Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria.

Abstract

Evolutionary conservation of an interspersed repetitive DNA sequence, BOX, from Streptococcus pneumoniae was investigated to explore the mosaic nature of these elements. BOX elements consist of various combinations of three subunits, boxA, boxB, and boxC. Eight oligonucleotide probes were designed based on consensus DNA sequences of boxA, boxB, and boxC subunits. DNA hybridization studies and PCR using these probes/primers demonstrate that oligonucleotide sequences within the boxA subunit appear to be conserved among diverse bacterial species. 1A). (7) For example, the boxB subunit was present alone as a single copy or as a variable n u m b e r of direct tandem repeats flanked by boxA and boxC. (7~ The DNA sequences of the BOX elements are entirely different from the prokaryotic interspersed repetitive DNA sequences REP and ERIC, although there are similarities to REP and ERIC with respect to size, copy number, and potential to form stable stem-loop structures. ~2-7~ Although the exact functions of interspersed repetitive DNA elements are unknown, the presence of these repetitive DNA sequences can be utilized for rapid physical mapping procedures (8~ and for DNA fingerprinting of prokaryotic genomes. (4'9-2a~ REP and ERIC sequences were used to design primers for PCR, in a technique known as repetitive sequence-based PCR (rep-PCR), to obtain DNA fingerprints from various microorganisms. Interspersed repetitive sequences can serve as primer binding sites that are separated by various distances in the bacterial chromosome. PCR of unique sequence located between interspersed repeats results in differently sized DNA amplification products. PCR products of different sizes constitute polymorphic DNA markers and yield DNA fingerprints that m a y be specific for individual bacterial strains or isolates. The apparent evolutionary conservation of these repetitive elements enables the use of a limited primer repertoire for DNA fingerprinting of a wide array of bacteria.

A new serotype 14 variant of the pneumococcal Spain9V-3 international clone detected in the central region of Argentina

“…The BOX elements were amplified from genomic DNA using the primer BOXA1R (Koeuth et al, 1995). The reaction conditions were 30 cycles of denaturation at 95 uC for 1 min, annealing at 52 uC for 2 min and elongation at 72 uC for 2 min.…”

Diversity and biofilm-forming capability of bacteria recovered from stainless steel pipes of a milk-processing dairy plant

“…The intraspecies genetic diversity of the isolates was assessed by independent PCR fingerprinting with primers BOXA2R (5′-ACGTGGTTTGAAGAGATTTTCG-3′), as reported by Koeuth et al (1995), and M13 (5′-GAGGGTGGCGGTTCT-3′), as described by Rossetti and Giraffa (2005). PCR reaction mixtures contained 5 μL of each DNA extract or purified genomic DNA, 25 μL of Taq master Mix (Ampliqon), 5 μL of primer (10 μmol L −1 ) and 15 μL of molecular-biology-grade water in a total volume of 50 μL.…”

PM 7/110 (1) Xanthomonas spp. (Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas vesicatoria) causing bacterial spot of tomato and sweet pepper

“…The comparison of genomic DNA between isolates and type strains by means of rep-PCR fingerprinting (Stern et al, 1984;Sharples & Lloyd, 1990;Louws et al, 1994;Koeuth et al, 1995), can be a useful identification test (see EPPO Standard PM 7/100(1) Rep-PCR tests for identification of bacteria (EPPO, 2010)).…”