Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology

Abstract

To address the long-standing need for more precise descriptions of natural microbial ecosystems, 16S rRNAs were used to track certain species and phylogenetically coherent groups of microorganisms in their natural setting without culturing. Speciesand group-specific 16S rRNA-targeted oligonucleotide hybridization probes were developed to enumerate various strains of Bacteroides succinogenes and Lachnospira multiparus-like organisms in the bovine rumen before, during, and after perturbation of that ecosystem by the addition of the ionophore antibiotic monensin. Based on probe hybridization, relative numbers of L. multiparus-like organisms were depressed about 2-fold during monensin addition and demonstrated a transient 5to 10-fold increase immediately after removal of the antibiotic from the diet. The most pronounced population changes were observed among different strains of B. succinogenes, as evaluated by three hybridization probes. One probe hybridized to all strains, whereas the other two identified genetically distinct groups represented by strains isolated from the rumen and from the ceca of nonruminants. The rumen-type strains predominated on most days (ca. 0.2 to 0.8% of total ribosome numbers). Their proportion transiently increased about fivefold immediately after the addition of monensin to the feed and then transiently fell below the average premonensin level. During this time (ca. 2 weeks after monensin addition) the cecal type predominated (ca. 0.1 to 0.2%). Cultural enumeration of B. succinogenes on nonselective agar and by observing clearings in cellulose agar media were largely unsuccessful due to the low number of organisms present and the predominance of other cellulolytic species. The results demonstrate the utility of rRNA-targeted hybridization to monitor complex microbial communities and are most consistent with the hypothesis that altered microbial metabolism, rather than altered population composition, is primarily responsible for changes in ruminal fermentation patterns resulting from the feeding of monensin.

Diversity responses of rumen microbial communities to Fusarium‐contaminated feed, evaluated with rumen simulating technology

“…However, PCR detections of Fibrobacter and its relatives tend to underestimate the actual abundance of this group, possibly due to low accessibility of the rRNA primer binding sites (Tajima et al, 2001;Firkins and Yu, 2006). The specificity of the chosen forward primer to detect Fibrobacter has already been demonstrated before (Stahl et al, 1988) and was confirmed by our own in silico analyses (data not shown). However, sequencing of bands from SSCP profiles targeting Fibrobacter revealed the Table 4.…”

Characterization and quantification of ammonia‐oxidizing bacteria in eutrophic coastal marine sediments using polyphasic molecular approaches and immunofluorescence staining

“…RNA was quantitatively extracted from approximately 0.6 g of wet sediment using the bead-beating disruption mentioned previously (Stahl et al, 1988). RNA concentration was determined spectrophotometrically, as described above.…”

Diversity and dynamics of microbial communities in soils from agro‐ecosystems

“…The RNA isolated from pure cultures [Ketogulonogenium vulgare DSM 4025, Nitrosomonas europaea ATCC 25978, Cytophaga johnsonae ATCC 17061, Arthrobacter globiformis ATCC 8010, Verrucomicrobium spinosum ATCC 43997, Planctomyces limnophilus ATCC 43296, Acidobacterium capsulatum ATCC 51196 and Saccharomyces cerevisiae American Ale Yeast 1056 (Wyeast Laboratories, INCS)] were included on all membranes as standards to control for differences in the specific activity of labelled probes and to account for the possibility of non-specific probe binding. Hybridization protocols for [ 32 P]-5¢-labelled oligonucleotide probes were previously described in detail (Stahl et al, 1988). Radiolabelled oligonucleotide probes that bind to the rRNA molecules from specific microbial groups were then used to determine the relative abundance of microbial group rRNA.…”

“…Within a soil sample, the relative abundance of rRNA derived from a specific group was measured as the ratio of the signal derived from a group-specific probe to the signal derived from the universal probe. This approach for determining microbial rRNA abundance has been used previously to describe aspects of microbial community structure (Stahl et al, 1988). Relating specific probe binding to universal probe binding controls for variability in the total amount of RNA recovered from each soil sample, and also controls for the presence of hybridization inhibitors that may co-purify with RNA from soil.…”