Studies of adaptive responses of the microaerophile Spirillum volutans to various stresses such as heat and peroxide shock have been hampered by an inability to obtain reliable colony counts of the organism by the spread plate method. Colony counts approaching direct microscopic counts (DMCs) were obtained by inoculating culture dilutions into a semisolid version of the medium and by supplementing the medium with pyruvate, which destroys hydrogen peroxide. Use of the new overlay/pyruvate method for colony counts revealed that exposure of S. volutans to 40 degrees C for 100 min results in a greater survival at 45 degrees C compared with cells having no prior exposure to 40 degrees C. Spirillum volutans is catalase-negative and is rapidly killed by levels of H2O2 greater than 10 micromolar. A mutant isolated by single step mutagenesis with diethyl sulfate was able to survive and grow after exposure to 40 micromolar H2O2 and was effective in eliminating H2O2 concentrations added to the medium. In addition, the mutant had high NADH peroxidase activity (0.072 I.U. mg-1) whereas the wild type had no detectable activity (<0.0002 I.U. mg-1). Nevertheless, the mutant was no more tolerant to O2 than the wild type. NADH peroxidase activity has not previously been reported in bacteria having a strictly respiratory type of metabolism. The peroxide-resistant mutant constitutively expresses a 21.5 kDa protein as determined by one and two-dimensional PAGE. This protein was undetectable and noninducible in the wild type cells. Part of the gene that encodes the protein was cloned by using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide showed high similarity to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris and to putative rubrerythrin proteins found in some anaerobic archeons. This is the first report of this type of protein in an organism that must respire with oxygen. This rubreythrin-like protein may play a role in the peroxide resistance of the mutant. The methodology may be useful for rapid cloning of genes in other bacteria. / Ph. D.

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 <i>Pseudomonas mendocina</i> type strain, indicating a similarity to the (Group I) pseudomonads. DNA-DNA reassociation was performed between <i>Pseudomonas</i> 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 <i>Pseudomonas mendocina</i> type strain, 10% homologous to <i>Pseudomonas alcaligenes</i>, and 5% homologous to <i>Pseudomonas aeruginosa</i>. Data from biochemical tests confirm the hypothesis that strain MR 100 is a novel species of <i>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. / Master of Science