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

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.

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S-Adenosylmethionine Transport in Rickettsia prowazekii

Journal of Bacteriology ◽

10.1128/jb.185.10.3031-3035.2003 ◽

2003 ◽

Vol 185 (10) ◽

pp. 3031-3035 ◽

Cited By ~ 47

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.

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Transformation of Rickettsia prowazekiito Rifampin Resistance

Journal of Bacteriology ◽

10.1128/jb.180.8.2118-2124.1998 ◽

1998 ◽

Vol 180 (8) ◽

pp. 2118-2124 ◽

Cited By ~ 58

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.

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

Journal of Bacteriology ◽

10.1128/jb.186.10.3262-3265.2004 ◽

2004 ◽

Vol 186 (10) ◽

pp. 3262-3265 ◽

Cited By ~ 13

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).

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mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

Applied and Environmental Microbiology ◽

10.1128/aem.01727-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6644-6649 ◽

Cited By ~ 50

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.

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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

Journal of Bacteriology ◽

10.1128/jb.00371-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6261-6268 ◽

Cited By ~ 37

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.

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Fishing in the (deoxyribonucleotide) pool

Biochemical Journal ◽

10.1042/bj20091194 ◽

2009 ◽

Vol 422 (3) ◽

pp. e3-e6 ◽

Cited By ~ 10

Author(s):

Ann Saada

Keyword(s):

De Novo ◽

Mitochondrial Respiratory Chain ◽

Building Blocks ◽

Nucleotide Metabolism ◽

De Novo Synthesis ◽

Inborn Errors ◽

Dntp Pool ◽

Adult Rat ◽

General Importance ◽

Biochemical Journal

Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA, and a constant supply is essential for the synthesis and maintenance of both the nuclear and mitochondrial genomes. Antiviral nucleoside analogues and inborn errors of nucleotide metabolism frequently cause dNTP pool imbalances, leading to depletion of mtDNA (mitochondrial DNA) in non-replicating tissues. mtDNA depletion, in turn, causes failure of the mitochondrial respiratory chain, resulting in cellular energy depletion and cell death. Accordingly, it is important to understand the origin and regulation of dNTPs in order to develop safe and effective treatments. In this issue of the Biochemical Journal, Morris et al. have pursued the origin of pyrimidines in perfused adult rat heart. They found no evident role for the nucleotide de novo synthesis pathway and also demonstrated that AZT (3′-azido-3′-deoxythymidine; also known as zidovudine) substantially decreased the TTP pool. Their results underscore the general importance of the mitochondrial deoxyribonucleoside salvage pathway in adult tissues, and particularly in AZT-mediated toxicity. Although the role of nucleoside salvaging versus de novo synthesis in humans remains unclear, the study of tissue cultures and animal models contribute to the understanding of the intricate network of biochemical pathways, maintaining the cellular dNTP supply.

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Whole-Genome Doubling Affects Pre-miRNA Expression in Plants

Plants ◽

10.3390/plants10051004 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1004

Author(s):

Salvatore Esposito ◽

Riccardo Aversano ◽

Pasquale Tripodi ◽

Domenico Carputo

Keyword(s):

Dna Repair ◽

Mirna Expression ◽

Building Blocks ◽

Nucleotide Metabolism ◽

Integrative Approach ◽

Whole Genome ◽

Solanum Commersonii ◽

Genome Doubling ◽

Whole Genome Doubling ◽

Micro Rnas

Whole-genome doubling (polyploidy) is common in angiosperms. Several studies have indicated that it is often associated with molecular, physiological, and phenotypic changes. Mounting evidence has pointed out that micro-RNAs (miRNAs) may have an important role in whole-genome doubling. However, an integrative approach that compares miRNA expression in polyploids is still lacking. Here, a re-analysis of already published RNAseq datasets was performed to identify microRNAs’ precursors (pre-miRNAs) in diploids (2x) and tetraploids (4x) of five species (Arabidopsis thaliana L., Morus alba L., Brassica rapa L., Isatis indigotica Fort., and Solanum commersonii Dun). We found 3568 pre-miRNAs, three of which (pre-miR414, pre-miR5538, and pre-miR5141) were abundant in all 2x, and were absent/low in their 4x counterparts. They are predicted to target more than one mRNA transcript, many belonging to transcription factors (TFs), DNA repair mechanisms, and related to stress. Sixteen pre-miRNAs were found in common in all 2x and 4x. Among them, pre-miRNA482, pre-miRNA2916, and pre-miRNA167 changed their expression after polyploidization, being induced or repressed in 4x plants. Based on our results, a common ploidy-dependent response was triggered in all species under investigation, which involves DNA repair, ATP-synthesis, terpenoid biosynthesis, and several stress-responsive transcripts. In addition, an ad hoc pre-miRNA expression analysis carried out solely on 2x vs. 4x samples of S. commersonii indicated that ploidy-dependent pre-miRNAs seem to actively regulate the nucleotide metabolism, probably to cope with the increased requirement for DNA building blocks caused by the augmented DNA content. Overall, the results outline the critical role of microRNA-mediated responses following autopolyploidization in plants.

