The 16S rRNA characterization of a novel "microaerophilic" Pseudomonas sp. from the oligotrophic deep subsurface environment

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Virginia Tech


A gram negative microaerophilic bacterium, designated Pseudomonas sp. strain MR 100, was isolated from a depth of 463 meters at the Savannah River DOE site and identified using 16S rDNA sequencing and DNA-DNA reassociation. Micro aerophiles from the Middendorf formation were isolated by use of a semi-solid agar assay, and constituted 10% of the plateable microorganisms. Genetic identification involved the isolation of genomic DNA and amplification of the gene encoding 16S rRNA by PCR, using universal primers. The amplified DNA was sequenced and compared to 16S rRNA sequences in Genbank. High sequence similarity (98.5%) was observed with the Pseudomonas mendocina type strain, indicating a similarity to the (Group I) pseudomonads. DNA-DNA reassociation was performed between Pseudomonas sp. strain MR 100 and 11 representative p seudomonads using the S 1 nuclease method. Strain MR 100 was found to be 20% homologous to the Pseudomonas mendocina type strain, 10% homologous to Pseudomonas alcaligenes, and 5% homologous to Pseudomonas aeruginosa. Data from biochemical tests confirm the hypothesis that strain MR 100 is a novel species of Pseudomonas</I. It was able to accumulate poly-β-hydroxybutyrate intracellularly, while it lacked the ability to produce cellular pigments, which is unique among the (Group I) pseudomonads. Growth occurred at oxygen concentrations of 20/0 and 21%, with similar growth rates and final cell densities.



DNA reassocation, 16S rRNA, microaerophile, subsurface, Pseudomonas