Vibrio vulnificus Has the Transmembrane Transcription Activator ToxRS Stimulating the Expression of the Hemolysin GenevvhA

ABSTRACT In an attempt to dissect the virulence regulatory mechanism inVibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS(toxRS Vc) homologs in V. vulnificus. By comparing the sequences of toxRS ofV. cholerae and V. parahaemolyticus(toxRS Vp), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kbBglII-HindIII fragment and a 1.2-kbHindIII fragment containing two complete open reading frames and one partial open reading frame attributable totoxR Vv, toxS Vv, andhtpG Vv were cloned. ToxRVv shared 55.0 and 63.0% sequence homology with ToxRVc and ToxRVp, respectively. ToxSVv was 71.5 and 65.7% homologous to ToxSVc and ToxSVp, respectively. The amino acid sequences of ToxRSVv showed transmembrane and activity domains similar to those observed in ToxRSVc and ToxRSVp. Western blot analysis proved the expression of ToxRVv in V. vulnificus. ToxRSVv enhanced, in an Escherichia coli background, the expression of the V. vulnificushemolysin gene (vvhA) fivefold. ToxRSVv also activated the ToxRVc-regulated ctx promoter incorporated into an E. coli chromosome. AtoxR Vv null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. ThetoxR Vv mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxRVv may regulate the virulence expression of V. vulnificus.

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A K+ Uptake Protein, TrkA, Is Required for Serum, Protamine, and Polymyxin B Resistance in Vibrio vulnificus

Infection and Immunity ◽

10.1128/iai.72.2.629-636.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 629-636 ◽

Cited By ~ 40

Author(s):

Yu-Chung Chen ◽

Yin-Ching Chuang ◽

Chun-Chin Chang ◽

Chii-Ling Jeang ◽

Ming-Chung Chang

Keyword(s):

Human Serum ◽

Vibrio Vulnificus ◽

Polymyxin B ◽

Wild Type ◽

Gene Encoding ◽

Reading Frame ◽

Isogenic Mutant ◽

Virulence Attenuation ◽

Insertional Inactivation ◽

Transposon Mutants

ABSTRACT Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.

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Cloning of two Bombyx homologues of the Drosophila rosy gene and their relationship to larval translucent skin colour mutants

Genetics Research ◽

10.1017/s0016672397003078 ◽

1998 ◽

Vol 71 (1) ◽

pp. 11-19 ◽

Cited By ~ 13

Author(s):

YUJI YASUKOCHI ◽

TOSHIO KANDA ◽

TOSHIKI TAMURA

Keyword(s):

Tissue Specificity ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Striking Similarity ◽

Wild Type ◽

Rt Pcr ◽

Significant Difference ◽

Phage Dna ◽

Polymerase Chain ◽

Reading Frames

To clone the Bombyx xanthine dehydrogenase (XDH) gene as a dominant marker for silkworm transgenesis, we performed nested reverse transcriptase–polymerase chain reaction (RT-PCR) using embryonic mRNA and primers designed from the conserved region of Drosophila and rat XDH genes. Sequencing of amplified 180 bp fragments showed that two different sequences were present in the fragments. Since both possessed striking similarity to XDH genes of other organisms, we considered these to be portions of silkworm XDH genes and designated them BmXDH1 and BmXDH2. Subsequently we cloned separately the entire region of the two cDNAs by PCR using phage DNA of an embryonic cDNA library and sequenced them. The two cDNAs were around 4 kb in size and possessed complete open reading frames. The deduced amino acid sequences of the two BmXDHs were very similar to each other and to those of other organisms. The expression pattern of wild-type larvae basically followed the tissue specificity of the enzyme and no significant difference was observed between the two XDH genes. The expression of both genes was detected in the XDH-deficient mutants, oq and og, but non-synonymous substitutions were specifically detected in the BmXDH1 of the oq mutant. In addition, a length polymorphism of the second intron of the BmXDH1 co-segregated with the oq translucent phenotype, suggesting that deficiency in BmXDH1 is the cause of the oq translucent phenotype.

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Identification of ccdA inParacoccus pantotrophus GB17: Disruption ofccdA Causes Complete Deficiency inc-Type Cytochromes

Journal of Bacteriology ◽

10.1128/jb.183.1.257-263.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 257-263 ◽

Cited By ~ 24

Author(s):

Frank Bardischewsky ◽

Cornelius G. Friedrich

Keyword(s):

Sulfur Oxidation ◽

Open Reading Frames ◽

Redox Mediators ◽

Transmembrane Helices ◽

Wild Type ◽

Structural Identity ◽

Paracoccus Pantotrophus ◽

Type Gene ◽

Extension Analysis ◽

Reading Frames

ABSTRACT A transposon Tn5-mob insertional mutant ofParacoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently. ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn5-mob was inserted into ccdA. The deduced ORFA product showed no similarity to any protein in databases. However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of P. pantotrophus is 42% identical to a protein predicted by ORF2, and its location within thesox gene cluster coding for lithotrophic sulfur oxidation suggested a different function.

