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ABSTRACTThe ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBAand the production of the siderophore anguibactin. Therefore, study of the expression of the angRgene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatAgene, is part of a polycistronic transcript that also includes the iron transport genesfatDCBAand angT, a gene located downstream ofangRwhich showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angRmutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angRmodifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5′-end region of the angRgene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms ofV. anguillarum. We also show in this study that theangTgene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.

The possession of specialized iron transport systems may be crucial for bacteria to override the iron limitation imposed by the host or the environment. One of the most commonly found strategies evolved by microorganisms is the production of siderophores, low-molecular-weight iron chelators that have very high constants of association for their complexes with iron. Thus, siderophores act as extracellular solubilizing agents for iron from minerals or organic compounds, such as transferrin and lactoferrin in the host vertebrate, under conditions of iron limitation. Transport of iron into the cell cytosol is mediated by specific membrane receptor and transport systems which recognize the iron-siderophore complexes. In this review I have analyzed in detail three siderophore-mediated iron uptake systems: the plasmid-encoded anguibactin system of Vibrio anguillarum, the aerobactin-mediated iron assimilation system present in the pColV-K30 plasmid and in the chromosomes of many enteric bacteria, and the chromosomally encoded enterobactin iron uptake system, found in Escherichia coli, Shigella spp., Salmonella spp., and other members of the family Enterobacteriaceae. The siderophore systems encoded by Pseudomonas aeruginosa, namely, pyochelin and pyoverdin, as well as the siderophore amonabactin, specified by Aeromonas hydrophila, are also discussed. The potential role of siderophore-mediated systems as virulence determinants in the specific host-bacteria interaction leading to disease is also analyzed with respect to the influence of these systems in the expression of other factors, such as toxins, in the bacterial virulence repertoire.

A 7.5-kilobase-pair multiresistance transposon, Tn1331, harboring amikacin resistance was identified as part of Klebsiella pneumoniae plasmid pJHCMW1. Restriction mapping, hybridization, and transposition complementation experiments demonstrated that Tn1331 belongs to the Tn3 family. Its structure is similar to that of Tn3 with the insertion of a DNA fragment encoding resistance to amikacin, kanamycin, and tobramycin.

The replication terminus of the plasmid R6K has been cloned into the single-stranded DNA phage vector M13mp5 and also into the plasmid vectors pBR313 and PBR322. By using single-stranded DNA templates prepared from the recombinant DNA clones, the sequence of 215 base pairs of DNA containing the replication terminus has been determined. The DNA sequence of the region of the terminus does not contain any 2-fold rotational symmetry. Therefore, folding of the DNA at the region of the terminus is unlikely to be a cause for replication termination. Interaction of a host-specified protein(s) with the sequence of the replication terminus is probably the basis of the mechanism of replicaion termination.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.

Deoxyribonucleic acid-deoxyribonucleic acid hybridization studies reveal that the plasmids coding for the production of heat stable and heat labile enteroxtoxins of Escherichia coli, regardless of their origin, have a majority of their polynucleotide sequences in common, but are not related in any significant way to those plasmids coding for the synthesis of only ST toxin. The heat stable and heat labile plasmids also share a significant degree of their polynucleotide sequences with plasmids of the FI and FII incompatibility groups, but not with R factors belonging to the I, N, W, P, or X incompatibility groups.

The negative regulation of the expression of iron transport genes fatA and fatB in Vibrio anguillarum is mediated by a chromosome-encoded Fur protein and a plasmid pJM1-derived antisense RNA (RNAalpha), which is preferentially expressed under iron-rich conditions. In this work, we characterized the RNAalpha promoter region, and by using promoter fusion and rifampicin experiments we were able to demonstrate that iron regulates RNAalpha synthesis posttranscriptionally by stabilizing RNAalpha half-life rather than enhancing transcription initiation. The Fur protein is also essential for RNAalpha synthesis at the transcription initiation level, independently of the iron status of the cell. From experiments assessing the relative contribution of Fur and RNAalpha, we were able to show that RNAalpha may indeed play an important role on the negative regulation of the expression of the iron transport genes under physiological conditions.

