PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis

Abstract

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.

Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart

“…Among the molecular techniques for the soil bacterial community analysis AR-ISA proved to be very effective. The ARISA (Fisher and Triplett, 1999) is a PCR-based fingerprinting approach targeting the 16S-23S intergenic transcribed spacers a region considered hypervariable respect to the adjacent genes and hence useful to discriminate bacteria at the subspecies level (Daffonchio et al, 1998(Daffonchio et al, , 2000. The advantage of ARISA respect to the traditional analysis of fingerprinting patterns in agarose/polyacrylamide gels is the separation of labelled fragments in an automated sequencer which guarantees high sensitivity also for poorly amplified fragments and high resolution even of hundreds of peaks per profile (Ranjard et al, 2001).…”

“…For the ARISA experiments (Fisher and Triplett, 1999), the intergenic transcribed spacers (ITS) between 16S and 23S rDNA were amplified using primers S-D-Bact-1494-a-S-20 and L-D-Bact-0035-a-A-15 as previously described (Daffonchio et al, 1998), except that primer S-D-Bact-1494-a-S-20 was 5 end labelled with the phosphoramidite dye 5-FAM. Aliquots of the PCR products (1 to 5 µL) were mixed with 1 µL of the GeneScan-1000 [ROX] internal size standard (Applied Biosystems), 20 µL of deionized formamide was added, the mixture was denatured at 95 • C for 5 min and cooled in an ice bath.…”

A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species

“…); and (iii) direct electrophoresis of PCR products. In the latter group, some techniques are based on ribosomal DNA spacers [1][2][3][4] and the results obtained have shown that, for most species, the PCR patterns are specific. Also, the existence of repetitive sequences in the bacterial genome has been widely used for typing bacteria, obtaining PCR patterns by direct electrophoresis of the PCR products generated.…”

Biological characterization of two Bacillus thuringiensis strains toxic against Spodoptera frugiperda

“…Additional PCR was carried out with specific primers in order to identify the presence of cry1Aa, cry1Ab, cry1Ac, cry1Ad, cry2Aa, cry2Ab and cry2Ac genes (Ben-Dov et al 1997;Bravo et al 1998). ITS-PCR was performed as previously described by Daffonchio et al (1998).…”