Isolation of mutants of Vibrio anguillarum defective in haeme utilisation and cloning of huvA, a gene coding for an outer membrane protein involved in the use of haeme as iron source.

The isolation of Vibrio anguillarum mutants lacking the ability to use haemin and haemoglobin as the only iron sources, as well as the identification of a gene involved in haeme utilisation are described. One of the isolated mutants defective in haeme utilisation lacked an iron-regulated outer membrane protein of 79-kDa. Although growth on haeme as iron source was completely abolished, the haemin and haemoglobin binding activities remained intact in the mutant, suggesting that the absent protein is not the only one involved in haeme binding. The wild-type phenotype in this mutant was restored by transformation with a cosmid clone (pML1) containing a 21-kb DNA fragment isolated from a gene library derived from the parental strain of V. anguillarum. Sequence analysis of pML1 subclones led to the finding of an ORF, huvA, that codes for a 79-kDa protein (HuvA) and whose sequence shows high identity with haeme receptors from Vibrio choleare (HutA) and Vibrio vulnificus (HupA). The sequence of huvA from the V. anguillarum haeme-utilisation mutant revealed a single mutation, leading to the synthesis of a truncated HuvA protein of 70 kDa. The parental strain and the cosmid-complemented mutant showed a higher degree of virulence for fish than the mutant strain in experimental infections in which fish were previously overloaded with haemin. This finding suggests that haeme uptake plays an important role in V. anguillarum multiplication in fish tissues when free haeme is available.