Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

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Functional-Genomics-Based Identification and Characterization of Open Reading Frames Encoding α-Glucoside-Processing Enzymes in the Hyperthermophilic Archaeon Pyrococcus furiosus

Applied and Environmental Microbiology ◽

10.1128/aem.01920-07 ◽

2007 ◽

Vol 74 (4) ◽

pp. 1281-1283 ◽

Cited By ~ 19

Author(s):

Donald A. Comfort ◽

Chung-Jung Chou ◽

Shannon B. Conners ◽

Amy L. VanFossen ◽

Robert M. Kelly

Keyword(s):

Glycoside Hydrolase ◽

Bioinformatics Analysis ◽

Pyrococcus Furiosus ◽

Transcriptional Response ◽

Open Reading Frames ◽

Processing Enzymes ◽

Response Information ◽

Identification And Characterization ◽

Reading Frames

ABSTRACT Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on α-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 β-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative α-glucan-processing enzymes.

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Identification and Characterization of a Novel Robigovirus Species from Sweet Cherry in Turkey

Pathogens ◽

10.3390/pathogens8020057 ◽

2019 ◽

Vol 8 (2) ◽

pp. 57 ◽

Cited By ~ 5

Author(s):

Kadriye Çağlayan ◽

Vahid Roumi ◽

Mona Gazel ◽

Eminur Elçi ◽

Mehtap Acioğlu ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sweet Cherry ◽

Prunus Avium ◽

Open Reading Frames ◽

Cherry Tree ◽

Coding Regions ◽

Sweet Cherries ◽

Identification And Characterization ◽

Reading Frames

High throughput sequencing of total RNA isolated from symptomatic leaves of a sweet cherry tree (Prunus avium cv. 0900 Ziraat) from Turkey identified a new member of the genus Robigovirus designated cherry virus Turkey (CVTR). The presence of the virus was confirmed by electron microscopy and overlapping RT-PCR for sequencing its whole-genome. The virus has a ssRNA genome of 8464 nucleotides which encodes five open reading frames (ORFs) and comprises two non-coding regions, 5′ UTR and 3′ UTR of 97 and 296 nt, respectively. Compared to the five most closely related robigoviruses, RdRp, TGB1, TGB2, TGB3 and CP share amino acid identities ranging from 43–53%, 44–60%, 39–43%, 38–44% and 45–50%, respectively. Unlike the four cherry robigoviruses, CVTR lacks ORFs 2a and 5a. Its genome organization is therefore more similar to African oil palm ringspot virus (AOPRV). Using specific primers, the presence of CVTR was confirmed in 15 sweet cherries and two sour cherries out of 156 tested samples collected from three regions in Turkey. Among them, five samples were showing slight chlorotic symptoms on the leaves. It seems that CVTR infects cherry trees with or without eliciting obvious symptoms, but these data should be confirmed by bioassays in woody and possible herbaceous hosts in future studies.

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Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium Agrobacterium radiobacter Strain S2

Journal of Bacteriology ◽

10.1128/jb.182.21.6123-6129.2000 ◽

2000 ◽

Vol 182 (21) ◽

pp. 6123-6129 ◽

Cited By ~ 21

Author(s):

Matthias Contzen ◽

Andreas Stolz

Keyword(s):

Amino Acid Sequences ◽

Open Reading Frames ◽

Agrobacterium Radiobacter ◽

Sequence Alignments ◽

Degrading Bacterium ◽

Regulator Protein ◽

Putative Operon ◽

Genes Encoding ◽

Reading Frames

ABSTRACT The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacteriumAgrobacterium radiobacter strain S2 (DSMZ 5681). ThepcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strainAgrobacterium tumefaciens A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.

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Molecular and Evolutionary Characterization of the cp32/18 Family of Supercoiled Plasmids in Borrelia burgdorferi297

Infection and Immunity ◽

10.1128/iai.68.3.1574-1586.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1574-1586 ◽

Cited By ~ 39

Author(s):

Melissa J. Caimano ◽

Xiaofeng Yang ◽

Taissia G. Popova ◽

Michael L. Clawson ◽

Darrin R. Akins ◽

...

