Freshwater Bacteria Can Methylate Selenium through the Thiopurine Methyltransferase Pathway

ABSTRACT Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 μM sodium selenite and 10 μM sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.

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Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/125.4.739 ◽

1990 ◽

Vol 125 (4) ◽

pp. 739-752 ◽

Cited By ~ 6

Author(s):

C A Woolford ◽

C K Dixon ◽

M F Manolson ◽

R Wright ◽

E W Jones

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mass ◽

Open Reading Frame ◽

Relative Molecular Mass ◽

Cell Fractionation ◽

Calculated Molecular Mass ◽

Reading Frame ◽

Dna Library ◽

Isolation And Characterization ◽

Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

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The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated

Biochemical Journal ◽

10.1042/bj3170089 ◽

1996 ◽

Vol 317 (1) ◽

pp. 89-95 ◽

Cited By ~ 30

Author(s):

Nélida BRITO ◽

Julio AVILA ◽

M. Dolores PEREZ ◽

Celedonio GONZALEZ ◽

José M. SIVERIO

Keyword(s):

Nitrate Reductase ◽

Nitrite Reductase ◽

Reductase Activity ◽

Hansenula Polymorpha ◽

Enzymic Activity ◽

Wild Type ◽

Reading Frame ◽

Dna Library ◽

Genomic Dna Library ◽

Nitrate And Nitrite

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages λNB5 and λJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the Δyni1::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

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Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4516-4525.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4516-4525

Author(s):

K G Coleman ◽

H Y Steensma ◽

D B Kaback ◽

J R Pringle

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Cloning ◽

Genomic Dna ◽

Shuttle Vector ◽

Blot Hybridization ◽

Yeast Genome ◽

Dna Library ◽

Isolation And Characterization ◽

Genomic Dna Library ◽

Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

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Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

Applied and Environmental Microbiology ◽

10.1128/aem.67.9.4064-4069.2001 ◽

2001 ◽

Vol 67 (9) ◽

pp. 4064-4069 ◽

Cited By ~ 120

Author(s):

Naeem Rashid ◽

Yuji Shimada ◽

Satoshi Ezaki ◽

Haruyuki Atomi ◽

Tadayuki Imanaka

Keyword(s):

Lipase Activity ◽

Lipase Gene ◽

Reading Frame ◽

Cold Environments ◽

Dna Library ◽

Gene Sequence Analysis ◽

Genomic Dna Library ◽

Lipase A ◽

Chain Lengths ◽

Sigmoidal Growth

ABSTRACT We have previously reported that a psychrotrophic bacterium,Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase fromPseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.

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Molecular Cloning, Sequencing, Expression, and Characterization of an Immunogenic 43-Kilodalton Lipoprotein of Bartonella bacilliformis That Has Homology to NlpD/LppB

Infection and Immunity ◽

10.1128/iai.68.9.4972-4979.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 4972-4979 ◽

Cited By ~ 8

Author(s):

Indira Padmalayam ◽

Timothy Kelly ◽

Barbara Baumstark ◽

Robert Massung

Keyword(s):

Bartonella Henselae ◽

High Titer ◽

Polyclonal Antiserum ◽

Open Reading Frame ◽

Bartonella Bacilliformis ◽

Reading Frame ◽

Dna Library ◽

Bacillary Angiomatosis ◽

Genomic Dna Library ◽

Entire Open Reading Frame

ABSTRACT A recombinant clone expressing an immunoreactive antigen ofBartonella bacilliformis was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis. The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein. Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame. The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa. The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence. Evidence has been provided to show that the 43-kDa antigen of B. bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins. This is the first report to date that characterizes a lipoprotein of B. bacilliformis. The immunogenicity of the B. bacilliformis LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis. Sera from patients who had a high titer forBartonella henselae, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in B. henselae. Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of B. bacilliformis was generated. This antiserum did not recognize the NlpD homologue of Escherichia coli or the 43-kDa antigen ofB. henselae.

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Cloning and analysis of the aroB gene encoding dehydroquinate synthase from Corynebacterium glutamicum

Canadian Journal of Microbiology ◽

10.1139/w99-076 ◽

1999 ◽

Vol 45 (10) ◽

pp. 885-890 ◽

Cited By ~ 1

Author(s):

Min-Ah Han ◽

Heung-Shick Lee ◽

Choong-Ill Cheon ◽

Kyung-Hee Min ◽

Myeong-Sok Lee

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Auxotrophic Mutant ◽

Gene Products ◽

Gene Encoding ◽

Reading Frame ◽

E Coli ◽

Dna Library ◽

Genomic Dna Library ◽

Dehydroquinate Synthase

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.Key words: Corynebacterium glutamicum, aromatic amino acid biosynthetic gene, dehydroquinate synthase, aroB gene.

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Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene ofHaemophilus ducreyi

Journal of Bacteriology ◽

10.1128/jb.182.8.2292-2298.2000 ◽

2000 ◽

Vol 182 (8) ◽

pp. 2292-2298 ◽

Cited By ~ 20

Author(s):

Shuhua Sun ◽

Birgit Schilling ◽

Laurie Tarantino ◽

Michael V. Tullius ◽

Bradford W. Gibson ◽

...

Keyword(s):

Sodium Dodecyl ◽

Etiologic Agent ◽

Pasteurella Haemolytica ◽

Published Data ◽

Reading Frame ◽

Ulcer Disease ◽

Dna Library ◽

Binding Epitope ◽

Galactose Residue ◽

Genomic Dna Library

ABSTRACT Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes theN-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases ofHaemophilus influenzae, Haemophilus somnus,Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in theN-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.

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CHARACTERIZATION OF THE GENE FOR HUMAN HISTIDINE-RICH GLYCOPROTEIN

10.1055/s-0038-1643599 ◽

1987 ◽

Author(s):

T Koide

Keyword(s):

Blot Hybridization ◽

Southern Blot Hybridization ◽

Single Chain ◽

Structural Domains ◽

Dna Library ◽

Genomic Dna Library ◽

Human Genomic ◽

Considerable Homology ◽

Amino Acid Segment

Human histidine-rich glycoprotein (HRG) is a single-chain glycoprotein in plasma which is considered to modulate a coagulation and fibrinolysis system with the ability to bind to heparin, plasminogen, fibrinogen, thrombospondin, etc. Recently we have elucidated the primary structure of HRG by determining the nucleotide sequence of its cDNA, and showed that HRG is composed of several different types of internal repeats, each one of which shows considerable homology with the functional and/or structural domains of other proteins including high molecular weight kininogen, antithrombin III, cystatins, and proline-rich protein and peptide. Thus, the multifunctional property of HRG was suggested to be due to its multi-domain structure. In the present studies, a human genomic DNA library, cloned in the bacteriophage vector Charon 4A, was screened for HRG gene using a full-length cDNA coding for human IMI as a probe. A total of 7 clones were isolated from 6 × 105 phage and each was plaque purified. The entire HRG gene is represented in 3 genomic inserts with overlapping sequences that carry human DNA spanning 30 kb. Overlapping gene fragments were subcloned into pUC9 and characterized by Southern blot hybridization using 5’ and 3’ end probes isolated from human HRG cDNA and by DNA sequencing. These studies have shown that the gene for human HRG spans about 9 kb and consists of at least 5 exons and 4 introns. The putative histidine-rich region consisted of 12 tandemly repeated sequences of a 5 amino acid segment and 2 proline-rich regions contiguous to it are likely to be involved within one exon.

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