Molecular Analysis of pDL10 from Acidianus ambivalens Reveals a Family of Related Plasmids from Extremely Thermophilic and Acidophilic Archaea

Abstract The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.

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Replication Specificity Elements of the Worland Strain of Beet Curly Top Virus Are Compatible with Those of the CFH Strain But Not Those of the Cal/Logan Strain

Phytopathology ◽

10.1094/phyto.1998.88.11.1174 ◽

1998 ◽

Vol 88 (11) ◽

pp. 1174-1178 ◽

Cited By ~ 30

Author(s):

Drake C. Stenger

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Separate Species ◽

Rep Protein ◽

Reading Frame ◽

Replication Assay ◽

Beet Curly Top Virus ◽

Rep Proteins ◽

Cis And Trans

Cloned genomes of the CFH, Worland, and Cal/Logan strains of beet curly top virus (BCTV) served as helper viruses to trans-replicate defective (D) DNAs that are incapable of self-replication due to deletions within the C1 open reading frame encoding the replication initiator (Rep) protein. The Logan Rep protein could trans-replicate a Logan-derived D DNA in a transient replication assay conducted in Nicotiana benthamiana leaf disks. However, the Logan Rep protein was unable to trans-replicate D DNAs derived from the CFH or Worland strains. In contrast, the Rep proteins of the CFH and Worland strains could trans-replicate CFH or Worland D DNAs, but not a Logan D DNA. These results indicate that the cis- and trans-acting replication specificity elements of the CFH and Worland strains are compatible and that the three strains of BCTV may be divided into two groupings based upon replication specificity determinants. A comparison of amino acid sequences of the Rep protein for the three BCTV strains suggests that the trans-acting replication specificity element may reside in one or more of 12 amino acid residues that are identical; in two amino acid residues that are chemically similar among the CFH and Worland Rep proteins, yet are different in the Logan Rep protein; or in both. Properties including replication specificity, nucleotide sequence identity, and symptom expression were used as criteria to propose separate species designations for each of the three BCTV strains. In this proposal, the Cal/ Logan strain retains the name BCTV, CFH and the closely related Iranian isolate are designated beet severe curly top virus, and Worland is designated beet mild curly top virus.

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Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria

Applied and Environmental Microbiology ◽

10.1128/aem.66.11.4688-4695.2000 ◽

2000 ◽

Vol 66 (11) ◽

pp. 4688-4695 ◽

Cited By ~ 43

Author(s):

Pornpimon Kiatpapan ◽

Yoshiteru Hashimoto ◽

Hisako Nakamura ◽

Yong-Zhe Piao ◽

Hisayo Ono ◽

...

Keyword(s):

Amino Acid ◽

Shuttle Vector ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Vector System ◽

Propionibacterium Acidipropionici ◽

Rep Protein ◽

Gram Positive Bacteria ◽

High Degree ◽

Reading Frames

ABSTRACT The complete nucleotide sequence of pRGO1, a cryptic plasmid fromPropionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4,orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containingorf1 (repA), orf2(repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichiisubsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp.freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

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Different Responses of Cytoplasmic and Endoplasmic Reticulum Hsp90 Genes from Eogystia hippophaecola (Lepidoptera: Cossidae) to Cold Stress

Forests ◽

10.3390/f10111039 ◽

2019 ◽

Vol 10 (11) ◽

pp. 1039

Author(s):

Qianqian Wang ◽

Jing Tao ◽

Yurong Li ◽

Yabei Xu ◽

Xinhai Liu ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Amino Acid ◽

Low Temperature ◽

Cold Stress ◽

Cold Tolerance ◽

Response Times ◽

Expression Patterns ◽

Amino Acid Sequences ◽

Sea Buckthorn ◽

Open Reading Frames

Eogystia hippophaecola Hua, Chou, Fang et Chen (Lepidoptera: Cossidae) is an important borer pest of the sea buckthorn forest (Hippophae rhamnoides L.) in China. Its larvae, which are highly cold tolerant, mainly overwinter in sea buckthorn roots. Heat shock proteins (Hsps) are important molecular chaperones that have been linked to cold tolerance in insects. In this study, we cloned the open reading frames (ORFs) of two Hsp90 genes from E. hippophaecola, EhHsp90-1 and EhHsp90-2, and analyzed their expression under cold stress by qRT-PCR. EhHsp90-1 and EhHsp90-2 are 2154 and 2346 bp in length, respectively, encoding 717 and 781 amino acids. The deduced amino acid sequences contain the conserved signature sequences of the Hsp90 family and the C-terminus characteristic sequence of cytoplasmic or endoplasmic reticulum Hsp90 protein. Phylogenetic analysis revealed the amino acid sequences of EhHsp90-1 and EhHsp90-2 were very similar to the corresponding proteins from Lepidoptera. Under various low-temperature treatments lasting 2 h, EhHsp90-1 and EhHsp90-2 exhibited similar expression patterns, increasing first and then decreasing. At −5 °C, EhHsp90-1 was significantly up-regulated after 12 h, whereas EhHsp90-2 was up-regulated after just 1 h and reached its highest level at 2 h; however, the overall degree of upregulation was greater for EhHsp90-1. Subsequently, the expression level of EhHsp90-2 fluctuated with time. Our results suggest that the two Hsp90s play important roles in E. hippophaecola larvae response to cold stress, but that their response times and the magnitudes of their responses to low-temperature stress differed significantly, providing a theoretical basis for further studying the molecular mechanism of cold tolerance in E. hippophaecola larvae.

