Production of Vibrio vulnificus metalloprotease VvpE begins during the early growth phase: usefulness of gelatin-zymography.
Recent studies have demonstrated that expression of the vvpE gene begins during the early growth phase albeit at low levels. However, we found that the traditional protease assay method that is used to measure caseinolytic activity in culture supernatants is not suitable for the measurement of extracellular VvpE that is produced at low levels during the early growth phase. By using gelatin-zymography in place of the protease assay, we could specifically detect only VvpE of several proteases produced by Vibrio vulnificus. Moreover, we could sensitively measure VvpE produced at low levels during the early growth phase, which was consistent with transcription of the vvpE gene. The extracellular production of VvpE was reduced or delayed by mutation of the pilD gene which encodes for the type IV leader peptidase/N-methyltransferase associated with the type II general secretion system; the delayed production of VvpE was recovered by in trans complementation of the wild-type pilD gene. These results indicate that VvpE begins to be produced during the early growth phase via the PilD-mediated type II general secretion system, and that the use of gelatin-zymography is recommended as a simple method for the sensitive and specific detection of VvpE production.
Kim
Choon-Mee
CM
Research Center for Resistant Cells, Chosun University Medical School, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea.
Kang
Sang-Mee
SM
Jeon
Ho-Jong
HJ
Shin
Sung-Heui
SH
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D005780:Gelatin / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D045726:Metalloproteases / Q000096:biosynthesis* / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D041261:Vibrio vulnificus / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D005780:Gelatin; D045726:Metalloproteases