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Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified (45) and is widely used for the biological control of insects in crop protection; B. mycoides has been recognized as a plant growth-promoting bacterium associated with conifer roots (38); B. weihenstephanensis, a psychrotolerant species frequently found in pasteurized milk, is a potential cause of spoilage problems (32). The six species are not easily distinguished on the basis of phenotypic or genetic traits (48). Recently, B. anthracis, B. cereus, and B. thuringiensis were found to be very closely related, and it has been proposed that they belong to a single species (24,33). This proposal has been based on multilocus enzyme electrophoresis data and sequencing of discrete genetic loci (24) and on the presence of an S-layer on the cell surface (33). The model considering B. anthracis, B. cereus, and B. thuringiensis as subspecies of a phylogenetically monomorphic group, differing mainly in characters linked to mobile genetic elements such as plasmids, is supported by very high sequence homology in the conserved molecular chronometers of the ribosomal operons, the 16S and 23S ribosomal DNA (rDNA), and the short intergenic spacer between them (3-5, 7, 21, 30). Considering the dangerousness of B. anthracis and the wide in-field application of B. thuringiensis as a biological insecticide, it would be opportune to further evaluate the phylogenetic relationship between the different clades of the B. cereus group. Whole-genome sequencebased analysis could give a definitive view of the genetic relationship between these species (29). However, an approach that is economically feasible, given current technology, is possible for few strains in a given species (42). Hence, for phylogenetic surveys based on a relatively large number of isolates of each species, permitting an assessment of the amount of variability and overlap within a species, the best means of approach remains the use of highly conserved molecules with no, or a low, horizontal gene transfer rate such as the ribosomal operon.In the prokaryote genome, the ribosomal operon can be present in multiple copies, up to 15 copies in Clostridium paradoxum (41). The 16S-23S rDNA intergenic transcribed spacers (ITS) are the most variable regions of the ribosomal operon, and, apart from interoperonic nucleotide substitutions, insertions, and deletions, such ITS can be differentiated, given the presence of the different numbers and types of tRNA genes (9,10,31,49). Since the ITS have fewer functional constraints than the adjacent ribosomal genes, which undergo concerted evolution (17-19), their sequences can contain traces of ribosomal operon rearrangements and species-specific or even strain-specific traits that are useful for strain typing.An analysis of ITS homoduplex-heteroduplex polymorphisms has shown that wide variability exists in the strains of the six species of the B. cereus group (14), indicating widely different length and sequence polymorphisms among the 8 t...

The antifoulant, zosteric acid, was synthesized using a non-patented process. Zosteric acid at 500 mg l(-1) caused a reduction of bacterial (Escherichia coli, Bacillus cereus) and fungal (Aspergillus niger, Penicillium citrinum) coverage by 90% and 57%, respectively. Calculated models allowed its antifouling activity to be predicted at different concentrations. Zosteric acid counteracted the effects of some colonization-promoting factors. Bacterial and fungal wettability was not affected, but the agent increased bacterial motility by 40%. A capillary accumulation test showed that zosteric acid did not act as a chemoeffector for E. coli, but stimulated a chemotactic response. Along with enhanced swimming migration of E. coli in the presence of zosteric acid, staining showed an increased production of flagella. Reverse transcriptase-PCR revealed an increased transcriptional level of the fliC gene and isolation and quantification of flagellar proteins demonstrated a higher flagellin amount. Biofilm experiments confirmed that zosteric acid caused a significant decrease in biomass (-92%) and thickness (-54%).

-This study aimed to evaluate the plant growth promoting (PGP) potential of Bacillus thuringiensis. In this context, several genetic determinants of factors implicated in PGP potential were investigated by polymerase chain reaction (PCR) in 16 B. thuringiensis strains of different origin and belonging to different subspecies. PCR screening was performed on acid phosphatase, phytase, siderophore biosynthesis protein, 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indolpyruvate decarboxylase (ipdC). Production of indol acetic acid (IAA)-like compounds and of ACC deaminase, and capability of solubilising mineral phosphate were investigated by phenotypic tests. All the strains were PCR positive for the presence of the siderophore biosynthesis protein, ACC deaminase and acid phosphatase genes. Five and seven strains gave an amplicon with the expected length for the phytase and ipdC genes respectively. All the strains produced IAA compounds and seven had a high capacity to solubilise inorganic phosphorous. Qualitative phenotypic test for ACC deaminase activity showed that seven strains are able to grow on salt minimal medium containing ACC as sole nitrogen source, indicating the expression of the accd genes. Our screening results in thirteen strains having more than one PGP trait and showed that B. thuringiensis harbours and expresses several PGP determinants that could be very interesting in field application to enhance the plant growth. To our knowledge, this is the first report on the multiple plant growth promoting potential of B. thuringiensis.

Genomic diversity in 21 strains of Bacillus cereus and 10 strains of Bacillus licheniformis was investigated by random amplified polymorphic DNA (RAPD) analysis, which samples the whole genome, and by two PCR fingerprinting techniques sampling the hypervariable spacers between the conserved 165 and 235 rRNA genes of the rRNA gene operon (ITS-PCR) and regions between tRNA genes (tDNA-PCR). RAPD analysis showed a remarkable diversity among strains of B. cereus that was not observed with the rRNA and tRNA intergenicspacer-targeted PCR, where all the strains showed practically identical fingerprints. A wide variability among the B. cereus strains was also observed in the plasmid profiles, suggesting that the genetic diversity within B. cereus species can arise from plasmid transfer. One contribution to the diversity detected by RAPD analysis was determined by the presence of large extrachromosomal elements that were amplified during RAPD analysis as shown by Southern hybridization experiments. In contrast to the strains of B. cereus, the 10 strains of B. licheniformis were grouped into two clusters which were the same with all the methods employed. The 165 rRNA genes were identical in all 10 strains when examined using single strand conformation polymorphism analysis after digestion with A M and Rsal. From these data w e hypothesize two different evolutionary schemes for the two species.