scholarly journals Yersinia pestis YbtU and YbtT Are Involved in Synthesis of the Siderophore Yersiniabactin but Have Different Effects on Regulation

2000 ◽  
Vol 68 (8) ◽  
pp. 4452-4461 ◽  
Author(s):  
Valerie A. Geoffroy ◽  
Jacqueline D. Fetherston ◽  
Robert D. Perry
Keyword(s):  
Yersinia Pestis ◽  
Iron Transport ◽  
Significant Loss ◽  
Gene Products ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Iron Deficient ◽  
High Pathogenicity ◽  
Mutant Strains ◽  
Culture Supernatants

ABSTRACT One prerequisite for the virulence of Yersinia pestis, causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of theY. pestis chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of ybtU and ybtT. In-frame mutations in ybtT or ybtU yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from a Ybt-producing strain of Y. pestis. TheybtU mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for otherybt biosynthetic mutants. In contrast, strains carrying mutations in ybtT or ybtS (a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of ybtT, -U, and -Smutations on transcription of ybt genes, reporter plasmids with ybtP or psn promoters controllinglacZ expression were introduced into these mutants. Normal iron-regulated β-galactosidase activity was observed in theybtT and ybtS mutants, whereas a significant loss of expression occurred in the ΔybtU strain. These results show that ybtT and ybtU genes are involved in the biosynthesis of the Ybt siderophore and that aybtU mutation but not ybtT or ybtSmutations affects transcription from the ybtP andpsn promoters.

scholarly journals Yersiniabactin Production Requires the Thioesterase Domain of HMWP2 and YbtD, a Putative Phosphopantetheinylate Transferase

2002 ◽  
Vol 70 (8) ◽  
pp. 4204-4214 ◽  
Author(s):  
Alexander G. Bobrov ◽  
Valerie A. Geoffroy ◽  
Robert D. Perry
Keyword(s):  
Iron Transport ◽  
Iron Status ◽  
Single Amino Acid ◽  
Gene Products ◽  
Bacterial Cells ◽  
High Molecular Weight Protein ◽  
Iron Deficient ◽  
Weight Protein ◽  
Nonribosomal Peptide Synthesis ◽  
Culture Supernatants

ABSTRACT One requirement for the pathogenesis of Yersinia pestis, the causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Nine gene products within the HPI have demonstrated functions in the nonribosomal peptide synthesis (NRPS)/polyketide (PK) synthesis or transport of Ybt. NRPS/PK synthetase or synthase enzymes are generally activated by phosphopantetheinylation. However, no products with similarities to known phosphopantetheinyl (P-pant) transferases were found within the pgm locus. We have identified a gene, ybtD, encoded outside the HPI and pgm locus, that is necessary for function of the Ybt system and has similarities to other P-pant transferases such as EntD of Escherichia coli. A deletion within ybtD yielded a strain (KIM6-2085+) defective in siderophore production. This strain was unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from Ybt-producing strains of Y. pestis. The promoter region of ybtD was fused to lacZ; β-galactosidase expression from this reporter was not regulated by the iron status of the bacterial cells or by YbtA, a positive regulator of other genes of the ybt system. The ybtD mutant failed to express indicator Ybt proteins (high-molecular-weight protein 1 [HMWP1], HMWP2, and Psn), a pattern similar to those seen with several other ybt biosynthetic mutants. In contrast, cells containing a single amino acid substitution (S2908A) in the terminal thioesterase domain of HMWP2 failed to exhibit any ybt regulatory defects but did not elaborate extracellular Ybt under iron-deficient conditions.

scholarly journals Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1407-1415 ◽  
Author(s):  
James E. A. Zlosnik ◽  
Gholam Reza Tavankar ◽  
Jacob G. Bundy ◽  
Dimitris Mossialos ◽  
Ronan O'Toole ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Toxic Agent ◽  
Stationary Phase Culture ◽  
Prolonged Incubation ◽  
Mutant Strains ◽  
Electron Sink ◽  
Culture Supernatants

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO. It was found that cyanide is produced in P. aeruginosa at similar levels irrespective of its complement of CIO, indicating that the CIO is not an obligatory electron sink for cyanide synthesis. However, MICs for cyanide and growth in its presence demonstrated that the CIO provides P. aeruginosa with protection against the effects of exogenous cyanide. Nevertheless, the presence of cyanide did not affect the viability of cio mutant strains compared to the wild-type during prolonged incubation in stationary phase. The detection of the fermentation end products acetate and succinate in stationary-phase culture supernatants suggests that P. aeruginosa, irrespective of its CIO complement, may in part rely upon fermentation for energy generation in stationary phase. Furthermore, the decrease in cyanide levels during incubation in sealed flasks suggested that active breakdown of HCN by the culture was taking place. To investigate the possibility that the CIO may play a role in pathogenicity, wild-type and cio mutant strains were tested in the paralytic killing model of Caenorhabditis elegans, a model in which cyanide is the principal toxic agent leading to nematode death. The CIO mutant had delayed killing kinetics, demonstrating that the CIO is required for full pathogenicity of P. aeruginosa in this animal model.

