scholarly journals S-Adenosylmethionine Transport in Rickettsia prowazekii

2003 ◽  
Vol 185 (10) ◽  
pp. 3031-3035 ◽  
Author(s):  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
Lonnie O. Driskell ◽  
David O. Wood
Keyword(s):  
Evolutionary Relationship ◽  
Host Cells ◽  
Transport Systems ◽  
Rickettsia Prowazekii ◽  
Gene Encoding ◽  
E Coli ◽  
Obligate Intracellular ◽  
Gene Coding ◽  
Epidemic Typhus ◽  
Methionine Transport

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a KT of 2.3 μM. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

scholarly journals mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

2007 ◽  
Vol 73 (20) ◽  
pp. 6644-6649 ◽  
Author(s):  
Zhi-Mei Liu ◽  
Aimee M. Tucker ◽  
Lonnie O. Driskell ◽  
David O. Wood
Keyword(s):  
Fluorescent Protein ◽  
Rickettsia Prowazekii ◽  
Obligate Intracellular ◽  
Transposon Insertion ◽  
Gene Coding ◽  
Epidemic Typhus ◽  
Intergenic Regions ◽  
Rifampin Resistance ◽  
Green Fluorescent ◽  
Random Transpositions

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFPUV) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFPUV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFPUV fluorescence.

scholarly journals Lawsonia intracellularis Contains a Gene Encoding a Functional Rickettsia-Like ATP/ADP Translocase for Host Exploitation

2008 ◽  
Vol 190 (17) ◽  
pp. 5746-5752 ◽  
Author(s):  
Stephan Schmitz-Esser ◽  
Ilka Haferkamp ◽  
Silvia Knab ◽  
Thomas Penz ◽  
Michelle Ast ◽  
...  
Keyword(s):  
Lawsonia Intracellularis ◽  
Swine Industry ◽  
Gene Encoding ◽  
Transfer Event ◽  
E Coli ◽  
Obligate Intracellular ◽  
Gene Coding ◽  
Obligate Intracellular Pathogen ◽  
Substrate Affinities ◽  
Energy Pool

ABSTRACT ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1 Li ]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1 Li were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1 Li transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (± 36.5) μM for ATP and 275.2 (± 28.1) μM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1 Li is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.

scholarly journals Rickettsia prowazekii Transports UMP and GMP, but Not CMP, as Building Blocks for RNA Synthesis

1999 ◽  
Vol 181 (10) ◽  
pp. 3238-3241 ◽  
Author(s):  
Herbert H. Winkler ◽  
Robin Daugherty ◽  
Fuquan Hu
Keyword(s):  
Rna Synthesis ◽  
De Novo ◽  
Building Blocks ◽  
Nucleotide Metabolism ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Rickettsia Prowazekii ◽  
Free Living ◽  
Obligate Intracellular ◽  
Epidemic Typhus

ABSTRACT Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekiiorganisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information.

scholarly journals Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

2014 ◽  
Vol 197 (5) ◽  
pp. 905-912 ◽  
Author(s):  
Yuriy A. Knirel ◽  
Nikolai S. Prokhorov ◽  
Alexander S. Shashkov ◽  
Olga G. Ovchinnikova ◽  
Evelina L. Zdorovenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Side Chain ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Content Type ◽  
Surface Receptors ◽  
E Coli ◽  
O Antigen ◽  
Phage Sensitivity

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmentalEscherichia coliisolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen ofE. coliO22, differing only in side-chain α-d-glucosylation in the former, mediated by agtrlocus on the chromosome. Spontaneous mutations ofE. coli4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions ofE. coli4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

scholarly journals VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

2005 ◽  
Vol 11 (1) ◽  
pp. 61-66
Author(s):  
Ira Djajanegara ◽  
Wayan Artama ◽  
Retno Lestari ◽  
Sabar Pambudi
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Restriction Site ◽  
Standard Procedure ◽  
Pcr Amplification ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Coding ◽  
Sds Page ◽  
Encoding Protein

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

scholarly journals Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

1998 ◽  
Vol 180 (22) ◽  
pp. 5997-6004 ◽  
Author(s):  
Karl Matussek ◽  
Patrick Moritz ◽  
Nina Brunner ◽  
Christoph Eckerskorn ◽  
Reinhard Hensel
Keyword(s):  
Substrate Affinity ◽  
Kinetic Properties ◽  
Gene Encoding ◽  
Enzyme Preparations ◽  
E Coli ◽  
Gene Coding ◽  
Key Enzyme ◽  
Methanothermus Fervidus ◽  
Synthetic Reaction ◽  
Sequencing And Expression

ABSTRACT Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the V max of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher V max of the enzyme from M. fervidus in the synthesizing direction.

scholarly journals Transformation of Rickettsia prowazekiito Rifampin Resistance

1998 ◽  
Vol 180 (8) ◽  
pp. 2118-2124 ◽  
Author(s):  
Lyudmila I. Rachek ◽  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
David O. Wood
Keyword(s):  
Genetic Manipulation ◽  
Single Point ◽  
Initial Step ◽  
Genetic System ◽  
Single Point Mutation ◽  
Rickettsia Prowazekii ◽  
Obligate Intracellular ◽  
Pcr Products ◽  
Epidemic Typhus ◽  
Obligate Intracellular Pathogen

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of theR. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifrregion of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.

scholarly journals The Nucleotide Transporter of Caedibacter caryophilus Exhibits an Extended Substrate Spectrum Compared to the Analogous ATP/ADP Translocase of Rickettsia prowazekii

2004 ◽  
Vol 186 (10) ◽  
pp. 3262-3265 ◽  
Author(s):  
Robin M. Daugherty ◽  
Nicole Linka ◽  
Jonathon P. Audia ◽  
Claude Urbany ◽  
H. Ekkehard Neuhaus ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Transport Systems ◽  
Human Pathogen ◽  
Rickettsia Prowazekii ◽  
Obligate Intracellular ◽  
Competitive Inhibitors ◽  
Cell Cytosol ◽  
Atp Analogs ◽  
Host Cell Cytosol ◽  
Adenine Base

ABSTRACT The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol. These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared. The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter. ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter. Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors. Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of [α-32P]ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter).

scholarly journals Study of the Five Rickettsia prowazekii Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides

2006 ◽  
Vol 188 (17) ◽  
pp. 6261-6268 ◽  
Author(s):  
Jonathon P. Audia ◽  
Herbert H. Winkler
Keyword(s):  
Heterologous Expression ◽  
Host Cell ◽  
De Novo ◽  
Open Reading Frames ◽  
Transport Systems ◽  
Heterologous Protein Expression ◽  
Rickettsia Prowazekii ◽  
E Coli ◽  
Nucleic Acid Biosynthesis ◽  
Reading Frames

ABSTRACT The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a Ki similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.

scholarly journals Rickettsial metK-Encoded Methionine Adenosyltransferase Expression in an Escherichia coli metK Deletion Strain

2005 ◽  
Vol 187 (16) ◽  
pp. 5719-5722 ◽  
Author(s):  
Lonnie O. Driskell ◽  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
David O. Wood
Keyword(s):  
Escherichia Coli ◽  
Stop Codon ◽  
Spotted Fever ◽  
Methionine Adenosyltransferase ◽  
Spotted Fever Group ◽  
Gene Products ◽  
E Coli ◽  
Obligate Intracellular ◽  
Gene Coding ◽  
Inactivating Mutations

ABSTRACT The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.

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