Identification and Functional Analysis of the putAP Genes Encoding Vibrio vulnificus Proline Dehydrogenase and Proline Permease

• Published in: Journal of microbiology and biotechnology Vol. 12; no. 2; pp. 318 - 326
• Main Authors: Hye Jin Kim, Jeong Hyun Lee, Jee Eun Rhee, Hye Sook Jeong, Hyun Kyung Choi, Hee Jong Chung, Sang Ryeol Ryu, Sang Ho Choi
• Format: Journal Article
• Language: Korean
• Published: 한국미생물생명공학회 30.04.2002
• Subjects: osmotolerance; putAP; V. vulnificus
• ISSN: 1017-7825

The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases such as life-threatening septicemia. To better understand this organism`s strategies to survive osmotic stress, a mutant that was more sensitive to high osmolarity was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, putAP genes encoding a proline dehydrogenase and a proline permease were identified and cloned from V. vulnificus. The amino acid sequences deduced from nucleotide sequences of putAP from V. vulnificus were 38 to 59% similar to those of PutA and PutP reported from other Enterobacteriaceae. Functions of putAP genes were assessed by the construction of mutants, whose putAP genes were inactivated by allelic exchanges. When proline as the sole carbon or nitrogen source was used, the putA mutant was not able to grow to the substantial level, revealing the proline dehydrogenase is the only enzyme for metabolic conversion of proline into other amino acids. Although the growth rate of the putP mutant on proline as the sole carbon or nitrogen source was significantly reduced, the mutant still grew. This indicated that at least one more proline permease is produced by V. vulnificus. The putP mutant decreased approximately 2-log10 CFU/ml after a hyperosmotic challenge, while the parent strain decreased approximately l-log10 CFU/ml. This result suggests that the gene product of putP contributes to the osmotic tolerance of V. vulnificus.