Shelley M. Payne

Title:	Professor	Video - PC  Mac
Education:	Ph.D.: 1977, The University of Texas Southwestern Medical Center at Dallas; B.A.: 1972, Rice University
Postdoc.:	University of California at Berkeley
Research:	Pathogenic mechanisms of Shigella, E. coli, and Vibrio cholerae; Genetics of bacterial iron transport systems
Office:	NMS 2.124
Phone:	(512) 471-9258     Fax: 471-9247
E-mail:	payne@mail.utexas.edu
Postal Address:	The University of Texas at Austin Molecular Genetics & Microbiology 1 University Station  A5000 Austin TX 78712-0162
Courses taught:
Laboratory home page: http://www.sbs.utexas.edu/paynelab/

Our studies focus on the genetics and regulation of iron acquisition systems and other virulence factors of Shigella and Vibrio species. The iron transport systems are of interest because the ability of potential pathogens to acquire iron from the host is an important determinant of microbial virulence. Iron is required for growth of bacteria, but little free iron is available in mammalian hosts. Many bacteria have been found to secrete high affinity iron-binding compounds, or siderophores, which may function to remove iron from host proteins and make it available to the microorganism for growth in vivo.

Vibrio species produce a variety of siderophores and iron-regulated outer membrane proteins. In response to iron deprivation, Vibrio cholerae synthesizes a norspermadine containing siderophore, and five cell surface proteins, including receptors for the siderophore and for heme. The Shigella species, a group of enteric pathogens, have at least three distinct iron transport systems. Two of these, the aerobactin and enterobactin systems, consist of siderophores and their associated outer membrane receptor proteins. Expression of these systems is regulated at the level of transcription by the concentration of iron within the cell. A third pathway is required for the transport and utilization of iron in the form of heme. Virulence assays of mutants defective in one or more of these systems suggest that siderophores provide iron in the extracellular environment while heme transport is utilized by Shigella growing intracellularly within epithelial cells. A heme-binding protein is found on the surface of wild-type Shigellae. Although this protein is not required for heme transport, it is required for invasion of intestinal epithelial cells by S. flexneri. The protein allows the bacteria to bind large amounts of heme on the surface of the bacteria and promotes the attachment of the bacteria to the host cells. This protein is encoded on a large 220 kilobase plasmid, and its synthesis is regulated by both temperature and pH. Analysis of regulatory mutants indicates that at least two loci, one chromosomal and one plasmid encoded, are responsible for the regulation of the structural gene for this cell surface protein. Genetic and recombinant DNA techniques are being used to characterize chromosomal and plasmid sequences involved in iron transport and invasion in these enteric pathogens. The genes have been cloned and gene fusions have been constructed to measure the expression of these genes under different environmental conditions. Techniques have been developed also to measure gene expression and synthesis of virulence-associated proteins by S. flexneri growing within host cells. These studies will allow us to determine the molecular mechanisms of iron acquisition and ultimately to assess the roles of these systems in bacterial infections.
 

Role of the Pst system in plaque formation by the intracellular pathogen Shigella flexneri.
Runyen-Janecky LJ, Boyle AM, Kizzee A, Liefer L, Payne SM.  
Infection and Immunity . 2005 Mar;73(3):1404-10.

ABSTRACT:  In response to the host cell environment, the intracellular pathogen Shigella flexneri induces the expression of numerous genes, including those in the pst operon which is predicted to encode a high-affinity phosphate acquisition system that is expressed under reduced phosphate conditions. An S. flexneri pst mutant forms smaller plaques in Henle cell monolayers than does the parental strain. This mutant exhibited normal production and localization of the S. flexneri IcsA protein. The pst mutant had the same growth rate as the parental strain in both phosphate-reduced and phosphate-replete media in vitro and during the first 3 h of growth in Henle cells in vivo. During growth in phosphate-replete media, the PhoB regulon was constitutively expressed in the pst mutant but not the parental strain. This suggested that the inability of the S. flexneri pst mutant to form wild-type plaques in Henle cell monolayers may be due to aberrant expression of the PhoB regulon. A mutation in phoB was constructed in the S. flexneri pst mutant, and the phoB mutation suppressed the small plaque phenotype of the pst mutant. Additionally, a specific mutation (R220Q) was constructed in the pstA gene of the pst operon that was predicted to eliminate Pst-mediated phosphate transport but allow normal PhoB-regulated gene expression, based on the phenotype of an Escherichia coli strain harboring the same mutation. Addition of this pstA(R220Q) mutation to a S. flexneri pst mutant, as part of the pst operon, restored normal plaque formation and regulation of phoA expression.

HutZ is required for efficient heme utilization in Vibrio cholerae.
Wyckoff EE, Schmitt M, Wilks A, Payne SM. 
Journal of Bacteriology. 2004 Jul;186(13):4142-51.

ABSTRACT: 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.