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Automatic simulation of RNA editing in plants for the identification of novel putative Open Reading Frames

10.7287/peerj.preprints.3362v1 ◽

2017 ◽

Author(s):

Fabio Fassetti ◽

Claudia Giallombardo ◽

Ofelia Leone ◽

Luigi Palopoli ◽

Simona E Rombo ◽

...

Keyword(s):

Rna Editing ◽

Dna Sequences ◽

Information Transfer ◽

Plant Mitochondria ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Reading Frame ◽

Novel Proteins ◽

Gene Search ◽

Novel Strategy

In plant mitochondria an essential mechanism for gene expression is RNA editing, often influencing the synthesis of functional proteins. RNA editing alters the linearity of genetic information transfer, intro- ducing differences between RNAs and their coding DNA sequences that hind both experimental and computational research of genes. Thus common software tools for gene search, successfully exploited to find canonic genes, often can fail in discovering genes encrypted in the genome of plants. In this work we propose a novel strategy useful to intercept candidate coding sequences resulting from some possible editing substitutions on the start and stop codons of a given input organism DNA. Our method is based on the simulation of the RNA editing mechanism, in order to generate candidate Open Reading Frame (ORF) sequences that could code for some, yet unknown, proteins. Results obtained on the mtDNA of Oryza sativa are promising, since we identified ORF sequences trascripted in Oriza, that do not cor- respond to already known proteins in this organism. Part of the corresponding amino acid sequences present high homologies with proteins already discovered in other organisms, the remaining ones could represent novel proteins not yet discovered in Oryza.

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An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby

Genome ◽

10.1139/gen-2021-0047 ◽

2022 ◽

Author(s):

Sakura Hayashi ◽

Konami Shimizu ◽

Yusuke Honda ◽

Yukako Katsura ◽

Akihiko Koga

Keyword(s):

Endogenous Retrovirus ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Length Variation ◽

Wild Type ◽

Long Terminal Repeats ◽

Recent Event ◽

Melanin Biosynthesis ◽

Dna Fragment ◽

Reading Frames

An albino infant wallaby was born to a mother with the wild-type body color. PCR and sequencing analyses of TYR (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Because the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named walb. We cloned other walb copies residing in the genomes of this species and another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of gag, pol, and env of retroviruses. It is not known through which of the following likely processes the walb copy was inserted into TYR: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into TYR is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances.

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A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3094 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3094-3103 ◽

Cited By ~ 55

Author(s):

C K Shih ◽

R Wagner ◽

S Feinstein ◽

C Kanik-Ennulat ◽

N Neff

Keyword(s):

Saccharomyces Cerevisiae ◽

Atp Synthase ◽

Resistance Mutation ◽

Amino Acid Sequences ◽

Wild Type ◽

Reading Frame ◽

F0f1 Atp Synthase ◽

General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

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Detection of potential suberinase-encoding genes in Streptomyces scabiei strains and other actinobacteria

Canadian Journal of Microbiology ◽

10.1139/cjm-2012-0741 ◽

2013 ◽

Vol 59 (5) ◽

pp. 294-303 ◽

Cited By ~ 11

Author(s):

Doaa Komeil ◽

Anne-Marie Simao-Beaunoir ◽

Carole Beaulieu

Keyword(s):

Esterase Activity ◽

Common Scab ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Open Reading Frames ◽

Streptomyces Scabiei ◽

Polymerase Chain ◽

Encoding Genes ◽

Reading Frames ◽

Important Disease

Streptomyces scabiei causes common scab, an economically important disease of potato tubers. Some authors have previously suggested that S. scabiei penetration into host plant tissue is facilitated by secretion of esterase enzymes degrading suberin, a lipidic biopolymer of the potato periderm. In the present study, S. scabiei EF-35 showed high esterase activity in suberin-containing media. This strain also exhibited esterase activity in the presence of other biopolymers, such as lignin, cutin, or xylan, but at a much lower level. In an attempt to identify the esterases involved in suberin degradation, translated open reading frames of S. scabiei 87-22 were examined for the presence of protein sequences corresponding to extracellular esterases of S. scabiei FL1 and of the fungus Coprinopsis cinerea VTT D-041011, which have previously been shown to be produced in the presence of suberin. Two putative extracellular suberinase genes, estA and sub1, were identified. The presence of these genes in several actinobacteria was investigated by Southern blot hybridization, and both genes were found in most common-scab-inducing strains. Moreover, reverse transcription – polymerase chain reaction performed with S. scabiei EF-35 showed that estA was expressed in the presence of various biopolymers, including suberin, whereas the sub1 gene appeared to be specifically expressed in the presence of suberin and cutin.

