CLONING AND CHARACTERIZATION OF AN OUTER-MEMBRANE PROTEIN OF VIBRIO-VULNIFICUS REQUIRED FOR HEME UTILIZATION - REGULATION OF EXPRESSION ANDDETERMINATION OF THE GENE SEQUENCE

Vibrio vulnificus is a halophilic, marine pathogen that has been assoc iated with septicemia and serious wound infections in patients with ir on overload and preexisting liver disease. For V. vulnificus, the abil ity to acquire iron from the host has been shown to correlate with vir ulence. V. vulnificus is able to use host iron sources such as hemoglo bin and heme, We previously constructed a fur mutant of V: vulnificus which constitutively expresses at least two iron-regulated outer membr ane proteins, of 72 and 77 kDa, The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vi brio cholerae heme receptor, HutA. In this report,we describe the clon ing, DNA sequence, mutagenesis, and analysis of transcriptional regula tion of the structural gene for HupA, the heme receptor of V. vulnific us. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequenc e of V. cholerae HutA and similar to those of other TonB-dependent out er membrane receptors, Primer extension analysis localized one promote r for the V, vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur b ox. Northern blot analysis showed that the transcript was strongly reg ulated by iron, An internal deletion in the V. vulnificus hupA gene, d one by using marker exchange, resulted in the loss of expression of th e 77-kDa protein and the loss of the ability to use hemin or hemoglobi n as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V, vulnificus pathogenesis.