The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur protein and the antisense RNA, RNAalpha. Here we report the identification of the promoter for the iron transport genes and studied the interaction between the V. anguillarum Fur protein and this promoter. The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1 mapping analysis. High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constitutive in the Fur-deficient strain. Gel retardation and DNase I footprint analysis showed that Fur binds specifically to two contiguous sites comprising the promoter region and the region downstream of the transcription start site. The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fur protein. DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hypersensitivity to the nuclease in the nontemplate strand. The periodicity of the hypersensitive sites suggested that the promoter DNA undergoes a structural change upon binding to Fur, which might play a role in the repression of gene expression.

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.

By using Southern blot hybridization procedures, we found that a specific sequence within a 16.3-kilobase HindIII restriction fragment of pColV-K30 was also present in at least three other pColV-K30 HindIII fragments. Restriction endonuclease mapping of these HindIII fragments indicated that two of these repeated sequences, identified as IS1-like, occur in reverse orientation adjacent to the ends of the aerobactin iron uptake region, also included in the 16.3-kilobase HindIII fragment. There are two distinct replication regions enclosing the IS1-flanked aerobactin genes. A pColV-K30 BamHI restriction endonuclease fragment, carrying one of these replication regions, showed homology with the F plasmid EcoRI fragment f5, which carries the F replication sequences.

The aerobactin-mediated iron uptake system encoded by pColV-K30 and other ColV plasmids has been associated with the ability of Escherichia coli strains to cause disease. We investigated whether the pColV-K30 aerobactin system is present in E. coli K1 VW187 isolated from a human neonate with meningitis. This strain exhibited a functional aerobactin-mediated iron uptake system, as assessed by a cross-feeding bioassay and by its sensitivity to cloacin, a bacteriocin that recognizes the outer membrane receptor for iron-aerobactin complexes. By using a variety of techniques, we could not find any plasmid harboring the aerobactin genes. Hybridization of restriction endonuclease-cleaved chromosomal DNA from strain VW187 with various clones containing subsets of the pColV-K30 aerobactin region showed that the aerobactin genes were located on a 10.5-kilobase-pair chromosomal HindIII restriction fragment which also contained IS1-like insertion sequences. The chromosomal aerobactin region showed a high degree of conservation when compared with the homologous region in plasmid pColV-K30, although it was located on a different restriction endonuclease site environment.

Covalently closed circular deoxyribonucleic acid molecules of RSF1030, a nonconjugative R-factor, initiate and complete rounds of semiconservative replication in the absence of protein synthesis long after the bacterial chromosome has ceased its replication. RSF1030 replication under these conditions is sensitive to the inhibitor of ribonucleic acid synthesis, rifampicin. The product of this replication, a covalently closed DNA molecule, shows, in contrast to those molecules produced during the replication in a logarithmically growing culture of Escherichia coli, a transition to the open circular form upon treatment with ribonucleases of alkali. Analysis of the product resulting from alkali treatment indicates that there is a single break in one strand of the original circular duplex. This alkali-sensitive site occurs with equal probability in either of the complementary strands. These results are interpreted as a requirement for an RNA primer for the initiation of RSF1030 DNA synthesis and as showing that its removal from the covalently closed molecule is inhibited in the absence of protein synthesis.

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

The virulence of the fish pathogen Vibrio anguillarum 775 is mediated by the pJM1 plasmid-specified iron uptake system which is expressed under conditions of iron limitation. Other V. anguillarum strains isolated from various geographical locations harbor plasmids that are highly related to pJM1 and that are also associated with the high-virulence phenotype of these strains. In this work, we found that a pJM1-like plasmid, pJHC1, from one of these virulent strains encoded an iron uptake system that resulted in an increased level of production of the siderophore anguibactin. The gene(s) responsible for increased anguibactin production was included within the iron uptake region of plasmid pJHC1. The cloned iron uptake regions of pJHC1 and pJM1 possessed identical restriction endonuclease maps, suggesting that the DNA region encoding those genes in pJHC1 may have diverged subtly from that in pJM1. Analysis of the iron uptake system from other V. anguillarum strains carrying pJM1-like plasmids demonstrated that strains originating from diseased fish from the Atlantic coast carry plasmids encoding an increased-siderophore-production phenotype, while strains isolated from Pacific Ocean locations behaved as the 775 strain.

