.7), were sequenced and analyzed for gene content and replication mode. Rolling-circle replication modules and mobilization proteins were found, very similar to those of other plasmids of the B. cereus group bacilli, mostly Bacillus thuringiensis living in the same ecosystem, suggesting active plasmid exchange in nature.     

Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis.

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.     

Bacillus anthracis pXO1 Plasmid Sequence Conservation among Closely Related Bacterial Species

The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.     

蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C(Phospholipase C,PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B.cereus基因组DNA为模板,PCR扩增得到磷脂酶C基因(bcplc),构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中,实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C(rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C,纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg,最适反应温度为80°C,最适pH为9.0。在低于40°C时,pH 7.0-8.0时,rbcPLC重组酶较稳定。Cu~(2+)和Co~(2+)对其有明显的抑制作用;Zn~(2+)、Mn~(2+)、Ca~(2+)、Mg~(2+)对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析,为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。     

蜡状芽孢杆菌群中规律成簇间隔短回文重复序列的生物信息学分析

规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)是最近发现针对噬菌体等外源遗传物质的获得性和可遗传性的新型原核生物防御系统。通过BLAST、多序列比对、RNA二级结构预测等生物信息学方法对已经完成全基因组测序的蜡状芽孢杆菌群24个菌株进行CRISPR的系统分析,结果表明:42%的菌株含有该结构;8个CRISPR座位的正向重复序列可以形成RNA二级结构,提示正向重复序列可能介导外源DNA或RNA与CAS编码蛋白的相互作用;31%的间区序列与噬菌体、质粒、蜡状芽孢杆菌群基因组序列具有同源性,进一步验证间区序列很可能来源于外源可移动遗传因子。由于大部分蜡状芽孢杆菌群菌株含有多个前噬菌体和质粒,通过对蜡状芽孢杆菌群CRISPR的分析,为揭示其对宿主菌与噬菌体,以及宿主菌与质粒间的关系奠定基础。     

The bcr1 DNA repeat element is specific to the Bacillus cereus group and exhibits mobile element characteristics

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor an approximately 155-bp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcr1 repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TTTAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis.

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.     

Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease

Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans; however, the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homologue of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbours bpsX-H, B. cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in Bacillus anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-d-γ-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease.     

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.