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Effects of Oligopeptide Permease in Group A Streptococcal Infection

Infection and Immunity ◽

10.1128/iai.73.5.2881-2890.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 2881-2890 ◽

Cited By ~ 37

Author(s):

Chih-Hung Wang ◽

Chia-Yu Lin ◽

Yueh-Hsia Luo ◽

Pei-Jane Tsai ◽

Yee-Shin Lin ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Wild Type ◽

Isogenic Mutant ◽

Transporter Family ◽

Streptolysin O ◽

Group A ◽

Oligopeptide Permease

ABSTRACT The oligopeptide permease (Opp) of group A streptococci (GAS) is a membrane-associated protein and belongs to the ATP-binding cassette transporter family. It is encoded by a polycistronic operon containing oppA, oppB, oppC, oppD, and oppF. The biological function of these genes in GAS is poorly understood. In order to understand more about the effects of Opp on GAS virulence factors, an oppA isogenic mutant was constructed by using an integrative plasmid to disrupt the opp operon and confirmed by Southern blot hybridization. No transcript was detected in the oppA isogenic mutant by Northern blot analysis and reverse transcriptase PCR. The growth curve for the oppA isogenic mutant was similar to that for wild-type strain A-20. The oppA isogenic mutant not only decreased the transcription of speB, speX, and rofA but also increased the transcription of speF, sagA (streptolysin S-associated gene A), slo (streptolysin O), pel (pleotrophic effect locus), and dppA (dipeptide permease). No effects on the transcription of emm, sda, speJ, speG, rgg, and csrR were found. The phenotypes of the oppA mutant were restored by the oppA revertant and by the complementation strain. The oppA mutant caused less mortality and tissue damage than the wild-type strain when inoculated into BALB/c mice via an air pouch. Based on these data, we suggest that the opp operon plays an important role in the pathogenesis of GAS infection.

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Isolation and characterization of mutants of the citrus canker pathogen Xanthomonas axonopodis pv. citri that induce a distinct pattern of disease

Canadian Journal of Botany ◽

10.1139/b00-078 ◽

2000 ◽

Vol 78 (8) ◽

pp. 1002-1009

Author(s):

Shu Yun Tung ◽

Tsong Teh Kuo

Keyword(s):

Blot Hybridization ◽

Random Mutagenesis ◽

Southern Blot Hybridization ◽

Citrus Canker ◽

Distinct Pattern ◽

Wild Type ◽

In Planta ◽

Xanthomonas Axonopodis ◽

Isolation And Characterization ◽

Indigenous Plasmids

Random mutagenesis with the transposon Tn5tac1 in Xanthomonas axonopodis pathovar citri, the causal agent of citrus canker, generated four mutants with altered pathogenicity. These mutants were classified into three groups: (i) the nonpathogenic (NP) mutants XT10 and XT122, which did not induce any visible symptoms in the host; (ii) the WS- mutant XT27, which induced a callus-like lesion but not a watersoaked lesion; and (iii) the CL- mutant XT37, which was unable to induce a callus-like eruption but did induce the formation of a watersoaked lesion around the infection site. The NP mutants failed to grow in planta, whereas the WS- and CL- mutants showed a reduced growth rate relative to that of the wild type. Co-inoculation of leaves with the WS- and CL- mutants did not result in complementation of their respective defects. The extent of extracellular accumulation of polysaccharide, protease, and amylase activities by each of the mutants was similar to that of the wild type. The extracellular activity of polygalacturonate lyase of XT27 was reduced relative to that of the wild type and other mutants. Unlike the wild type and other mutants, XT27 also required glutamic acid for growth in culture. Southern blot hybridization revealed that each of the mutants resulted from transposon insertion at a single site; the insertion sites for XT10 and XT27 were located in the chromosome, whereas those for XT37 and XT122 were located in the indigenous plasmids. These results provide evidence that bacterial genes contribute independently to the pathogenesis of citrus canker.Key words: citrus canker, pathogenicity genes, transposon mutagenesis.

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An efficient gene deletion system for Bacillus thuringiensis

Biologia ◽

10.2478/s11756-013-0184-4 ◽

2013 ◽

Vol 68 (3) ◽

Cited By ~ 1

Author(s):

Tugrul Doruk ◽

Sedef Gedik

Keyword(s):

Bacillus Thuringiensis ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Bacillus Thuringiensis Israelensis ◽

Kinase Gene ◽

E Coli ◽

Efficient Gene ◽

Specific Restriction ◽

Type Gene ◽

Deletion Cassette

AbstractIt is not easy to manipulate biosynthetic genes of Bacillus thuringiensis since there is a powerful methyl-specific restriction system in this microorganism. In this study, a PCR-based system was used to delete polyphosphate kinase gene (ppk) of Bacillus thuringiensis israelensis (Bti) by replacing the wild-type gene with a cassette containing the apramycin resistance gene as selectable marker. λ-Red was used to promote recombination in Escherichia coli between a PCR-amplified apramycin resistance cassette (linear deletion cassette selectable in E. coli and Bti) and Bti DNA on a plasmid. The isolated mutant plasmid was transferred to Bti by conjugation. Double cross-over transformants were screened for their antibiotic resistance and the mutation was proven by PCR, southern blot hybridization and RT-PCR. The described method, which uses the advantage of quick plasmid construction in E. coli and simple transformation of linear deletion cassette, is very useful to delete entire gene/genes of Bti without any polar effects on genes transcriptionally downstream.

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