Keyword(s):

Sequence Analysis ◽

Genomic Sequence ◽

Complete Sequence ◽

Open Reading Frames ◽

Exogenous Dna ◽

Variable Regions ◽

Different Types ◽

Sequence Relatedness ◽

Reading Frames

ABSTRACT In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526–1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.

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Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5’-G(5mC)^NG(5mC)-3’

Acta Naturae ◽

10.32607/20758251-2016-8-1-117-125 ◽

2016 ◽

Vol 8 (1) ◽

pp. 117-125 ◽

Cited By ~ 1

Author(s):

V. A. Chernukhin ◽

D. A. Gonchar ◽

M. A. Abdurashitov ◽

O. A. Belichenko ◽

V. S. Dedkov ◽

...

Keyword(s):

Dna Sequence ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Site Specific ◽

Pcr Screening ◽

Methylated Dna ◽

Chromatographic Techniques ◽

Dna Fragment ◽

Reading Frames

Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5-GCNGC- 3 before the central nucleotide N if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

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Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

Journal of Bacteriology ◽

10.1128/jb.181.10.3155-3163.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3155-3163 ◽

Cited By ~ 229

Author(s):

M. Gita Bangera ◽

Linda S. Thomashow

Keyword(s):

Genomic Dna ◽

Plant Pathogens ◽

Polyketide Synthase ◽

Open Reading Frames ◽

Fungal Plant Pathogens ◽

Repressor Proteins ◽

Flanking Regions ◽

Identification And Characterization ◽

Reading Frames

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescensQ2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipientPseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlEand phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, andphlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.

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Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8

Microorganisms ◽

10.3390/microorganisms8010044 ◽

2019 ◽

Vol 8 (1) ◽

pp. 44 ◽

Cited By ~ 1

Author(s):

Daisuke Miyazawa ◽

Le Thi Ha Thanh ◽

Akio Tani ◽

Masaki Shintani ◽

Nguyen Hoang Loc ◽

...

Keyword(s):

Amino Acid ◽

Thermophilic Bacterium ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Pcr Analysis ◽

Naphthalene Degradation ◽

Isolation And Characterization ◽

Dihydrodiol Dehydrogenase ◽

Reading Frames

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

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Identification and Characterization of Phytoplasmal Genes, Employing a Novel Method of Isolating Phytoplasmal Genomic DNA

Journal of Bacteriology ◽

10.1128/jb.185.22.6513-6521.2003 ◽

2003 ◽

Vol 185 (22) ◽

pp. 6513-6521 ◽

Cited By ~ 8

Author(s):

Sharon Melamed ◽

Edna Tanne ◽

Raz Ben-Haim ◽

Orit Edelbaum ◽

David Yogev ◽

...

Keyword(s):

Genomic Dna ◽

Plant Pathogens ◽

Open Reading Frames ◽

Rrna Genes ◽

Unique Method ◽

Novel Method ◽

Identification And Characterization ◽

Reading Frames ◽

Large Background

ABSTRACT Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.

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Genetic Characterization of vanG, a Novel Vancomycin Resistance Locus of Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3224-3228.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3224-3228 ◽

Cited By ~ 111

Author(s):

Stuart J. McKessar ◽

Anne M. Berry ◽

Jan M. Bell ◽

John D. Turnidge ◽

James C. Paton

Keyword(s):

Enterococcus Faecalis ◽

Amino Acid Sequence Identity ◽

Vancomycin Resistance ◽

Open Reading Frames ◽

Pcr Analysis ◽

Resistance Locus ◽

Gene Products ◽

The Individual ◽

Reading Frames

ABSTRACT Enterococcus faecalis strain WCH9 displays a moderate level of resistance to vancomycin (MIC = 16 μg/ml) and full susceptibility to teicoplanin but is negative by PCR analysis using primers specific for all known enterococcal vancomycin resistance genotypes (vanA, vanB,vanC, vanD, and vanE). We have isolated and sequenced a novel putative vancomycin resistance locus (designated vanG), which contains seven open reading frames, from this strain. These are organized differently from those of all the other enterococcal vanloci, and, furthermore, the individual vanG gene products exhibit less than 50% amino acid sequence identity to othervan gene products.

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