scholarly journals Role of the ssu and seu Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources

2005 ◽  
Vol 71 (10) ◽  
pp. 6104-6114 ◽  
Author(s):  
D. J. Koch ◽  
C. Rückert ◽  
D. A. Rey ◽  
A. Mix ◽  
A. Pühler ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Flavin Mononucleotide ◽  
Gene Products ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
Similarity Searches

ABSTRACT Corynebacterium glutamicum ATCC 13032 was found to be able to utilize a broad range of sulfonates and sulfonate esters as sulfur sources. The two gene clusters potentially involved in sulfonate utilization, ssuD1CBA and ssuI-seuABC-ssuD2, were identified in the genome of C. glutamicum ATCC 13032 by similarity searches. While the ssu genes encode proteins resembling Ssu proteins from Escherichia coli or Bacillus subtilis, the seu gene products exhibited similarity to the dibenzothiophene-degrading Dsz monooxygenases of Rhodococcus strain IGTS8. Growth tests with the C. glutamicum wild-type and appropriate mutant strains showed that the clustered genes ssuC, ssuB, and ssuA, putatively encoding the components of an ABC-type transporter system, are required for the utilization of aliphatic sulfonates. In C. glutamicum sulfonates are apparently degraded by sulfonatases encoded by ssuD1 and ssuD2. It was also found that the seu genes seuA, seuB, and seuC can effectively replace ssuD1 and ssuD2 for the degradation of sulfonate esters. The utilization of all sulfonates and sulfonate esters tested is dependent on a novel putative reductase encoded by ssuI. Obviously, all monooxygenases encoded by the ssu and seu genes, including SsuD1, SsuD2, SeuA, SeuB, and SeuC, which are reduced flavin mononucleotide dependent according to sequence similarity, have SsuI as an essential component. Using real-time reverse transcription-PCR, the ssu and seu gene cluster was found to be expressed considerably more strongly during growth on sulfonates and sulfonate esters than during growth on sulfate.

scholarly journals Generation of Yersinia pestis Attenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates

2004 ◽  
Vol 72 (2) ◽  
pp. 908-915 ◽  
Author(s):  
Yehuda Flashner ◽  
Emanuelle Mamroud ◽  
Avital Tidhar ◽  
Raphael Ber ◽  
Moshe Aftalion ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Wide Spectrum ◽  
Wild Type ◽  
Bacterial Physiology ◽  
Vaccine Candidates ◽  
Time To Death ◽  
Mutant Strains ◽  
Antibody Titers ◽  
Virulence Regulator

ABSTRACT In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10−5 to 10−7 to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >107 CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.

scholarly journals Quorum-Sensing Signal Synthesis by the Yersinia pestis Acyl-Homoserine Lactone Synthase YspI

2006 ◽  
Vol 188 (2) ◽  
pp. 784-788 ◽  
Author(s):  
J. Paul Kirwan ◽  
Ty A. Gould ◽  
Herbert P. Schweizer ◽  
Scott W. Bearden ◽  
Robert C. Murphy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Yersinia Pestis ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Liquid Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Physical Chemical ◽  
Signal Synthesis ◽  
Chemical Techniques ◽  
Culture Supernatants

ABSTRACT The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-l-homoserine lactone and N-3-oxo-hexanoyl-l-homoserine lactone.

scholarly journals Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

2005 ◽  
Vol 187 (20) ◽  
pp. 7018-7026 ◽  
Author(s):  
Hamid Manyani ◽  
Luis Rey ◽  
José M. Palacios ◽  
Juan Imperial ◽  
Tomás Ruiz-Argüeso
Keyword(s):  
Rhizobium Leguminosarum ◽  
Hydrogenase Activity ◽  
Gene Products ◽  
Structural Genes ◽  
Wild Type ◽  
Free Living ◽  
Iron Sulfur ◽  
Specific Antisera ◽  
Mutant Strains ◽  
Isogenic Mutants

ABSTRACT In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (ΔhupG, ΔhupH, ΔhupI, and ΔhupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The ΔhupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the ΔhupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from ΔhupH, ΔhupI, and ΔhupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the ΔhupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3857-3866 ◽  
Author(s):  
Michelle R. Rondon ◽  
Katie S. Ballering ◽  
Michael G. Thomas
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Biosynthetic Gene Cluster ◽  
Filamentous Cyanobacterium ◽  
Wild Type ◽  
Reactive Material ◽  
Iron Deficient ◽  
Mutant Strains

Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.

