Vibrio vulnificus has proven difficult to culture from water or shellfish during winter months, which is attributed to the viable but nonculturable (VBNC) state. Because reactive oxygen species were found to be involved in the low temperature-induced entrance of V. vulnificus into this state, we generated an oxyR mutant which lacks catalase activity. This strain is nonculturable on solid media even at ambient temperature, due to the presence of H(2)O(2) in such media. Low temperature incubation of the parent resulted in loss of catalase activity, making the cells H(2)O(2) sensitive, and paralleling the loss of culturability (entry into the VBNC state). Thus, cells of V. vulnificus in the VBNC state are likely exhibiting this response to low in situ temperature and only when the artificial condition of laboratory culture is attempted are the cells nonculturable due to cold-induced loss of catalase activity. To our knowledge, this is the first study providing direct evidence for the metabolic basis of nonculturability and the viable but nonculturable state.

Acetyl-coenzyme A carboxylase (Ac-CoA carboxylase; EC 6.4.1.2) catalyzes the rate-limiting reaction in long-chain fatty acid biosynthesis. To investigate the mechanism of genetic control of expression of Ac-CoA carboxylase and the relationship between its structure and function, cDNA clones for Ac-CoA carboxylase were isolated. The complete coding sequence contains 7035 bases; it encodes a polypeptide chain of 2345 amino acids having a Mr of 265,220. The sequences of several CNBr peptides of Ac-CoA carboxylase were localized within the predicted protein sequence as were those peptides that contain the sites for phosphorylation. The deduced protein contains one putative site for biotinylation in the NH2-terminal half. The "conserved" biotinylation site peptide, Met-Lys-Met, is preceded by valine, whereas alanine is found in a similar position in all other known biotincontaining proteins. The primary sequences of Ac-CoA carboxylase and carbamoyl phosphate synthetase exhibit substantial identity.

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. In order to understand the mechanisms that regulate human acetyl-CoA carboxylase at the gene level, and the relationship between its structure and function, cDNA clones for human acetyl-CoA carboxylase have been isolated and sequenced. Human acetyl-CoA-carboxylase cDNA contains 7020 nucleotides encoding a protein of 2340 amino acids with a calculated relative molecular mass of 264575. The human enzyme shows approximately 85 % identity in nucleotide sequence with previously cloned rat acetyl-CoA carboxylase, and shows 90% identity in the amino acid sequence. Two human acetyl-CoA-carboxylase mRNA species, which differ in the 5' untranslated region with the same coding sequence, have been identified. The sequence analysis reveals that type I and type I1 acetyl-CoA-carboxylase mRNA contain 31 3-and 173-base-long 5' untranslated regions, respectively. The first 240 nucleotides in the 5' untranslated region of type I acetyl-CoA-carboxylase mRNA replace the first 100 nucleotides of the (G+C)-rich region of the 5' untranslated region of the type I1 mRNA. These two species of mRNAs are the only species of human ACC mRNA which have been detected compared to at least five species in rat tissues, and they are expressed in a tissue-specific manner.Acetyl-CoA carboxylase (ACC) catalyzes the fate-limiting reaction in the biogenesis of long-chain fatty acids, namely the ATP-dependent carboxylation of acetyl-CoA into malonyl-CoA [l -31. The subunit relative molecular mass of ACC from animal sources is approximately M, 260000; thus, it represents one of the largest proteins with enzyme activity. The protomer contains two identical subunits which undergo polymerization in the presence of activator molecules, such as citrate, into aggregates with M, values of several million [4]. The extremely large size of the subunit and its property of aggregation into different polymeric states have hindered our progress toward understanding the primary structure of the enzyme as well as its structure/function relationship. Since the enzyme is readily broken down by proteolysis, the real size of the enzyme was also a matter of dispute for a long time. Recent success in cloning the rat [5] and chicken enzyme [6] has provided some information of the size, primary structure, biotin-binding site [7] and other putative functional sites of the protein. However, there has not been any similar information on the human enzyme, largely because of the lack of suitable human tissues in addition to the difficulties involved in studying the enzyme itself, as indicated above. Regulatory mechanisms for the rat liver enzyme include covalent modification of ACC protein ; phosphoryla-

Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.

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