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Definition of a Second Bacillus subtilis pur Regulon Comprising the pur and xpt-pbuX Operons plus pbuG, nupG (yxjA), and pbuE (ydhL)

Journal of Bacteriology ◽

10.1128/jb.185.17.5200-5209.2003 ◽

2003 ◽

Vol 185 (17) ◽

pp. 5200-5209 ◽

Cited By ~ 54

Author(s):

Lars Engholm Johansen ◽

Per Nygaard ◽

Catharina Lassen ◽

Yvonne Agersø ◽

Hans H. Saxild

Keyword(s):

Bacillus Subtilis ◽

Amino Acid ◽

Transcription Initiation ◽

Indirect Evidence ◽

Amino Acid Sequences ◽

Common Mechanism ◽

Nucleoside Transporter ◽

Independent Manner ◽

Stem Loop ◽

Transcription Terminator

ABSTRACT In Bacillus subtilis expression of genes or operons encoding enzymes and other proteins involved in purine synthesis is affected by purine bases and nucleosides in the growth medium. The genes belonging to the PurR regulon (purR, purA, glyA, guaC, pbuO, pbuG, and the pur, yqhZ-folD, and xpt-pbuX operons) are controlled by the PurR repressor, which inhibits transcription initiation. Other genes are regulated by a less-well-described transcription termination mechanism that responds to the presence of hypoxanthine and guanine. The pur operon and the xpt-pbuX operon, which were studied here, are regulated by both mechanisms. We isolated two mutants resistant to 2-fluoroadenine in which the pur operon and the xpt-pbuX operon are expressed at increased levels in a PurR-independent manner. The mutations were caused by deletions that disrupted a potential transcription terminator structure located immediately upstream of the ydhL gene. The 5′ part of the ydhL leader region contained a 63-nucleotide (nt) sequence very similar to the 5′ ends of the leaders of the pur and xpt-pbuX operons. Transcripts of these regions may form a common tandem stem-loop secondary structure. Two additional genes with potential leader regions containing the 63-nt sequence are pbuG, encoding a hypoxanthine-guanine transporter, and yxjA, which was shown to encode a purine nucleoside transporter and is renamed nupG. Transcriptional lacZ fusions and mutations in the 63-nt sequence encoding the possible secondary structures provided evidence that expression of the pur and xpt-pbuX operons and expression of the ydhL, nupG, and pbuG genes are regulated by a common mechanism. The new pur regulon is designated the XptR regulon. Except for ydhL, the operons and genes were negatively regulated by hypoxanthine and guanine. ydhL was positively regulated. The derived amino acid sequence encoded by ydhL (now called pbuE) is similar to the amino acid sequences of metabolite efflux pumps. When overexpressed, PbuE lowers the sensitivity to purine analogs. Indirect evidence indicated that PbuE decreases the size of the internal pool of hypoxanthine. This explains why the hypoxanthine- and guanine-regulated genes are expressed at elevated levels in a mutant that overexpresses pbuE.

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Molecular Characterization of Plasmid pBM300 from Bacillus megaterium QM B1551

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.3068-3076.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 3068-3076 ◽

Cited By ~ 12

Author(s):

Muthusamy Kunnimalaiyaan ◽

Patricia S. Vary

Keyword(s):

Bacillus Megaterium ◽

Protein A ◽

Coding Region ◽

Rolling Circle ◽

Rep Protein ◽

Reading Frame ◽

Germination Ability ◽

Rep Proteins ◽

A Cell ◽

Complete Sequencing

ABSTRACT Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.

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Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3321-3329.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3321-3329 ◽

Cited By ~ 40

Author(s):

Kengo Inoue ◽

Hiroshi Habe ◽

Hisakazu Yamane ◽

Hideaki Nojiri

Keyword(s):

Amino Acid ◽

Gene Clusters ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Class Iii ◽

Rt Pcr ◽

Gram Positive ◽

Mononuclear Iron ◽

Reverse Transcription Pcr ◽

Genes Encoding

ABSTRACT Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

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The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins

Science ◽

10.1126/science.3289117 ◽

1988 ◽

Vol 240 (4860) ◽

pp. 1759-1764 ◽

Cited By ~ 1936

Author(s):

WH Landschulz ◽

PF Johnson ◽

SL McKnight

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Binding Proteins ◽

Leucine Zipper ◽

Regulatory Protein ◽

Alpha Helix ◽

Dna Binding Proteins ◽

Amino Acid Sequences ◽

Helical Conformation ◽

Hypothetical Structure

A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.

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Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses

Journal of Virology ◽

10.1128/jvi.73.2.939-947.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 939-947 ◽

Cited By ~ 74

Author(s):

Ursula Bantel-Schaal ◽

Hajo Delius ◽

Rainer Schmidt ◽

Harald zur Hausen

Keyword(s):

Amino Acid ◽

Virus Type ◽

Acid Level ◽

Amino Acid Level ◽

Open Reading Frames ◽

Nucleotide Level ◽

Adeno Associated Virus ◽

Exterior Surface ◽

Rep Proteins ◽

The Right

ABSTRACT We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.

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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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Genes Involved in Anaerobic Metabolism of Phenol in the Bacterium Thauera aromatica

Journal of Bacteriology ◽

10.1128/jb.182.20.5849-5863.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5849-5863 ◽

Cited By ~ 71

Author(s):

Sabine Breinig ◽

Emile Schiltz ◽

Georg Fuchs

Keyword(s):

Gene Product ◽

Anaerobic Metabolism ◽

Regulatory Protein ◽

Gene Clusters ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Chromosomal Dna ◽

Two Dimensional Gel Electrophoresis ◽

Thauera Aromatica ◽

Induced Proteins

ABSTRACT Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor (“phenylphosphate synthase”) and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate (“phenylphosphate carboxylase”). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcusstrains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.

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