Elizabeth E. Wyckoff

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set (tonB1, exbB1, exbD1) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high‐affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild‐type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild‐type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system (tonB2, exbB2, exbD2). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes (hutBCD), and all six genes appeared to be expressed from a single Fur‐regulated promoter upstream of tonB1. A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae, the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae.

Iron and Fur Regulation in Vibrio cholerae and the Role of Fur in Virulence

ABSTRACT Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli. We found that many of these haem‐utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA. These shuA‐positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem‐utilizing pathogenic E. coli strains did not hybridize with shuA, so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae. The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA. The haem transport locus is not present in E. coli K‐12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K‐12. The junctions and flanking sequences in the shuA‐positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae, indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae, and it is located in the same relative position on the chromosome.

Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging

The mechanism of DNA packaging by dsDNA viruses is not well understood in any system. In bacteriophage P22 only five genes are required for successful condensation of DNA within the capsid. The products of three of these genes, the portal, scaffolding, and coat proteins, are structural components of the precursor particle, and two, the products of genes 2 and 3, are not. The scaffolding protein is lost from the structure during packaging, and only the portal and coat proteins are present in the mature virus particle. These five genes map in a contiguous cluster at the left end of the P22 genetic map. Three additional genes, 4, 10, and 26, are required for stabilizing of the condensed DNA within the capsid. In this report we present the nucleotide sequence of 7461 bp of P22 DNA that contains the five genes required for DNA condensation, as well as a nonessential open reading frame (ORF109), gene 4, and a portion of gene 10. N-terminal amino acid sequencing of the encoded proteins accurately located the translation starts of six genes in the sequence. Despite the fact that most of these proteins have striking analogs in the other dsDNA bacteriophage groups, which perform highly analogous functions, no amino acid sequence similarity between these analogous proteins has been found, indicating either that they diverged a very long time ago or that they are the products of spectacular convergent evolution.

Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II

We have determined the nucleotide sequence of the Drosophila DNA topoisomerase II gene. Data from primer extension and S1 nuclease protection experiments were combined with comparisons of genomic and cDNA sequences to determine the structure of the mature messenger RNA. This message has a large open reading frame of 4341 nucleotides. The length of the predicted protein is 1447 amino acids with a molecular weight of 164,424. Topoisomerase II can be divided into three domains: (1) an N-terminal region with homology to the B (ATPase) subunit of the bacterial type II topoisomerase, DNA gyrase; (2) a central region with homology to the A (breaking and rejoining) subunit of DNA gyrase; (3) a C-terminal region characterized by alternating stretches of positively and negatively charged amino acids. DNA topoisomerase II from the fruit fly shares significant sequence homology with those from divergent sources, including bacteria, bacteriophage T4 and yeasts. The location and distribution of homologous stretches in these sequences are analyzed.

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.

Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid. In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid. To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants. The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined. The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22. Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22. We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.

Characterization of Ferric and Ferrous Iron Transport Systems in Vibrio cholerae

Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

HutZ Is Required for Efficient Heme Utilization in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, requires iron for growth. One mechanism by which it acquires iron is the uptake of heme, and several heme utilization genes have been identified in V. cholerae. These include three distinct outer membrane receptors, two TonB systems, and an apparent ABC transporter to transfer heme across the inner membrane. However, little is known about the fate of the heme after it enters the cell. In this report we show that a novel heme utilization protein, HutZ, is required for optimal heme utilization. hutZ (open reading frame [ORF] VCA0907) is encoded with two other genes, hutW (ORF VCA0909) and hutX (ORF VCA0908), in an operon divergently transcribed from the tonB1 operon. A hutZ mutant grew poorly when heme was provided as the sole source of iron, and the poor growth was likely due to the failure to use heme efficiently as a source of iron, rather than to heme toxicity. Heme oxygenase mutants of both Corynebacterium diphtheriae and C. ulcerans fail to use heme as an iron source. When the hutWXZ genes were expressed in the heme oxygenase mutants, growth on heme was restored, and hutZ was required for this effect. Biochemical characterization indicated that HutZ binds heme with high efficiency; however, no heme oxygenase activity was detected for this protein. HutZ may act as a heme storage protein, and it may also function as a shuttle protein that increases the efficiency of heme trafficking from the membrane to heme-containing proteins.

Iron and Pathogenesis of Shigella: Iron Acquisition in the Intracellular Environment

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.