scholarly journals H-NS and StpA Proteins Stimulate Expression of the Maltose Regulon in Escherichia coli

1998 ◽  
Vol 180 (23) ◽  
pp. 6117-6125 ◽  
Author(s):  
Jörgen Johansson ◽  
Björn Dagberg ◽  
Evelyne Richet ◽  
Bernt Eric Uhlin
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Receptor Protein ◽  
Gene Products ◽  
Wild Type ◽  
Positive Role ◽  
Homologous Protein ◽  
Expression Of Genes ◽  
Mutant Strains

ABSTRACT The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in Escherichia coli. Its role in gene regulation is manifested by the increased expression of several gene products in hns mutant strains. Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon. In studies with hns mutant strains and derivatives also deficient in the stpA gene, we found that expression of the LamB porin was decreased. Our results showed that the amounts of both LamB protein and lamB mRNA were greatly reduced inhns and hns-stpA mutant strains. The same results were obtained when we monitored the amount of transcription from the malEFG operon. The lamB gene is situated in the malKlamBmalM operon, which forms a divergent operon complex together with the malEFG operon. The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein. Using a malT-lacZ translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in hns andhns-stpA mutant strains in comparison with the wild-type strain. Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon.

scholarly journals Genetic interactions among floral homeotic genes of Arabidopsis

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 1-20 ◽  
Author(s):  
J.L. Bowman ◽  
D.R. Smyth ◽  
E.M. Meyerowitz
Keyword(s):  
Field Model ◽  
Homeotic Genes ◽  
Gene Products ◽  
Wild Type ◽  
Allelic Series ◽  
Mature Flower ◽  
Mutant Strains ◽  
In The Wild ◽  
Mutant Lines ◽  
New Mutations

We describe allelic series for three loci, mutations in which result in homeotic conversions in two adjacent whorls in the Arabidopsis thaliana flower. Both the structure of the mature flower and its development from the initial primordium are described by scanning electron microscopy. New mutations at the APETALA2 locus, ap2-2, ap2-8 and ap2-9, cause homeotic conversions in the outer two whorls: sepals to carpels (or leaves) and petals to stamens. Two new mutations of PISTILLATA, pi-2 and pi-3, cause second and third whorl organs to differentiate incorrectly. Homeotic conversions are petals to sepals and stamens to carpels, a pattern similar to that previously described for the apetala3-1 mutation. The AGAMOUS mutations, ag-2 and ag-3, affect the third and fourth whorls and cause petals to develop instead of stamens and another flower to arise in place of the gynoecium. In addition to homeotic changes, mutations at the APETALA2, APETALA3 and PISTILLATA loci may lead to reduced numbers of organs, or even their absence, in specific whorls. The bud and flower phenotypes of doubly and triply mutant strains, constructed with these and previously described alleles, are also described. Based on these results, a model is proposed that suggests that the products of these homeotic genes are each active in fields occupying two adjacent whorls, AP2 in the two outer whorls, PI and AP3 in whorls two and three, and AG in the two inner whorls. In combination, therefore, the gene products in these three concentric, overlapping fields specify the four types of organs in the wild-type flower. Further, the phenotypes of multiple mutant lines indicate that the wild-type products of the AGAMOUS and APETALA2 genes interact antagonistically. AP2 seems to keep the AG gene inactive in the two outer whorls while the converse is likely in the two inner whorls. This field model successfully predicts the phenotypes of all the singly, doubly and triply mutant flowers described.

Identification of a baculovirus polyhedron formation mutant with a novel phenotype

1996 ◽  
Vol 54 ◽  
pp. 796-797
Author(s):  
James M. Slavicek ◽  
Melissa J. Mercer ◽  
Mary Ellen Kelly
Keyword(s):  
Viral Gene ◽  
Gene Products ◽  
Type Number ◽  
Infection Cycle ◽  
Wild Type ◽  
Protein Matrix ◽  
Insect Midgut ◽  
Viral Particles ◽  
Budded Virus ◽  
Viral Form

Nucleopolyhedroviruses (NPV, family Baculoviridae) produce two morphological forms, a budded virus form and a viral form that is occluded into a paracrystalline protein matrix. This structure is termed a polyhedron and is composed primarily of the protein polyhedrin. Insects are infected by NPVs after ingestion of the polyhedron and release of the occluded virions through dissolution of the polyhedron in the alkaline environment of the insect midgut. Early after infection the budded virus form is produced. It buds through the plasma membrane and then infects other cells. Later in the infection cycle the occluded form of the virus is generated (reviewed by Blissard and Rohrmann, 1990).The processes of polyhedron formation and virion occlusion are likely to involve a number of viral gene products. However, only two genes, the polyhedrin gene and 25K FP gene, have been identified to date that are necessary for the wild type number of polyhedra to be formed and viral particles occluded.

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