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Prospective enzymes for omega-3 PUFA biosynthesis found in endoparasitic classes within the phylum Platyhelminthes

Journal of Helminthology ◽

10.1017/s0022149x20000954 ◽

2020 ◽

Vol 94 ◽

Author(s):

D. Babaran ◽

M.T. Arts ◽

R.J. Botelho ◽

S.A. Locke ◽

J. Koprivnikar

Keyword(s):

Parasite Species ◽

De Novo ◽

Genomic Data ◽

Wide Distribution ◽

Omega 3 ◽

Free Living ◽

Chain Pufa ◽

Pufa Biosynthesis ◽

Aquatic Food ◽

Long Chain

Abstract The free-living infectious stages of macroparasites, specifically, the cercariae of trematodes (flatworms), are likely to be significant (albeit underappreciated) vectors of nutritionally important polyunsaturated fatty acids (PUFA) to consumers within aquatic food webs, and other macroparasites could serve similar roles. In the context of de novo omega-3 (n-3) PUFA biosynthesis, it was thought that most animals lack the fatty acid (FA) desaturase enzymes that convert stearic acid (18:0) into ɑ-linolenic acid (ALA; 18:3n-3), the main FA precursor for n-3 long-chain PUFA. Recently, novel sequences of these enzymes were recovered from 80 species from six invertebrate phyla, with experimental confirmation of gene function in five phyla. Given this wide distribution, and the unusual attributes of flatworm genomes, we conducted an additional search for genes for de novo n-3 PUFA in the phylum Platyhelminthes. Searches with experimentally confirmed sequences from Rotifera recovered nine relevant FA desaturase sequences from eight species in four genera in the two exclusively endoparasite classes (Trematoda and Cestoda). These results could indicate adaptations of these particular parasite species, or may reflect the uneven taxonomic coverage of sequence databases. Although additional genomic data and, particularly, experimental study of gene functionality are important future validation steps, our results indicate endoparasitic platyhelminths may have enzymes for de novo n-3 PUFA biosynthesis, thereby contributing to global PUFA production, but also representing a potential target for clinical antihelmintic applications.

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Natural variation in yolk fatty acids, but not androgens, predicts offspring fitness in a wild bird

Frontiers in Zoology ◽

10.1186/s12983-021-00422-z ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Lucia Mentesana ◽

Martin N. Andersson ◽

Stefania Casagrande ◽

Wolfgang Goymann ◽

Caroline Isaksson ◽

...

Keyword(s):

Fatty Acids ◽

Polyunsaturated Fatty Acids ◽

Maternal Effects ◽

Natural Variation ◽

De Novo ◽

Natural Populations ◽

Egg Laying ◽

Interactive Effects ◽

Free Living ◽

Offspring Fitness

Abstract Background In egg-laying animals, mothers can influence the developmental environment and thus the phenotype of their offspring by secreting various substances into the egg yolk. In birds, recent studies have demonstrated that different yolk substances can interactively affect offspring phenotype, but the implications of such effects for offspring fitness and phenotype in natural populations have remained unclear. We measured natural variation in the content of 31 yolk components known to shape offspring phenotypes including steroid hormones, antioxidants and fatty acids in eggs of free-living great tits (Parus major) during two breeding seasons. We tested for relationships between yolk component groupings and offspring fitness and phenotypes. Results Variation in hatchling and fledgling numbers was primarily explained by yolk fatty acids (including saturated, mono- and polyunsaturated fatty acids) - but not by androgen hormones and carotenoids, components previously considered to be major determinants of offspring phenotype. Fatty acids were also better predictors of variation in nestling oxidative status and size than androgens and carotenoids. Conclusions Our results suggest that fatty acids are important yolk substances that contribute to shaping offspring fitness and phenotype in free-living populations. Since polyunsaturated fatty acids cannot be produced de novo by the mother, but have to be obtained from the diet, these findings highlight potential mechanisms (e.g., weather, habitat quality, foraging ability) through which environmental variation may shape maternal effects and consequences for offspring. Our study represents an important first step towards unraveling interactive effects of multiple yolk substances on offspring fitness and phenotypes in free-living populations. It provides the basis for future experiments that will establish the pathways by which yolk components, singly and/or interactively, mediate maternal effects in natural populations.

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