A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis

Abstract

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5' end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR-single-stranded DNA approach for microbial community analysis.

FIG. 1

Analysis of PCR-amplified 16S rRNA genes by SSCP on 0.6× MDE polyacrylamide gels, comparing band positions obtained from nondenatured PCR products (lanes 2, 5, and 8), denatured PCR products (lanes 3, 6, and 9), and PCR products after removal of one single strand (lanes 4, 7, and 10). Template DNA for PCRs was obtained from pure cultures of B. subtilis (lanes 2 to 4), S. meliloti (lanes 5 to 7), and P. fluorescens (lanes 8 to 10). Size standard VI (double-stranded DNA; Boehringer) is shown in lane 1.

FIG. 2

SSCP patterns obtained from different bacterial species and model communities after PCR amplification of the 16S rRNA genes and removal of one strand of the double-stranded PCR product. The following species were analyzed: B. subtilis (lane 1), C. glutamicum (lane 2), P. denitrificans (lane 3), R. leguminosarum subsp. trifolii (lane 4), S. meliloti (lane 5), G. terrae (lane 6), A. radiobacter (lane 7), A. beijerinckii (lane 8), P. fluorescens (lane 9), P. stutzeri (lane 10), and E. coli (lane 11). Lanes 12 to 14, PCR products obtained from mixed template DNA with strains shown in lanes 1 to 5 (lane 12), lanes 6 to 10 (lane 13), and lanes 1 to 10 (lane 14).

FIG. 3

Comparative SSCP analysis of PCR-amplified 16S rRNA genes with DNA obtained from pure cultures and from DNA single strands, extracted after SSCP separation from polyacrylamide gels and reamplified by using a second PCR (the original gels from which single-stranded DNA molecules were extracted are not shown). To analyze the regeneration of both DNA strands by reamplification, pure-culture PCR products of both single strands are shown. PCR products from template DNA obtained from S. meliloti (reamplified [lane 1] and from pure culture [lane 2]), C. glutamicum (lane 3, reamplified; lane 4, pure culture), B. subtilis (lane 5, reamplified; lane 6, pure culture), P. fluorescens (lane 7, reamplified; lane 8, pure culture), P. stutzeri (lane 9, reamplified; lane 10, pure culture), P. fluorescens (lane 11, reamplified from a mixed community with 10 different species; lane 12, pure culture), and P. stutzeri (lane 13, reamplified from the mixed community indicated for lane 11; lane 14, pure culture) are shown. The arrows in lanes 2, 4, 6, 8, 10, 12, and 14 indicate the respective DNA single strands which were used as templates for reamplifications.

FIG. 4

SSCP patterns obtained with single-stranded PCR products of 16S rRNA genes amplified from rhizosphere-extracted bacterial communities. Template community DNA was obtained from M. sativa (location 1, lanes 2 to 4; location 2, lanes 5 to 7) and C. album (location 1, lanes 8 to 10; location 2, lanes 11 to 13). Initial template amounts were 10 ng (lanes 2, 5, 8, and 11), 2 ng (3, 6, 9, and 12), and 0.2 ng (4, 7, 10, and 13). Lane 1, single-stranded products obtained from P. fluorescens (P.f.), P. stutzeri (P.s.), and A. radiobacter (A.r.) as standards.