Complete Genome Sequence and Comparative Genomics of Shigella flexneri Serotype 2a Strain 2457T. Wei J, Goldberg MB, Burland V, Venkatesan MM, Deng W, Fournier G, Mayhew GF, Plunkett III G, Rose DJ, Darling A, Mau B, Perna NT, Payne SM, Runyen-Janecky LJ, Zhou S, Schwartz DC, Blattner FR. (2003).
Infection and Immunity 71(5):2775-2786.

ABSTRACT: We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.

Contribution of the Shigella flexneri Sit, Iuc, and Feo Iron Acquisition Systems to Iron Acquisition In Vitro and in Cultured Cells.
Runyen-Janecky LJ, Reeves SA, Gonzales EG, Payne SM. (2003). 
Infection and Immunity 71(4):1919-28.

ABSTRACT: Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.

Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors.
Mey AR and Payne SM. 2003.
Journal of Bacteriology. 185: 1195-207.

ABSTRACT: In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex. Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V. cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors. To investigate the receptor specificity exhibited by V. cholerae TonB1, chimeras were created between V. cholerae TonB1 and E. coli TonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus of V. cholerae TonB1 were identified that allowed TonB1 to recognize E. coli receptors and at least one V. cholerae TonB2-dependent receptor. This indicates that the very C-terminal end of V. cholerae TonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V. cholerae and E. coli receptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box in E. coli receptors with an aromatic residue allowed these receptors to use V. cholerae TonB1. Further, site-directed mutagenesis of the TonB box -1 residue in a V. cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V. cholerae TonB1. These data suggest that the TonB box -1 position controls productive interactions with V. cholerae TonB1.
 

Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread.
Purdy GE, Hong M, Payne SM. 2002.  
Infection and Immunity 70:6355-64.
 

Identification of the Vibrio cholerae Enterobactin Receptors VctA and IrgA: IrgA Is Not Required for Virulence. 
Mey AR, Wyckoff EE, Oglesby AG, Rab E, Taylor RK, Payne SM. 2002. 
Infection and Immunity 70:3419-3426.

Identification of chromosomal Shigella flexneri genes induced by the eukaryotic intracellular environment.
Runyen-Janecky LJ, Payne SM. (2002). 
Infection and Immunity 70(8):4379-88.

ABSTRACT: Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S. flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp). Bacteria containing promoter fusions that had a higher level of gfp expression when S. flexneri was intracellular (in Henle cells) than when S. flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (uhpT, bioA, and lysA) were involved in metabolic processes. The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified. The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S. flexneri uhpT and pstS genes by allelic exchange. The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line.

Haem utilization in Vibrio cholerae involves multiple TonB-dependent haem receptors.
A.R. Mey and S.M. Payne. 2001. 
Molecular Microbiology. 42: 835-849.
 

TonB-dependent systems of uropathogenic Escherichia coli: aerobactin and heme transport and TonB are required for virulence in the mouse.
Torres AG, Redford P, Welch RA, Payne SM. 2001.
Infection and Immunity. 69(10):6179-85.

The SHI-3 Iron Transport Island of Shigella boydii 0-1392 Carries the Genes for Aerobactin Synthesis and Transport. 
Purdy, G.E. and S.M. Payne. 2001.
Journal of Bacteriology. 183:4176-4182.

ABSTRACT: In Shigella boydii 0-1392, genes encoding the synthesis and transport of the hydroxamate siderophore aerobactin are located within a 21-kb iron transport island between lysU and the pheU tRNA gene. DNA sequence analysis of the S. boydii 0-1392 island, designated SHI-3 for Shigella island 3, revealed a conserved aerobactin operon associated with a P4 prophage-like integrase gene and numerous insertion sequences (IS). SHI-3 is present at the pheU tRNA locus in some S. boydii isolates but not in others. The map locations of the aerobactin genes vary among closely related species. The association of the aerobactin operon with phage genes and mobile elements and its presence at different locations within the genomes of enteric pathogens suggest that these virulence-enhancing genes may have been acquired by bacteriophage integration or IS element-mediated transposition. An S. boydii aerobactin synthesis mutant, 0-1392 iucB, was constructed and was similar to the wild type in tissue culture assays of invasion and intercellular spread.

VibD and VibH Are Required for Late Steps in Vibriobactin Biosynthesis in Vibrio cholerae. 
Wyckoff, E.E., S.L. Smith, and S. M. Payne. 2001. 
Journal of Bacteriology. 183(5):1830-1834.

ABSTRACT: Vibrio cholerae synthesizes the catechol siderophore vibriobactin. In this report, we present the complete map of a vibriobactin gene region containing two previously unreported vibriobactin biosynthetic genes. vibD encodes a phosphopantetheinyl transferase, and vibH encodes a novel nonribosomal peptide synthase. Both VibD and VibH are required for vibriobactin biosynthesis.

