Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures

Abstract

Molecular information about the bacterial composition of a coculture capable of sulfate reduction after exposure to oxic and microoxic conditions was used to identify and subsequently to isolate the components of the mixture in pure culture. PCR amplification of 16S ribosomal DNA fragments from the coculture, analyzed by denaturing gradient gel electrophoresis, resulted in two distinct 16S ribosomal DNA bands, indicating two different bacterial components. Sequencing showed that the bands were derived from a Desulfovibrio strain and an Arcobacter strain. Since the phylogenetic positions of bacteria are often consistent with their physiological properties and culture requirements, molecular identification of the two components of this coculture allowed the design of specific culture conditions to separate and isolate both strains in pure culture. This approach facilitates the combined molecular and physiological analysis of mixed cultures and microbial communities.

Growth of a bacterial consortium on triclosan

“…Other molecular-genetic techniques such as ARDRA and REP-PCR have also been used to guide the isolation of refractory organisms from mixed cultures [15]. This work adds to the evidence demonstrating the value of molecular ecology techniques in the characterization of microbial consortia [15,45].…”

“…The isolation of strain Rd1 was guided in large part by the sequence information obtained through random cloning and sequencing of partial 16S rRNA gene fragments from the consortium DNA extract. Teske et al [45] used 16S rDNA sequence information to design selective enrichment conditions for the isolation of individual organisms from a mixed culture. Other molecular-genetic techniques such as ARDRA and REP-PCR have also been used to guide the isolation of refractory organisms from mixed cultures [15].…”

Molecular characterization of a sulfate-reducing consortium which mineralizes benzene

“…Variations of these techniques have been used to study the microbial communities in hot springs [36], marine bacterioplankton [37], soils [38], marine sediments [39], oil ¢elds [12], activated sludge [40], and freshwater lakes [41]. The information gained by phylogenetically identifying the members of a consortium can be used to make inferences about the microbially mediated processes occurring in an environment [12,33] or to design isolation schemes for speci¢c bacteria [13].…”

“…Given all these reasons, alternative methods to examine the microorganisms would be useful and molecular tools can provide information about the organisms that we would not otherwise be able to determine. The approach of using 16S rRNA gene sequences has been applied to characterize the sulfate-reducing communities present at oil ¢elds [12] and to identify bacteria present in a co-culture capable of reducing sulfate after exposure to oxygen [13]. In the latter study, the information from analyses of 16S rRNA genes was used to devise an isolation scheme which successfully separated the two strains.…”

Identification and characterization of humic substances-degrading bacterial isolates from an estuarine environment

“…Cell debris was removed by centrifugation at 11 750Ug for 5 min. 5 Wl of the supernatant was used to amplify a portion of the 16S rDNA gene using primers 341F (CCTACGGGAAGGCAGCAG) and 907R (CCG-TCAATTCCTTTRAGTT) [10,11]. The PCR ampli¢cation mixture contained 0.1 mM (each) deoxynucleoside triphosphate, 100 nM (each) primer, 5 mM MgCl 2 , and 1 U of Thermalase rec-Thr (Amresco, Solon, OH) polymerase in a ¢nal volume of 50 Wl.…”