K-072. Proline Catabolism in Staphylococcus saprophyticus

C. E. Deutch;
Arizona State Univ., Phoenix, AZ.

Genomic sequencing of the Gram-positive urinary tract pathogen Staphylococcus saprophyticus indicates it has genes encoding multiple transporters for L-proline as well as the catabolic enzymes L-proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase. While S. saprophyticus accumulates L-proline during osmotic stress, it is not known if the genes for the enzymes are expressed and how the proteins compare to the multifunctional PutA protein found in Gram-negative bacteria. The goals of this study were to determine if L-proline is utilized by S. saprophyticus and to describe the properties of the enzymes involved in proline catabolism. S. saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 formed small red colonies on proline/tetrazolium plates, indicative of proline catabolism. However, addition of L-proline to tryptic soy broth or a defined medium did not increase the growth rate or final yield of bacteria. Whole cell assays indicated that all three strains had low but measurable L-proline dehydrogenase activity. Addition of L-proline alone or in combination with 0.5 M or 1.0 M NaCl to the growth media did not increase the specific activity. To characterize the L-proline dehydrogenase activity, exponential-phase cells of strain ATCC 49907 were treated with lysostaphin in a hypertonic buffer and lysed by passage through a French pressure cell. The membrane fraction obtained after centrifugation at 105,000 x g had low levels of activity as measured both by reaction of Δ1-pyrroline-5-carboxylate with 2-aminobenzaldehyde and by reduction of iodonitrotetrazolium in the presence of L-proline, FAD, and phenazine methosulfate. To characterize the Δ1-pyrroline-5-carboxylate dehydrogenase activity, exponential-phase cells were disrupted with a “bead breaker” and the soluble fraction recovered by centrifugation at 105,000 x g. This fraction had measurable levels of activity as determined by the NAD+-dependent oxidation of Δ1-pyrroline-5-carboxylate. These results indicate that S. saprophyticus can degrade L-proline, but unlike Gram-negative bacteria, does so through separable enzymatic proteins.