Cells of the marine fish pathogen Vibrio anguillarum 775 harboring a plasmid associated with virulence can grow unaffected in the presence of iron-binding compounds such as transferrin. In contrast, the growth of isogenic plasmidless derivatives is inhibited by the presence of iron chelators. Radioactive from (55Fe3+) uptake experiments indicate that this plasmid-linked ability of V. anguillarum cells to grow under conditions of iron limitation is indeed due to a more rapid and efficient iron uptake mediated by the virulence plasmid. In addition, V. anguillarum cells growing under iron limitation show at least two novel outer membrane proteins. One of them, a 86,000-dalton protein we called OM2, is inducible only in those cells in which the virulence plasmid is present.

Analysis of the plasmid deoxyribonucleic acid complement of high- and low- virulent strains of the fish pathogen Vibrio anguillarum showed a correlation between enhanced virulence and the presence of a 50-megadalton plasmid class. All 50-megadalton plasmids isolated from different high-virulent V. anguillarum strains were homologous as judged by the analysis of plasmid deoxyribonucleic acid-deoxyribonucleic acid hybridization. The 50-megadalton plasmid class did not have polynucleotide sequences in common with plasmids of different incompatibility groups.

Representative strains of marine vibrios pathogenic for fish were shown to be resistant to the bactericidal activity of normal (nonimmmune) rainbow trout (Salmo gairdneri) serum, and loss of this resistance coincided with a marked reduction in virulence. Thermal lability and a requirement for Mg2+, but not for Ca2+, suggested that a mechanism for serum killing was the alternative complement pathway. In the case of Vibrio anguillarum, serum resistance was not coded for by the virulence plasmid pJM1. Additional testing showed that these pathogenic vibrios were able to agglutinate a variety of eucaryotic cells and that selected strains agglutinated trout erythrocytes; however, a correlation between strain virulence and the ability to agglutinate fish erythrocytes was not apparent. Moreover, whereas mannose was found to inhibit the agglutinating activity of several strains, two of the high-virulence strains displayed a transient, mannose-resistant hemagglutinating activity. No relationship between the carriage of pJM1 by the V. anguillarum strains and hemagglutinating activity was demonstrable.

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.

We have identified a 110-kDa polypeptide that has a trans-acting regulatory activity on the expression of the pJM1 plasmid iron-uptake genes in Vibrio anguillarum. This protein is encoded by the angR gene and maps in a 3.6-kilobase-pair pJM1 DNA region located downstream of the iron transport genes. Full expression of this gene occurs under iron-limiting conditions and requires a 2.9-kilobase-pair upstream region in cis that maps within the coding region of the OM2 outer membrane protein, essential for the transport of iron into the cell cytosol. Determination of the siderophore anguibactin levels as well as analysis of specific transcripts for anguibactin biosynthetic genes demonstrated that AngR and another transcriptional activator, Taf, regulate in a synergistic fashion the level of anguibactin production by activation of transcription of the anguibactin biosynthetic genes under iron-limiting conditions.

On the basis of cultural and biochemical properties as well as DNA homology assays, 81 Vibrio strains isolated from diseased striped bass and from Chesapeake Bay water were assigned to eight distinct groups. All organisms belonging to two of the groups were pathogenic for striped bass and were identified as Vibrio anguillarum, whereas organisms classified in the other six groups were nonpathogenic and were designated as Vibrio spp. Unlike the pathogenic V. anguillarum strain 775 isolated in the Pacific Northwest, strains pathogenic for striped bass did not contain any plasmids; however, they were similar to the Northwest isolates in that virulence was correlated with their ability to grow in the presence of nonimmune striped bass serum or under conditions of iron limitation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of outer membranes showed that additional proteins were induced in those organisms capable of growth under conditions of iron limitation. It was of interest that 22 of the nonpathogenic isolates harbored one or more plasmids which, by restriction endonuclease analyses, were shown to be clearly different from the virulence plasmid pJM1.

