Characterization of Heme Uptake Cluster Genes in the Fish PathogenVibrio anguillarum

Abstract

Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources. In previous work we identified HuvA, the V. anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone (pML1) isolated from a genomic library of V. anguillarum. In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor; HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the putative periplasmic binding protein; HuvC, the putative inner membrane permease; and HuvD, the putative ABC transporter ATPase. A V. anguillarum strain with an in-frame chromosomal deletion of the nine-gene cluster was impaired for growth with heme or hemoglobin as the sole iron source. Single-gene in-frame deletions were constructed, demonstrating that each of the huvAZBCD genes are essential for utilization of heme as an iron source in V. anguillarum, whereas huvX is not. When expressed in Escherichia coli hemA (strain EB53), a plasmid carrying the gene for the heme receptor, HuvA, was sufficient to allow the use of heme as the porphyrin source. For utilization of heme as an iron source in E. coli ent (strain 101ESD), the tonB exbBD and huvBCD genes were required in addition to huvA. The V. anguillarum heme uptake cluster shows some differences in gene arrangement when compared to homologous clusters described for other Vibrio species.Iron is an essential element for most bacteria, serving as a cofactor in key metabolic processes such as nucleotide biosynthesis, electron transfer, and energy transduction. Most bacterial pathogens require iron for growth and to establish an infection, and thus they have developed efficient mechanisms to obtain iron from the host (32). The small amounts of extracellular iron are quickly bound by high-affinity carrier proteins such as transferrin in serum and lactoferrin in secretions. Vibrio anguillarum is the etiological agent of a septicemic disease known as vibriosis, which affects a large number of marine fish species. Within the 10 O serogroups described by Sørensen and Larsen (35), only serotypes O1 and O2 and, to a lesser extent, serotype O3 are considered important fish pathogens. Although V. anguillarum is the best known fish pathogen of the genus Vibrio, the nature of its virulence mechanisms is not thoroughly understood. Strains of pathogenic V. anguillarum serotypes can acquire iron by the production and secretion of siderophores (2,4,16,19,38). Heme and some heme-containing proteins, including hemoglobin and hemoglobin-haptoglobin, can also be used as iron sources by a siderophore-independent mechanism (23,24).In this way, many gram-negative pathogens have the ability to obtain iron through utilization of free heme or heme proteins from the host tissues (7, 18), and heme utilization genes have been identified in numerous species, including Yersinia enterocolitica (36, 37), Vibrio cholerae (13,26,29), Escherichia coli O157 (39), Vibrio vulnificus (20), Plesiomonas shigelloides (1...

Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii

“…The TonB1 and TtpC-TonB2 clusters, which are essential for iron transport, are encoded on Chr1 (67,68). The fur gene is located on Chr1, which also harbors the heme determinants, although we have also identified a heme-related V. cholerae hutR homologue on Chr2 (42,48,49,70). Genes involved in ferric vanchrobactin and/or ferric enterobactin transport are located on Chr1, although vanchrobactin biosynthesis in 775 is abrogated by a mutation in vabF and there are no biosynthesis genes for enterobactin (51).…”

Heme Utilization in Campylobacter jejuni

“…The potential existence of such genes is indicated by the apparent semiredundancy of chuBCD described here and also by comparison of the Cj1613c-17 gene cluster with known heme utilization gene clusters from other bacterial species (21,35,36). Most notable is the absence of a linked TonB-ExbB-ExbD system, which is expected to be required for energizing the outer membrane receptor(s).…”

Shigella dysenteriaeShuS Promotes Utilization of Heme as an Iron Source and Protects against Heme Toxicity

“…Although it has no sequence identity with ShuS, HutZ is required for efficient utilization of heme (9,18,39), and the absorbance spectra produced when heme is bound to ShuS or HutZ are nearly identical (37,39). Interestingly, many of the heme transport loci contain either a hutZ or a shuS homologue, and we are not aware of any loci that contain both.…”