Twenty-eight clinical isolates of Yersinia enterocolitica were investigated for their abilities to produce heat-stable enterotoxin (YST). All 21 invasive strains (serogroup 03 biotype 4) canied the previously described gene for YST (yst), with toxin detectable in culture supernatants from 20 strains. One of seven noninvasive, biotype 1A strains also had enterotoxic activity, despite failure to hybridize with a probe for yst. The toxin produced by this noninvasive (serogroup 06) strain resembled YST in terms of molecular size, heat stability, and solubility in methanol. It differed from YST, however, with respect to regulation of its production by temperature and its mechanism of action, which did not appear to involve cyclic GMP. on July 31, 2020 by guest http://iai.asm.org/ Downloaded from on July 31, 2020 by guest http://iai.asm.org/ Downloaded from on July 31, 2020 by guest http://iai.asm.org/ Downloaded from on July 31, 2020 by guest

Summary DNA colony hybridization with a polynucleotide clonal DNA probe for heat‐stable enterotoxin of Vibrio cholerae non‐01 (NAG‐ST) was used to screen 197 isolates of V. cholerae 01. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae 01 GP156, heated at 100°C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely netralized by an anti‐NAG‐ST monoclonal antibody (mAb2F). The preparation from V. cholerae 01 GP156 also inhibited the binding of mAb2F to NAG‐ST in a competitive ELISA. V. cholerae 01 GP156 was confirmed to possess a gene encoding cholera toxin (CT). The results indicate that a heat‐stable enterotoxin is produced by certain strains of CT‐producing V. cholerae 01.

The biological activity of the heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was found to be predominantly associated with the periplasmic extract (about four-fold higher than the culture supernatant) of a recombinant E. coli (JM109) strain carrying the NAG-ST toxin gene. Four molecular species of NAG-ST, two each from the periplasmic extract and culture supernatant of JM109, were purified. Amino acid sequence analysis of the four NAG-ST peptides isolated by HPLC revealed that they all differed from that of the mature 17-amino acid residue NAG-ST released by V. cholerae non-O1. The M(r)-values of the peptides obtained from the periplasmic extract were 4331 and 2785, while those recovered from the culture supernatant were 3154 and 2785. It thus appears that V. cholerae NAG-ST is synthesized as larger molecules in the recombinant E. coli strain. The differences in sizes of the exported NAG-ST molecule could relate to differences in the enzyme cleavage system between E. coli and V. cholerae.