Distribution and abundance of Gram-positive bacteria in the environment: development of a group-specific probe

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Abstract

We developed a 16S rRNA-targeted oligonucleotide probe (S-P-GPos-1200-a-A-13) for the Gram-positive bacteria, confirmed its specificity by database searches and hybridization studies, and investigated the effects of humic acids on membrane hybridizations with this probe. S-P-GPos-1200-a-A-13 was used to estimate the abundance of Gram-positive populations in the bovine rumen and Lake Michigan sediments. This probe should be useful for studies of the environmental distribution of Gram-positive bacteria and the detection of uncultured, phylogenetically Gram-positive bacteria with variable or negative Gram staining reactions, and could serve for Gram staining in some diagnostic settings.

Introduction

Oligonucleotide probes and primers targeting small-subunit ribosomal RNA (SSU rRNA) and the genes encoding it (rDNA) are the basis of molecular methods in microbial ecology including slot–blot hybridization, fluorescent in situ hybridization (FISH), and various methods based on PCR amplification (reviewed in Jansson and Prosser, 1997, Head et al., 1998, MacGregor, 1999). These methods allow the detection and (within limits) quantification of microorganisms without isolation, and have greatly increased appreciation for the tremendous number of microbial species. Since ribosome abundance is generally correlated with growth rate, rRNA-based methods can also yield information about the relative activity of particular phylogenetic groups.

Identification of the Gram-positive bacteria has traditionally relied on the thick peptidoglycan cell wall found in most members of this group Bone and Balkwill, 1988, Buck, 1982, Doetsch, 1981. However, some species that are Gram-positive by phylogenetic criteria (Woese, 1987) lack typical cell walls and have variable or negative Gram stain reactions (Stackebrandt et al., 1985). Branched-chain fatty acids have been considered as a marker for actinomycetes and Gram-positive bacteria in natural samples (Zelles and Bai, 1994), but branched-chain fatty acids are also produced by several other bacterial groups (Zelles et al., 1995). A probe for the Gram-positive bacteria will allow classification of uncultured organisms that might otherwise be misidentified.

Probes encompassing large groups of bacteria are also useful for constructing nested sets of hybridization probes (Lin et al., 1994), to check for the presence of populations not detected by more specific probes. For example, a set of three SSU rRNA-targeted probes has been designed for the low-G+C Gram-positive bacteria (Meier et al., 1999), but the only probe specific for the high-G+C Gram positives that we are aware of targets the large-subunit ribosomal RNA (Roller et al., 1994). The sequence database of large-subunit sequences is as yet relatively limited, so probe specificity cannot be determined with as much certainty as for the small subunit. The high-G+C Gram positive group might also be estimated indirectly as the difference between total Gram-positive and low-G+C Gram-positive probe hybridization, although it would be more satisfactory to have a complete set of probes for one (or both) types of molecule.

Section snippets

Probe design

S-P-GPos-1200-a-A-13 (5′-AAGGGGCATGATG-3′) complements positions 1200–1212 of Escherichia coli rDNA (Brosius et al., 1978). Of 2289 near-complete Gram-positive SSU rDNA sequences examined, 2120 had perfect matches to the probe; 147 had single mismatches; and only 22 had two or more mismatches to the probe (Fig. 1A). Only 33 of 3649 phylogenetically Gram-negative sequences had perfect matches. These included 12 sequences affiliated with Fusobacterium. Although the fusobacteria are classified as

Optimization of wash temperature

The wash temperature for S-P-GPos-1200-a-A-13 was determined using bacteria with perfect matches (Streptococcus bovis ATCC 33317 and Ruminococcus albus ATCC 27753), a single mismatch (Syntrophobacter wolfeii (Zhao et al., 1989)), or two mismatches (Fibrobacter succinogenes ATCC 19169 (Amann et al., 1992)) to the probe. Total nucleic acids were recovered by phenol–chloroform extraction and ethanol precipitation (Stahl et al., 1988) and their concentrations measured spectrophotometrically. RNA

Probe specificity study

The binding specificity of S-P-GPos-1200-a-A-13 was further tested using membranes blotted with RNA (25 ng/well) from organisms representing all three domains of life and the major recognized bacterial groups. Only Gram-positive bacteria hybridized detectably to the probe (Fig. 3). The two Gram-positive species that did not hybridize, Syn. wolfeii and Mycoplasma neurolyticum (Weisburg et al., 1989), both have single mismatches with the probe sequence. Complete target-region sequences for

Effect of humic acids

To evaluate the effects of humic acids in RNA extracts on membrane hybridizations, RNA extracted from Clostridium innocuum by the low-pH, hot-phenol method (Stahl et al., 1988) was mixed with various amounts of humic acids (Aldrich, Milwaukee, WI cat# 1675-2). The RNA/humic acid samples were denatured with glutaraldehyde, applied to membranes (Magna Charge, Micron Separations, Westboro, MA) at 10 ng RNA/slot, and hybridized as previously described (Raskin et al., 1994). The C. innocuum 16S rRNA

Lake Michigan sediment

S-P-GPos-1200-a-A-13 has also been used in studies of the microbial population of Lake Michigan sediments (MacGregor et al., 1996). S-P-GPos-1200-a-A-13 hybridization relative to S-*-Univ-1390-a-A-18 hybridization generally increases with depth in the sediment below, rising to considerably over 100% (solid lines in Fig. 6). Hybridization to single-mismatch species (Fig. 1B) may account for some of the discrepancy. Hybridization to humic acids may also contribute. To ensure that probe

Conclusion

S-P-GPos-1200-a-A-13 is highly specific both by data base searches and by hybridization with RNA isolated from pure cultures, detecting over 90% of known Gram-positive species with very few false positive results. Since it can detect uncultured bacteria that are phylogenetically Gram-positive but have Gram-negative staining reactions, it may be particularly useful for studies of environments like the rumen, which have a high proportion of such organisms. The human gut microflora, which is as

Nucleotide sequence database deposition

The S-P-GPos-1200-a-A-13 sequence has been submitted to the oligonucleotide probe database (Alm et al., 1996). The Lake Michigan SSU rDNA sequences have been deposited with GenBank, accession numbers A012518–A012534.

Acknowledgements

This work was supported by NSF Grant DEB-9615356. The Lake Michigan sediment samples were collected during cruises on the R/V Neeskay, for which we thank Captain Ron Smith, First Mate Greg Stamatelakys, Duane Moser, and the crew. We also thank the Center for Great Lakes Studies of the University of Wisconsin-Milwaukee for laboratory facilities.

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    Current address: Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany.

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    Current address: Environmental Microbiology, CSIRO Land and Water, Private Bag No. 5, PO Wembley, WA, 6014, Australia.

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    Current address: School of Applied Sciences, South Bank University, London SE1 0AA, UK

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    Current address: National Animal Disease Center, ARS, USDA, 2300 Dayton Road, Ames, IA 50010, USA.

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