Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution

Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.     

Characterization of ferric-anguibactin transport in Vibrio anguillarum

The fish pathogen Vibrio anguillarum is the causative agent of a fatal hemorrhagic septicemia in salmonid fish. Many serotype O1 strains harbors a 65 Kbp plasmid (pJM1 encoding an iron sequestering system essential for virulence. The genes involved in the biosynthesis of the indigenous siderophore anguibactin are encoded by both the pJM1 plasmid and the chromosome, while those involved in the transport of the ferric-siderophore complex, including the outer membrane receptor, are plasmid-encoded. This work describes the role of specific amino acid residues of the outer membrane receptor FatA in the mechanism of transport of ferric-anguibactin. FatA modeling indicated that this protein has a 22 stranded ß-barrel blocked by the plug domain, the latter being formed by residues 51–54. Deletion of the plug domain resulted in a receptor unable to act as an open channel for the transport of the ferric anguibactin complex.     

鳗弧菌毒力相关基因的研究进展

鳗弧菌是引起多种海水鱼类出血性败血症的病原菌。其致病机理与各个毒力基因的协同作用密切相关。文中综述了鳗弧菌的主要毒力基因,包括编码外毒素、粘附因子、侵袭因子、细胞表面成分以及铁吸收系统的基因和部分检测方法。     

鳗弧菌毒力质粒DNA序列的测定

采用亚克隆法与引物步移法相结合的测序战略 ,对海洋鱼类重要病原菌鳗弧菌毒力质粒pEIB1进行序列测定 ,测得整个质粒序列长度为 6 6 16 4bp。序列的初步分析结果表明 ,G C含量为 4 2 .7% ,共有 4 4个可读框 (ORF) ,其中包括与铁载体合成、调节、运输以及质粒复制相关的基因。     

Plasmid-mediated iron uptake and virulence in Vibrio anguillarum

The plasmid pJM1 of Vibrio anguillarum harbors genes encoding proteins that enable the bacterial cell to survive under iron limiting conditions. A subset of these proteins are involved in the biosynthesis of the siderophore anguibactin and in the internalization of the ferric-siderophore into the cell cytosol. We have identified several genes encoding non-ribosomal peptide synthetases that catalyze the synthesis of anguibactin, these genes are: angB/G, angM, angN, angR, and angT. In addition, the genes fatA, fatB, fatC, and fatD are involved in the transport of ferric-anguibactin complexes. These transport genes, together with the biosynthesis genes angR and angT, are included in the iron transport biosynthesis operon (ITBO). Both the biosynthesis and the transport genes are under tight positive as well as negative control. We have identified four regulators; two of them, a chromosomally encoded Fur and a plasmid-mediated antisense RNA, RNAbeta, act in a negative fashion, while positive regulation is facilitated by AngR and TAFr. We also have evidence that the siderophore itself plays a positive role in the regulatory mechanism of the expression of both transport and biosynthesis genes.     

Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.     

Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system

The iron-transport genes from the pJM1 plasmid of Vibrio anguillarum have been cloned and sequenced. Five open-reading frames have been identified, one of which encodes the outer membrane receptor for ferric anguibactin, OM2. This coding region corresponds to a protein of 726 amino acids with a Mr of 78,777. The protein has a hydrophobic signal sequence of 35 amino acids and a potential membrane-associated hydrophobic region at the carboxyl terminus. A 2.3-kilobase iron-regulated mRNA was transcribed from this region in vivo. The four other open-reading frames were shown to be involved in the regulation of OM2 expression and in iron transport by the use of insertion mutagenesis and complementation analysis. One of these open-reading frames, ORF3, encodes a 40-kDa polypeptide which, as deduced from the amino acid sequence and the hydropathy plot, is likely to be membrane-associated and together with OM2 may play a role in the transport of iron into the cell cytosol.     

Iron uptake mechanisms as key virulence factors in bacterial fish pathogens

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.     

Phylogenetic relationships among Vibrio anguillarum plasmids

Results of restriction endonuclease analysis and Southern blot hybridization suggest that the R-plasmids from Vibrio anguillarum strains isolated in Japan can be divided into at least four groups of homology depending on the time of their isolation and geographical source. Molecular cloning experiments allowed identification of specific restriction endonuclease fragments carrying the genes for either Cm^r or Tc^r as the common sequences between some of these groups of R-plasmids. The Cm^r region from the V. anguillarum R-plasmids was homologous to the Cm^r sequences of an R-plasmid isolated from another fish pathogen, Aeromonas salmonicida. The plasmid pJM1 from V. anguillarum strains isolated in the Pacific Northwest region of the United States, which encodes an iron transport system associated with the high-virulence phenotype of these strains, showed homology with two of the Japanese R-plasmids.     

Reactivation of the vanchrobactin siderophore system of Vibrio anguillarum by removal of a chromosomal insertion sequence originated in plasmid pJM1 encoding the anguibactin siderophore system

A chromosomal gene cluster encoding vanchrobactin biosynthesis and transport genes was identified in the Vibrio anguillarum serotype O1 strain, 775(pJM1), harbouring the anguibactin biosynthetic genes in the pJM1 plasmid. In this strain only anguibactin is produced as the vanchrobactin chromosome cluster has a RS1 transposition insertion into vabF , one of the vanchrobactin biosynthesis genes. Removal of this RS1 generating 775(pJM1)Δ tnp , still resulted in the detection of only anguibactin in specific bioassays. Surprisingly, when the pJM1 plasmid was not present as in the plasmidless strain H775-3, removal of the RS1 resulted in the detection of only vanchrobactin. These results thus can be interpreted as if presence of the pJM1 plasmid or of anguibactin itself is associated with the lack of detection of the vanchrobactin siderophore in bioassays. As high-performance liquid chromatography (HPLC) and mass spectrometry analysis demonstrated that both vanchrobactin and anguibactin were indeed produced in 775(pJM1)Δ tnp , it is clear that the pJM1-encoded anguibactin siderophore has higher affinity for iron than the vanchrobactin system in strains in which both systems are expressed at the same time. Our results underscore the importance of the anguibactin system in the survival of V. anguillarum 775 under conditions of iron limitation.     

Using bacteria to analyze sequences involved in chloroplast gene expression

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.Abbreviations bp base pair - kDa kilodalton - IR inverted repeat - ORF open reading frame - DALA -aminolevulinate     

Iron acquisition in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.     

Iron-binding proteins and heme compounds as iron sources forVibrio anguillarum

Utilization of several iron sources available from the host was investigated in different strains ofVibrio anguillarum. We tested the ability to use transferrins, heme, hemoglobin, and haptoglobin-hemoglobin as iron sources in strains ofV. anguillarum possessing different iron uptake systems mediated by siderophores. Only the wild-type pathogenic strains with an intact siderophore-mediated iron transport system were able to obtain iron from transferrins. None of the low-virulence derivatives lacking siderophore production could grow in the presence of transferrins. However, all strains, wild-type and iron-deficient derivatives, could utilize heme, hemoglobin, and haptoglobin-hemoglobin as iron sources when added to iron-deficient media. The ability to grow in fish serum was also evaluated. Although only wild-type strains could grow in fresh serum, derivative strains lacking siderophore production also were able to grow when serum was heat inactivated or when a utilizable siderophore was present in serum. The results indicate that besides the siderophore-mediated mechanism,V. anguillarum can also obtain iron from other sources presumably available from the host, although its importance for growth in vivo is so far unknown.     

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.