Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.

Comparative secretome analyses of three Bacillus anthracis strains with variant plasmid contents.

Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1(+) pXO2(+)) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1(+) pXO2(-)) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1(-) pXO2(-)). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1(+). This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization.

BACKGROUND: Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis. RESULTS: Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. CONCLUSIONS: Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

Genetic diversity among Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis strains using repetitive element polymorphism-PCR.

Repetitive element polymorphism-PCR (REP-PCR) is one of the tools that has been used to elucidate genetic diversity of related microorganisms. Using the MB1 primer, REP-PCR fingerprints from 110 Bacillus strains within the "B. cereus group" have identified eighteen distinct categories, while other more distantly related bacterial species fell within six additional categories. All Bacillus anthracis strains tested were found to be monomorphic by fluorophore-enhanced REP-PCR (FERP) fingerprinting using the MB1 primer. In contrast, other non- B. anthracis isolates displayed a high degree of polymorphism. Dendrogramic analysis revealed that the non- B. anthracis strains possessing the Ba813 chromosomal marker were divided into two clusters. One of the clusters shared identity with the B. cereus strains examined.

Molecular characterization of Brucella abortus chromosome II recombination.

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.

The genome of Brucella melitensis.

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3,198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other alpha-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.

Rapid genotyping of Bacillus anthracis strains by real-time polymerase chain reaction.

Rapid and accurate identification of Bacillus anthracis is critical for patient care as well as outbreak control. We have developed 3 separate PCR based assays using fluorescence resonance energy transfer (FRET) to detect the presence of pXO1, pXO2 plasmids and a chromosomal marker. A set of amplification primers and probes were used in each assay. The probes were ad jacently placed inside the primer sites and were 1-bp apart. The upstream probe was labeled with fluorescein at the 3' end, and the downstream probe had Cy5 attached at the 5' end. The probes are included in the PCR reactions and hybridize with the PCR products as they are formed. Binding of probes to PCR products results in transfer of energy from fluorescein to Cy5, resulting in emission from Cy5. Increase in fluorescence, indicating amplification, was monitored in real time on a LightCycler((TM)) LC24. Initial denaturation of target sequences was accomplished at 95 degrees C for 1 min, followed by 28 cycles of denaturation at 95 degrees C for 0 sec, annealing at 58 degrees C for 15 sec, and elongation at 72 degrees C for 5 sec. These assays are specific and can be performed on as little as 25 ng of total DNA or crude cell lysate a from fresh colony. It is thus possible to deter mine the genotype of B. anthracis strains in less than 1 hour.

Variation in rRNA operon number as revealed by ribotyping of Bacillus anthracis strains.

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.

The genome sequence of the facultative intracellular pathogen Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.