rpoB -Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity

Abstract

Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.

Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov.

“…Previous studies have suggested that microbial community analysis with a low-copy-number gene may be more accurate than analysis with high-copy-number genes, such as the 16S rRNA gene (Dahllof et al, 2000;Peixoto et al, 2002). Preliminary analysis of the Geobacter sulfurreducens and Geobacter metallireducens genomes indicated that several of the conserved genes used in this study occur as single-copy or low-copy-number genes in the Geobacteraceae.…”

Genetic diversity of total, active and culturable marine bacteria in coastal seawater

“…Despite the heterogeneity of 16S rDNA reported previously for other marine isolates (Dahllof et al 2000), all the DILs strains gave a single band on DGGE gel (Fig. 4).…”

“…A total of 7 different strains was found and referenced DILs-1 to DILs-7 (Table 2). Some of the strains were not culturable onto R2Am in primary culture but all were able to grow on this medium in secondary culture (Table 2).Despite the heterogeneity of 16S rDNA reported previously for other marine isolates (Dahllof et al 2000), all the DILs strains gave a single band on DGGE gel (Fig. 4).…”

16S rRNA gene-based identification of bacteria in postoperative endophthalmitis by PCR- Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprinting

“…(6,24). This result can be explained by the intraspecies heterogeneity displayed in a DGGE banding pattern as the result of multiple copies of the ribosomal genes and by the fact that the gene copies have evolved differently (6,24).…”

“…(6,24). This result can be explained by the intraspecies heterogeneity displayed in a DGGE banding pattern as the result of multiple copies of the ribosomal genes and by the fact that the gene copies have evolved differently (6,24). In order to provide a solution to that issue, some authors had successfully used the rpoB gene, encoding the beta subunit of RNA polymerase which exists in a single copy within the chromosome (6,19).…”