Identification of the Bacillus anthracis (gamma) phage receptor

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. It belongs to the Bacillus cereus group, which also contains Bacillus cereus and Bacillus thuringiensis. Most B. anthracis strains are sensitive to phage gamma, but most B. cereus and B. thuringiensis strains are resistant to the lytic action of phage gamma. Here, we report the identification of a protein involved in the bacterial receptor for the gamma phage, which we term GamR (Gamma phage receptor). It is an LPXTG protein (BA3367, BAS3121) and is anchored by the sortase A. A B. anthracis sortase A mutant is not as sensitive as the parental strain nor as the sortase B and sortase C mutants, whereas the GamR mutant is resistant to the lytic action of the phage. Electron microscopy reveals the binding of the phage to the surface of the parental strain and its absence from the GamR mutant. Spontaneous B. anthracis mutants resistant to the phage harbor mutations in the gene encoding the GamR protein. A B. cereus strain that is sensitive to the phage possesses a protein similar (89% identity) to GamR. B. thuringiensis 97-27, a strain which, by sequence analysis, is predicted to harbor a GamR-like protein, is resistant to the phage but nevertheless displays phage binding.     

The infection of bacilli by Mu cts phage integrated into the plasmid

The infection of Bacillus thuringiensis, B. cereus, B. mesentericus and B. polymyxa strains with temperate E. coli bacteriophage Mu cts62 integrated into plasmid RP4 under conditions of conjugative transfer is shown possible. The investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. Gel electrophoresis and blot hybridization of DNA have confirmed the transfer of Mu cts62 genome or a part of it in the investigated strains of bacilli.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group (B. cereus, B. mycoides and B. thuringiensis) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10 degrees C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group ( B. cereus, B. mycoides and B. thuringiensis ) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10°C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure

Bacillus anthracis , Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus , in some B. thuringiensis strains and in B. anthracis , an S-layer has been described. We investigated how the S-layer is distributed in B. cereus , and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non- B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis . Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis , except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.     

Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.     

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.     

Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus

Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.     

Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation

During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus were isolated from four individuals (including one with Norwalk virus) and spice. Phage typing confirmed all Bacillus clinical isolates were phage type 2. All clinical isolates were subsequently identified as B. thuringiensis when tested as a result of a related study (L. Leroux, personal communication). Eight of 10 spice isolates were phage type 4. All B. cereus and B. thuringiensis isolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus . An additional 20 isolates each of B. cereus and B. thuringiensis from other sources were tested for cytotoxicity. With the exception of one B. cereus , all showed characteristic cytotoxic patterns.     

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.     

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

蜡状芽胞杆菌群菌株中部分病原相关因子的检测

对26株蜡状芽胞杆菌群菌株进行了肠毒素基因及其它病原相关因子的检测。PCR结果表明,17株蜡状芽胞杆菌群菌株中含有病原调控因子plcR的同源序列。采用3组溶血肠毒素hbl基因和3组非溶血肠毒素nhe基因特异性引物,分别可从73%的菌株中至少扩增出一个与预期DNA片段大小一致的片段,其中,苏云金芽胞杆菌菌株中溶血素hbl基因和非溶血素nhe基因的阳性检出率为83%。蜡状芽胞杆菌DBt248完全没有溶血活性,而且在溶血素hbl和非溶血素nhe基因的3个亚基以及病原调控因子plcR的PCR检测中均为阴性,有望作为宿主菌用于苏云金芽胞杆菌晶体蛋白的表达和应用。     

A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis

Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.     

Transduction in Bacillus thuringiensis.

Bacteriophage CP-51, originally reported as a generalized transducing phage for Bacillus cereus and B. anthracis, has been shown to carry out generalized transduction in several strains of B. thuringiensis. A newly isolated phage, CP-54, which has a broader host range than CP-51, also mediates generalized transduction in B. thuringiensis. CP-51 and CP-54 are similar in size and morphology and are related serologically, but they are not identical. CP-54 is more cold labile than CP-51, and, as with CP-51, its stability both at 0 and 15 degrees C is enhanced by the presence of 0.02 M Mg2+. Some examples of cotransduction of linked markers in B. thuringiensis are presented, demonstrating the feasibility of chromosomal mapping in this organism. The rare occurrence of cross-transduction among strains of B. thuringiensis is probably a reflection of nonhomology rather than restriction, since phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicator.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.