Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera

Abstract

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.

FIG. 1.

Schematic diagram showing how heteroduplex cross-hybridization products may be formed during PCR and how these products may be eliminated from the pattern by mung bean nuclease treatment. Steps 1 to 3 show the procedure used to facilitate cloning of long ITS containing tRNA genes from B. anthracis strain 282. The two lanes in the silver-stained MDE gel show the ITS-HHP profile of strain 282 (left lane) and the same products after mung bean nuclease treatment (right lane). When a standard procedure consisting of excision of bands from the ITS-HHP profile and a PCR to generate a sufficient amount of DNA for cloning was used, short ITS without tRNA genes were always coamplified, which led to their preferential cloning. Band excision from a mung bean nuclease-treated profile completely eliminated heteroduplex forms, which facilitated exclusive recovery of the long ITS by PCR and successive cloning of this ITS.

FIG. 2.

ITS-PCR patterns of B. anthracis strains resolved by polyacrylamide gel electrophoresis (A) and by Southern hybridization with the short ITS cloned from B. anthracis strain Cepanzo as the probe (B). Lane M contained a 50-bp ladder. Lanes 1 to 6, B. anthracis strains 300, 376, 779, 832, 170, and 663, respectively. The position of the 250-bp band of the ladder is indicated on the left.

FIG. 3.

ITS-PCR electrophoretic profiles of B. cereus group strains obtained under denaturing conditions. (A) Denaturing 6% polyacrylamide gels (denaturation with 40% formamide and 7 M urea). Lanes M contained a 50-bp ladder. Lanes 1 to 18, B. anthracis strains 282, 582, 846, 376, 663, 832, 779, 300, 170, 227, 957, Cepanzo, 6602, 4229, 7702, Davis TE702, 7700, and ANTmi2522, respectively; lanes 19 to 22, B. mycoides strains G2, DSMZ 2048^T, DSMZ 303, and NOV1, respectively; lane 23, B. pseudomycoides strain NRRL 617^T; lane 24, B. weihenestephanensis strain 10204^T; lanes 25 to 27, B. cereus strains DSMZ 31^T, DSMZ 626, and DSMZ 6127, respectively; lanes 28 to 32, B. thuringiensis strains DSMZ 2046^T, HD1, BMG1.7, and BMG1.1, respectively. The solid arrows on the gel indicate the short and long ITS homoduplex PCR products, while the open arrow indicates homoduplex fragments of intermediate-length ITS present in strains Davis TE702 and G2. The positions of the 250- and 450-bp bands of the ladder are indicated on the left and on the right. (B) Electropherogram showing the ARISA profile of strain B. cereus DSMZ 31^T. The dotted arrows connect the short and long ITS peaks of the ARISA profile with the corresponding ITS homoduplex products in the agarose gel in panel C. (C) Agarose gel electrophoresis of strain B. cereus DSMZ 31^T (lanes 1 and 2). The open arrowhead indicates the position of the heteroduplex products. Lane L contained a 100-bp ladder. The positions of the 200-, 400-, 600-, and 800-bp bands of the ladder are indicated on the right.

FIG. 4.

Silver-stained MDE gel showing ITS heteroduplex reconstruction by PCR amplification of the mixture of short and long tDNA-containing ITS clones of B. anthracis strain 282. Lanes M contained a 50-bp ladder. Lanes 1 and 5, ITS-PCR profile of strain B. anthracis 282; lane 2, PCR product of the short ITS obtained by nested PCR from clone ITS282.1; lane 4, PCR product of the long tRNA-containing ITS obtained by nested PCR from clone ITS282.6; lane 3, heteroduplex reconstruction obtained by PCR amplification of mixed clones ITS282.1 and ITS282.6. The open arrows indicate the positions of the heteroduplex products The positions of the 250- and 500-bp bands of the ladder are indicated on the left and on the right.

FIG. 5.

Effect of the number of cycles of the PCR on the formation of heteroduplex products. Lanes 1 to 4 show the ITS-PCR patterns obtained after 25, 30, 35, and 40 cycles, respectively. The strains used were Bacillus cereus DSMZ 31^T (B.ce. 31^T), Bacillus subtilis 8633 (B.su. 8633), Bacillus megaterium DSMZ 32^T (B.me. 32^T), Bacillus coagulans DSMZ 1^T (B.co. 1^T), Geobacillus stearothermophilus DSMZ 22^T (G.st. 22^T), and Bacillus sphaericus DSMZ 461 (B.sp. 461). (A) Negative images of agarose gels stained with ethidium bromide. (B) Silver-stained MDE gel.

FIG. 6.

ITS-HHP pattern variability observed in the collection examined. Lanes M contained a 50-bp ladder. Lanes 1 to 6, Bacillus maroccanus CCM 671, Bacillus pacificus CCM 689, Bacillus sphaericus DSMZ 1087, Bacillus sphaericus DSMZ 461, Bacillus smithii DSMZ 4216, and Bacillus amyloliquefaciens DSMZ 7^T, respectively; lanes 7 to 13, Bacillus licheniformis strains 3.2, 283, 75.2, 17.1, 6.1, ATCC 14580^T, and DSMZ 13^T, respectively; lanes 14 to 16, Bacillus subtilis strains 357, 42, and 8633, respectively; lanes 17 and 18, Bacillus anthracis strains Davis TE702 and 7700, respectively; lane 19, Geobacillus thermocatenulatus DSMZ 730; lanes 20 to 24, Geobacillus stearothermophilus strains ATCC 12016, ATCC 21365, ATCC 29609, ATCC 12980, and DSMZ 22^T, respectively; lanes 25 to 36, Geobacillus thermodenitrificans DSMZ 465, Bacillus caldovelox DSMZ 411, Bacillus flavothermus DSMZ 2641, Geobacillus thermoleovorans ATCC 43513^T, Bacillus caldolyticus DSMZ 405, Bacillus caldotenax DSMZ 406, Bacillus sp. strain OI170, Bacillus sp. strain 1459, Paenibacillus polymyxa DSMZ 36^T, Bacillus coagulans DSMZ 1^T, Bacillus megaterium DSMZ 32^T, and Brevibacillus brevis DSMZ 30^T, respectively. The positions of the 250- and 500-bp bands of the ladder are indicated on the left and on the right.