Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

Abstract

A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a "standard") to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples.

Microbial diversity in production waters of a low-temperature biodegraded oil reservoir

“…Aerobic biodegradation of hydrocarbons has long been believed to be the dominant mechanism of subsurface oil degradation. This view has been modified since the evidence for the widespread occurrence of anaerobic microorganisms in deep petroleum systems [5,9,28,62], the demonstration of microbial anaerobic degradation of saturated and aromatic hydrocarbons [30,51] and the occurrence of metabolites characteristic of anaerobic hydrocarbon degradation in samples from biodegraded petroleum reservoirs [3]. The Pelican Lake oil field is shallow and the chemistry of its formation waters suggests that it has been flushed with meteoric waters [46].…”

“…Enrichments and isolation experiments performed with samples collected from low-and high-temperature reservoirs revealed the presence of physiologically diverse microbial communities and led to the characterization of numerous microorganisms, including both lithoautotrophic and chemoorganoheterotrophic strains. The resident anaerobic bacteria include sulfate-reducers [4][5][6][7][8], fermentative bacteria [9][10][11][12][13][14][15], iron reducers [16][17][18], acetogens [19,20] and methanogens [21][22][23][24][25][26] (for a review, see [27]). Recent applications of molecular techniques allowed more complete characterizations of the microbial assemblages inhabiting a low-temperature waterflooded Canadian reservoir [28] and high-temperature, sulfur-rich oil-bearing formations in California and in Siberia [15,29].…”

Bacterial chromosomal painting for in situ monitoring of cultured marine bacteria

“…Rare cells are difficult to count by microscopic methods. For this reason, we screened the isolates to determine which cultured isolates were the most abundant at the study site, using a hybridization protocol based on reverse sample genome probing (Voordouw et al, 1991) (Fig. 1).…”

“…< 20% similarity) (Lanoil and Giovannoni, 1997). Therefore, unlike blot-based methods (Voordouw et al, 1991), low levels of cross hybridization will not affect the results of this in situ approach. BCP is less sensitive to permeability issues than rRNA-based methods because a balance between permeabilization and retention of the rRNA target is not required.…”

“…In this study, we tested BCP in a natural system for the first time by using it to determine the vertical distribution and abundance of an alpha proteobacterium cultured from the western Sargasso Sea (WSS). Screening procedures based on reverse sample genome probing (Voordouw et al, 1991), as well as phylogenetic analyses of SSU rRNA gene sequences, were applied to a set of isolates to maximize the likelihood that the isolate chosen would be abundant enough to detect in samples from the WSS by BCP.…”