We sequenced the minimum REPI replication region and the incompatibility determinants of pColV-K30. The minimum replication region contains an open reading frame which corresponds to a 35-kilodalton (kDa) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis with maxicells transformed with a number of deletion derivatives demonstrated that this replication region encodes a 39-kDa protein and also established the direction of transcription of the RepI protein gene. The 39-kDa polypeptide was identified as the trans-acting factor essential for replication of REPI-containing plasmids. A translated region of the nucleotide sequence of the RepI protein gene showed homology with the helix-turn-helix binding domains of a number of DNA-binding proteins and also with other plasmid replication proteins. Further nucleotide analysis of the REPI region revealed the presence of direct and inverted repeat sequences in the incE, incF, and ori regions. The REPI ori also contained a perfect DnaA-binding site in addition to a high frequency of occurrence of the DNA adenine methylation (dam) site 5'GATC3'.

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.

Clones carrying the iron uptake region of the Vibrio anguillarum plasmid pJM1 were subjected to insertion mutagenesis, using transposon Tn3::HoHo1 which carries a promoterless lacZ gene and can thus generate lacZ transcriptional fusions if inserted downstream from an indigenous promoter. Four classes of insertion mutants were obtained based on the level of expression of components of the iron uptake system, and six genetic units were defined according to the phenotype of the mutants. Five of the six genetic units were crucial for biosynthesis of the siderophore anguibactin. Insertions in the remaining genetic unit led to an iron uptake-deficient phenotype and showed either reduced levels of the outer membrane protein OM2 as well as anguibactin activity or a complet shutoff of both OM2 and anguibactin biosyntheses. Analysis of beta-galactosidase production by cells carrying the lacZ fusion derivatives identified iron-regulated and constitutive transcriptional units as well as their orientation in the genetic units. Molecular cloning of pJM1 plasmid DNA noncontiguous to the iron uptake region also identified genetic determinants for a trans-acting factor required for full expression of anguibactin activity. Evidence obtained from bioassays, spectrophotometric measurements, and the lacZ fusion mutants suggested that the trans-acting factor is a novel activator of siderophore biosynthesis at the transcriptional level.

The incidence of the aerobactin system and the genetic location of aerobactin genes were investigated in Escherichia coli K1 neonatal isolates belonging to different clonal groups. A functional aerobactin system was found in all members of the O7 MP3, O1 MP5, O1 MP9, and O18 MP9 clonal groups examined and also in K1 strains having O6, O16, and O75 lipopolysaccharide types, which are less frequently associated with neonatal infections. In contrast, the aerobactin system was not detected in strains from the O18 MP6 clone. The combined results of plasmid and colony hybridization experiments showed that the aerobactin genes were located on the chromosome in the majority (75%) of the aerobactin-producing K1 isolates, the genetic location of the aerobactin genes was closely correlated with the outer membrane protein profile rather than the O lipopolysaccharide type, the K1 strains harboring a chromosome-mediated aerobactin system did not possess colicin V genes, and five of six K1 isolates possessing a plasmid-borne aerobactin system contained colicin V genes which were located on the same plasmids carrying the aerobactin genes. The comparison of hemolysin production with possession of the aerobactin system in virulent clones of E. coli K1 strains showed that all of the aerobactin-producing strains from the O18 MP9 and O7 MP3 clonal groups did not synthesize hemolysin, whereas 11 of 12 aerobactin-nonproducing O18 MP6 isolates were hemolytic. Of the K1 strains examined, 92.5% possessed either the aerobactin system or the ability to produce hemolysin or both.

A multiresistant Klebsiella pneumoniae strain isolated from neonates in Mendoza, Argentina, harbored a 48-kilobase-pair (kbp) plasmid, pMET1, with genetic determinants for resistance to amikacin and also ampicillin, kanamycin, streptomycin, and tobramycin. This plasmid was compared with pJHCMW1, a previously isolated 11-kbp plasmid carrying transposon Tn1331, which encodes resistance to amikacin, as well as ampicillin, kanamycin, streptomycin, and tobramycin, and which was originally present in a K. pneumoniae strain that caused an outbreak in a hospital in Buenos Aires, Argentina. The comparison demonstrated that the replication regions of the two plasmids are unrelated. However, in pMET1 an 11-kbp transposition element, Tn1331.2, was identified; it was closely related to Tn1331, with the difference that a 3-kbp BamHI DNA fragment carrying the aminoglycoside resistance genes was duplicated in tandem.