ABSTRACT: The two TonB systems in Vibrio cholerae were found to have unique as well as common functions. Both systems can mediate transport of haemin and the siderophores vibriobactin and ferrichrome. However, TonB1 specifically mediates utilization of the siderophore schizokinen, whereas TonB2 is required for utilization of enterobactin by V. cholerae. Although either TonB system was sufficient for the use of haemin as an iron source, in vitro competition between TonB1 and TonB2 system mutants indicates a preferential role for TonB1 in haemin utilization. This was most pronounced in conditions of high osmolarity, in which TonB1 system mutants were unable to grow with haemin as the sole iron source. Sequence analysis predicted that the two TonB proteins differ in both amino acid sequence and protein size. An internal deletion in TonB1 was constructed in order to generate a protein of approximately the same size as TonB2. A strain expressing the TonB1 deletion protein, and no other TonB, used haemin as the iron source in low-osmolarity medium, but could not use haemin in high osmolarity. This is the same phenotype as a strain expressing only TonB2 and suggests that TonB1, but not TonB2, can span the increased periplasmic space in high osmolarity and thus mediate haemin transport. Mouse colonization assays indicated a role for both TonB systems, and mutations in either system resulted in reduced ability to compete with the wild type in vivo.

TonB Is Required for Intracellular Growth and Virulence of Shigella dysenteriae.
Reeves,S.A., A.G. Torres, and S.M. Payne. 2000. 
Infection and Immunity 68(11):6329-6336.

ABSTRACT: To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.
 

ABSTRACT: Upon exposure to UV radiation, Shigella flexneri SA100 displayed survival and mutation frequencies comparable to those of Escherichia coli AB1157, which contains a functional UmuDC error-prone DNA repair system. Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP. This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in Salmonella typhimurium, and ImpC, encoded upstream of ImpA and ImpB. Although the impB gene was present in representatives of all four species of Shigella, not all isolates tested contained the gene. Shigella isolates that lacked impB were more sensitive to UV radiation than isolates that contained impB. The nucleotide sequence of a 2.4-kb DNA fragment containing the imp operon from S. flexneri SA100 pVP was 96% identical to the imp operon from the plasmid TP110. An SA100 derivative with a mutation in the impB gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain. We also found that S. flexneri contained a chromosomally encoded umuDC operon; however, the umuDC promoter was not induced by exposure to UV radiation. This suggests that the imp operon but not the umuDC operon contributes to survival and induced mutagenesis in S. flexneri following exposure to UV radiation.

ABSTRACT: Genes encoding the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence of enteric bacteria, were mapped within a pathogenicity island in Shigella flexneri. The island, designated SHI-2 for Shigella pathogenicity island 2, was located downstream of selC, the site of insertion of pathogenicity islands in several other enteric pathogens. DNA sequence analysis revealed the presence of multiple insertion sequences upstream and downstream of the aerobactin genes and an integrase gene that was nearly identical to an int gene found in Escherichia coli O157:H7. SHI-2 sequences adjacent to selC were similar to sequences at the junction between selC and pathogenicity islands found in E. coli O157:H7 and in enteropathogenic E. coli, but the junctions between the island and downstream yic genes were variable. SHI-2 also encoded immunity to the normally plasmid-encoded colicins I and V, suggesting a common origin for the aerobactin genes in both S. flexneri and E. coli pColV. Polymerase chain reaction and Southern hybridization data indicate that SHI-2 is present in the same location in Shigella sonnei, but the aerobactin genes are not located within SHI-2 in Shigella boydii or enteroinvasive E. coli. Shigella dysenteriae type 1 strains do not produce aerobactin but do contain sequences downstream of selC that are homologous to SHI-2. The presence of the aerobactin genes on plasmids in E. coli pColV and Salmonella, on a pathogenicity island in S. flexneri and S. sonnei and in a different chromosomal location in S. boydii and some E. coli suggests that these virulence-enhancing genes are mobile, and they may constitute an island within an island in S. flexneri.

ABSTRACT: Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [(3)H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.

ABSTRACT: Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae.

 

ABSTRACT: 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.

ABSTRACT: 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.

ABSTRACT: The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.

ABSTRACT: The expression of the Shigella flexneri chromosomal aerobactin genes during growth of the bacterium within tissue culture cells was assayed. During intracellular growth, aerobactin promoter activity was repressed relative to the level observed in bacteria grown extracellularly, even when the bacteria had been starved for iron prior to infection. Similarly, the level of one of the proteins encoded by this operon, the aerobactin outer membrane receptor, Iut, was reduced in the intracellular environment. These studies indicate that the aerobactin system is not highly expressed by bacteria within host cells, suggesting that siderophore-independent iron acquisition systems can provide essential iron during intracellular multiplication.

ABSTRACT: In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene (chuA) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependent, Fur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis. A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.