Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

Abstract

Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.Plesiomonas shigelloides is a gram-negative bacterium associated with diarrheal disease in humans (4). The organism has been reported to cause several types of gastroenteritis, including acute secretory gastroenteritis (33), an invasive shigellosislike disease (35), and a cholera-like illness (55). Extraintestinal infections, such as meningitis, bacteremia (2), and pseudoappendicitis (13), are also associated with P. shigelloides infection.Many bacterial pathogens have iron transport systems that play a critical role in allowing the organism to establish an infection (for reviews, see references 31 and 41). Several bacterial pathogens, including P. shigelloides (9), obtain iron from heme or heme-containing compounds. Heme iron utilization systems have been examined in many gram-negative pathogens, including Vibrio cholerae (20,21,38), Vibrio vulnificus (30), Shigella dysenteriae (37, 60), Escherichia coli O157:H7 (54), Serratia marcescens (16, 26, 27), yersiniae (22, 50, 51, 53), Haemophilus (8,11,24,32), neisseriae (7,29,52,64), Pseudomonas (23,28,39), and Porphyromonas gingivalis (47). Many heme iron utilization systems studied to date require an outer membrane receptor which binds heme from the environment (for a review, see reference 58). TonB, with the aid of ExbBD, is thought to interact with the receptor to allow movement of heme into the periplasm. A periplasmic binding protein then moves the heme across th...

Characterization of Heme Uptake Cluster Genes in the Fish PathogenVibrio anguillarum

“…huvZ encodes a 176-amino-acid protein with homology to proteins linked to heme transport systems ( Table 3). The function of HuvZ homologues remains uncharacterized, though some observations suggest that P. shigelloides HugZ could be involved in preventing heme toxicity (14). Interestingly, database comparisons evidence that HuvZ has homology to proteins containing a flavin mononucleotide-binding split barrel (data not shown).…”

“…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 (14), and Shigella dysenteriae (28) among others. Specific receptors are involved in heme binding and transport.…”

HutZ Is Required for Efficient Heme Utilization inVibrio cholerae

“…This large family of proteins uses a radical-based mechanism to catalyze diverse chemical reactions. HutW has the highest homology with proteins encoded by genes linked to heme transport genes of Vibrio parahaemolyticus (26), Plesiomonas shigelloides (16), Yersinia pestis (28), and E. coli O157:H7 (Table 2). HutW has lower homology with HemN, the oxygen-independent form of the heme biosynthetic enzyme coproporphyrinogen oxidase (54), and also with yggW, an ORF of unknown function present in the genome of E. coli K-12 (2) and several other bacterial species.…”

“…When the P. shigelloides heme transport genes were used to reconstruct heme transport in E. coli, bacteria carrying clones that contained the heme receptor, the tonB system, and the hutWXZ homologues (called hugWXZ) used heme efficiently as an iron source. When the hugWXZ genes were deleted from the plasmid, the strain only used heme weakly and was sensitive to high levels of heme (16). The ORF(s) required was not identified.…”

“…An ORF with homology with hemN was first identified within the heme transport loci of S. dysenteriae and E. coli O157:H7 (50), and additional hemN homologues were later found linked to heme transport genes in V. parahaemolyticus (26), V. vulnificus (3), P. shigelloides (16), Y. pestis (28), and V. cholerae (12). Each of the loci listed above also contains a homologue of hutX, but only the P. shigelloides and the other Vibrio species loci contain a hutZ homologue.…”