Vibrio fischeri genes hvnA and hvnB encode secreted NAD(+)-glycohydrolases

HvnA and HvnB are proteins secreted by Vibrio fischeri ES114, an extracellu lar light organ symbiont of the squid Euprymna scolopes, that catalyze the transfer of ADP-ribose from NAD(+) to polyarginine. Based on this activity, HvnA and HvnB were presumptively designated mono-ADP-ribosyltransferases ( ARTases), and it was hypothesized that they mediate bacterium-host signalin g. We have cloned hvnA and hvnB from strain ES114. hvnA appears to be expre ssed as part of a four-gene operon, whereas hvnB is monocistronic. The pred icted HvnA and HvnB amino acid sequences are 46% identical to one another a nd share 44% and 34% identity, respectively, with an open reading frame pre sent in the Pseudomonas aeruginosa genome. Four lines of evidence indicate that HvaA and HvnB mediate polyarginine ADP-ribosylation not by ARTase acti vity, but indirectly through an NAD(+)-glycohydrolase (NADase) activity tha t releases free, reactive, ADP-ribose: (i) like other NADases, and in contr ast to the ARTase cholera toxin, HvnA and HvnB catalyzed ribosylation of no t only polyarginine but also polylysine and polyhistidine, and ribosylation was inhibited by hydroxylamine; (ii) HvnA and HvnB cleaved 1-N-6-etheno-NA D(+) and NAD(+); (iii) incubation of HvnA and HvnB with [P-32] NAD(+) resul ted in the production of ADP-ribose; and (iv) purified HvnA displayed an NA Dase V-max of 400 mol min(-1) mol(-1), which is within the range reported f or other NADases and 10(2)- to 10(4)-fold higher than the minor NADase acti vity reported in bacterial ARTase toxins. Construction and analysis of an h vnA hvnB mutant revealed no other NADase activity in culture supernatants o f V. fischeri, and this mutant initiated the light organ symbiosis and trig gered regression of the light organ ciliated epithelium in a manner similar to that for the wild type.