Gene cloning,expression and functional characterization of a phosphopantetheinyl transferase from <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> serotype O1

Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.     

Complete sequence of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region

AIMS: The aim of this study was to determine the whole DNA sequence of pEIB1, one pJM1-like virulence plasmid from Vibrio anguillarum MVM425 and locate the replication region. METHODS AND RESULTS: DNA sequence of virulence plasmid pEIB1 from V. anguillarum MVM425 was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole nucleotide sequence of pEIB1 comprises 66,164 bp, encoding 44 open reading frames (>400 bp) containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. With no demonstrated replication origin, the Sau3AI partial digested plasmid DNA fragments of pEIB1 were ligated into the BamHI-fragment containing the kanamycin-resistance gene (Kmr). For there is no effective transformation in V. anguillarum, the ligated DNA was first introduced into E. coli JM83, and the transfomants were selected for resistance to kanamycin. It was demonstrated with southern blotting and DNA sequencing that plasmid pEIB7 containing the Sau3AI DNA fragment of pEIB1 (from 12516 to 13957) has the ability to replicate in E. coli JM83 and V. anguillarum MVM425sh. The segregational stability of plasmid pEIB7 kept in 100 and 4% in E. coli JM83 and V. anguillarum MVM425sh respectively when the cells were cultured in 200th generation. In following experiments, we also found that plasmid pEIB7 replicated at a middle-copy number of 10-40 in JM83, while at a high-copy number of 100-300 in MVM425sh. Moreover, pEIB7 can survive in V. alginolyticus, another fish pathogenic. CONCLUSIONS: With the whole DNA sequence of pEIB1 determining, it was found that pEIB1 showed microheterogeneity in its restriction endonuclease patterns with pJM1 though their DNA sequences had slight difference. According to the complete DNA sequence of pEIB1, its replication region was located from 12516 to 13957. And this replication region is compatible to pUC18 (pMB1), pKA3 (pSC101) and p15A: caiE (p15A). SIGNIFICANCE AND IMPACT OF THE STUDY: The worldwide vibriosis marine pathogen V. anguillarum strains contain common virulence, pJM1-like plasmids, independent on the geographical source. The pEIB1 was the second common virulence plasmid, which sequence was determined. Its sequence is highly homologous to pJM1 as they both encode biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. Some interesting features as in pJM1 were also identified, such as transposon-like structures. So it can be deferred that the whole DNA sequences of virulent plasmid pEIB1 will be great helpful to future revealing these V. anguillarum virulence-related genes derived during evolution from transposition events or horizontal transfer of genes potentially originating in other organisms. Another result, replication region of pEIB1 locating is the first report about replication of pJM1-like plasmid. This work will be useful for researching pJM1-like plasmid replication mechanism in V. anguillarum.     

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.     

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.     

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.     

Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1.

Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.     

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.     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins.

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species.

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Iron uptake system medicated by Vibrio anguillarum plasmid pJM1.

Plasmid pJM1 from an invasive strain of Vibrio anguillarum mediates an iron-sequestering system that is associated with the ability of this bacterium to cause septicemia in marine fishes. This plasmid-mediated iron uptake system was analyzed by using mutations caused by transposon Tnl. Restriction endonuclease analysis of iron uptake-deficient and -proficient derivatives generated by insertion of Tnl and molecular cloning experiments permitted us to localize the plasmid regions involved in the process of iron sequestration to a stretch of about 20 kilobase pairs. In addition, the existence of two plasmid-mediated components involved in the process of iron uptake in V. anguillarum was defined: a diffusible substance which functions as a siderophore and a nondiffusible receptor for complexes of iron-siderophore, which we have tentatively identified as the pJM1 plasmid-mediated outer membrane protein OM2 of V. anguillarum.     

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.     

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">anguillarum</Emphasis> strain 775

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.     

Molecular cloning and expression of genetic determinants for the iron uptake system mediated by the Vibrio anguillarum plasmid pJM1

Plasmid pJM1 from an invasive strain of Vibrio anguillarum encodes an iron uptake system which mediates the biosynthesis of a siderophore and a membrane receptor for the iron-siderophore complex. This system has been associated with the ability of V. anguillarum to cause hemorrhagic septicemic disease in marine fish. Recombinant derivatives containing essential regions of the pJM1-mediated iron uptake system cloned into cosmid vector pVK102 were introduced into low-virulence iron uptake-deficient V. anguillarum strains by using a trifactor mating procedure with helper plasmid pRK2013. Three recombinant clones, pJHC-T7, pJHC-T11, and pJHC-T2612, possessed genetic determinants for receptor activity. Production of receptor activity was correlated in all three cases with the presence of OM2, an 86-kilodalton outer membrane protein which was induced under iron-limiting conditions. Two of the clones, pJHC-T7 and pJHC-T2612, also coded for the production of siderophore activity, although at a much lower level than the wild type. Strains harboring either of these two clones were still unable to grow under iron-limiting conditions. This inability was overcome only when other indigenous pJM1 derivatives were present in the cells in addition to the recombinant cosmids. This restoration of high siderophore production and ability to grow under iron-limiting conditions was achieved even when the indigenous plasmids possessed lesions in genes involved in siderophore activity or in both siderophore and receptor production. Thus, another function mediated by plasmid pJM1, possibly a transacting factor, may play a role in the regulation of siderophore production. Results of experimental infections demonstrated that restoration of the ability to grow under conditions of iron limitations by introduction of an recombinant clone into one of the low-virulence V. anguillarum strains was correlated with an increase in bacterial pathogenicity.     

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.     

Subcloning, expression, and purification of the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase: demonstration of enzyme-bound (2,3-dihydroxybenzoyl)adenylate product

The gene coding for the enzyme 2,3-dihydroxybenzoate-AMP ligase (2,3DHB-AMP ligase), responsible for activating 2,3-dihydroxybenzoic acid in the biosynthesis of the siderophore enterobactin, has been subcloned into the multicopy plasmid pKK223-3 and overproduced in a strain of Escherichia coli. The protein is an alpha 2 dimer with subunit molecular mass of 59 kDa. The enzyme catalyzes the exchange of [32P]pyrophosphate with ATP, dependent upon aromatic substrate with a turnover number of 340 min-1. The enzyme also releases pyrophosphate upon incubation with 2,3-dihydroxybenzoic acid and ATP; an initial burst corresponding to 0.7 nmol of pyrophosphate released per nanomole of enzyme is followed by a slower, continuous release with a turnover number of 0.41 min-1. The 1000-fold difference in rates observed between ATP-pyrophosphate exchange and continuous pyrophosphate release, as well as the close to stoichiometric amount of pyrophosphate released, suggests that intermediates are accumulating on the enzyme surface. Such intermediates have been observed and correspond to enzyme-bond (2,3-dihydroxybenzoyl)adenylate product.