Reassessment of Sequence-Based Targets for Identification of Bacillus Species

Abstract

The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B. cereus and B. anthracis as well as nonpathogenic B. psychrotolerans and B. psychrodurans. Consequently, 27 type and clinical strains from the B. cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification. The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B. cereus and B. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/.The Bacillus genus is an extensive heterogeneous group encompassing 83 validly described species to date (http://www .bacterio.cict.fr/b/bacillus.html). Many species in this taxon are of major clinical importance, such as the B. cereus group (comprised of B. cereus, B. anthracis, B. thuringiensis, B. mycoides, and B. weihenstephanensis), but unfortunately, members of this group share a great deal of morphological and biochemical similarities (3,8,16). In contrast, the environmental and nonpathogenic species of this genus exhibit a wide range of physiology, DNA base content, and nutritional requirements (2,4,15). Since the biochemical approach for species identification can be tedious, expensive, and inaccurate, a rapid, definitive method is greatly needed. Molecular procedures are increasingly being used for rapid species identification. However, some methods used for this genus such as restriction digests of a target gene (i.e., 16S rRNA gene) (11) or randomly amplified polymorphic DNA analysis (22) are limiting in discriminating between a large group of species (6). Sequencing of the 16S rRNA gene and select housekeeping genes has shown to be particularly useful, generating large public sequence databases due to the tangible, exact nature of sequence data. With the increasing use of these methods and decreased expense of running sequencing reactions after the initial equipment investment, more laboratories are relying on sequence data for species identification (21).A previous study using the 16S rRNA gene for rapid identification of the Bacillus genus was undertaken by Goto et al. (6). At this time, the validity of using a hypervariable region (nucleotides [nt] 70 to 344) of the gene was proven adequate to discrimina...

Isolation and Molecular Characterization of Chitinase-Deficient Bacillus licheniformis Strains Capable of Deproteinization of Shrimp Shell Waste To Obtain Highly Viscous Chitin

“…Physiological tests (API) (14) and 16S rRNA gene comparison (9), as well as microscopic and macroscopic investigations, revealed only faint differences between the bacterial isolates F5 and F11. It was suggested that 16S rRNA gene sequence comparisons should be performed only in conjunction with a number of phenotypic and phylogenetic properties because two distinct species may exist with identical 16S rRNA gene sequences (2,20,27). To inspect whether the different extracellular enzymatic activities are due to nucleotide or amino acid exchanges, a number of loci encoding degradative extracellular enzymes, as well as an operon influencing secretion (degS-degU), were analyzed in addition to the pga locus.…”

“…Pasteurized (15 min 80°C) dilutions (in 0.9% NaCl) of the culture were spread onto shrimp protein agar plates and incubated again as described above. Shrimp protein medium consisted of 5 g of yeast extract, 5 g of KH 2 PO 4 , 5 of g NaCl, 1 g of Mg 2 SO 4 · 7H 2 O, and 100 g of freshly ground, dried shrimp shell waste, which was washed and separated from insoluble material by filtration and filled up to a total volume of 1 liter; the pH was adjusted to 7.0. Material from single colonies was transferred to calcium-caseinate agar plates (Merck, Darmstadt, Germany) and incubated for 24 h to determine the proteolytic activity.…”

Ability of indigenous Bacillus licheniformis and Bacillus subtilis in microbial enhanced oil recovery

“…The graphs had been drawn by special software recognized as the unknown nucleotides. Eventually, the results were compared with gene bank sequences (Blackwood and Turenne, 2004). Nutrient broth (Merck) was used for preparation of the inoculums.…”

Phylogenetic analysis of Xanthomonas based on partial rpoB gene sequences and species differentiation by PCR-RFLP

“…enterica (Kwon et al, 2001), Legionella pneumophila (Ko et al, 2002(Ko et al, , 2003, Staphylococcus species (Drancourt & Raoult, 2002), non-pathogenic and clinical Bacillus species (Blackwood et al, 2004) In a study of phylogenetic relationships within the genus Pseudomonas, by comparing partial rpoB gene sequences (about 1000 nt), it was found that the phylogenetic resolution of the rpoB tree was approximately three times greater than that of the 16S rRNA gene tree (Ait Tayeb et al, 2005); i.e., the use of the rpoB gene allowed better separation of closely related species in comparison with data from the 16S rRNA gene.…”