scholarly journals Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1

2000 ◽  
Vol 66 (3) ◽  
pp. 904-908 ◽  
Author(s):  
Matthew M. Bresler ◽  
Susan J. Rosser ◽  
Amrik Basran ◽  
Neil C. Bruce
Keyword(s):  
Rhodococcus Erythropolis ◽  
Sole Source ◽  
Tropane Alkaloid ◽  
Nucleotide Sequencing ◽  
Serine Esterase ◽  
Gene Encoding ◽  
Genomic Libraries ◽  
Reading Frame ◽  
Apparent Homogeneity ◽  
Cocaine Esterase

ABSTRACT A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized byRhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned fromRhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocEcorresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with anM r of approximately 65,000. The apparentKm of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

scholarly journals Characterization of a flavocytochrome that is induced during the anaerobic respiration of Fe3+ by Shewanella frigidimarina NCIMB400

1999 ◽  
Vol 342 (2) ◽  
pp. 439-448 ◽  
Author(s):  
Paul S. DOBBIN ◽  
Julea N. BUTT ◽  
Anne K. POWELL ◽  
Graeme A. REID ◽  
David J. RICHARDSON
Keyword(s):  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Nucleotide Sequencing ◽  
Amino Acid Residues ◽  
Sequencing Data ◽  
Gene Encoding ◽  
Terminal Electron Acceptor ◽  
Reading Frame ◽  
Ferric Pyrophosphate ◽  
Reduction Potentials

A 63.9 kDa periplasmic tetrahaem flavocytochrome c3, designated Ifc3, was found to be expressed in Shewanellafrigidimarina NCIMB400 grown anaerobically with ferric citrate or ferric pyrophosphate as the sole terminal electron acceptor, but not in anaerobic cultures of the bacterium with other respiratory substrates. Ifc3 was purified to homogeneity and revealed by biochemical, spectroscopic and primary structure analyses to contain four low-spin bis-His-ligated c3-haems, with midpoint reduction potentials of -73, -141, -174 and -259 mV. A low-potential flavin was present in the form of non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity. Disruption of the chromosomal gene encoding Ifc3, ifcA, did not lead to a significant change in the rate of Fe3+ reduction in batch culture. However, during such growth the Ifc3-deficient mutant produced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-associated c-type cytochrome at markedly higher levels than did the parent strain. Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane β-barrel protein of 324 amino acid residues.

scholarly journals Expression of hurP, a Gene Encoding a Prospective Site 2 Protease, Is Essential for Heme-Dependent Induction of bhuR in Bordetella bronchiseptica

2007 ◽  
Vol 189 (17) ◽  
pp. 6266-6275 ◽  
Author(s):  
Natalie D. King-Lyons ◽  
Kelsy F. Smith ◽  
Terry D. Connell
Keyword(s):  
Membrane Protein ◽  
Sigma Factor ◽  
Cytoplasmic Membrane ◽  
Regulatory System ◽  
Sole Source ◽  
Bordetella Bronchiseptica ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Reading Frame ◽  
Regulatory Cascade

ABSTRACT Expression of the hurIR bhuRSTUV heme utilization locus in Bordetella bronchiseptica is coordinately controlled by the global iron-dependent regulator Fur and the extracytoplasmic function sigma factor HurI. Activation of HurI requires transduction of a heme-dependent signal via HurI, HurR, and BhuR, a three-component heme-dependent regulatory system. In silico searches of the B. bronchiseptica genome to identify other genes that encode additional participants in this heme-dependent regulatory cascade revealed hurP, an open reading frame encoding a polypeptide with homology to (i) RseP, a site 2 protease (S2P) of Escherichia coli required for modifying the cytoplasmic membrane protein RseA, and (ii) YaeL, an S2P of Vibrio cholerae required for modification of the cytoplasmic membrane protein TcpP. A mutant of B. bronchiseptica defective for hurP was incapable of regulating expression of BhuR in a heme-dependent manner. Furthermore, the hurP mutant was unable to utilize hemin as a sole source of nutrient Fe. These defects in hemin utilization and heme-dependent induction of BhuR were restored when recombinant hurP (or recombinant rseP) was introduced into the mutant. Introduction of hurP into a yaeL mutant of V. cholerae also complemented its S2P defect. These data provided strong evidence that protease activity and cleavage site recognition was conserved in HurP, RseP, and YaeL. The data are consistent with a model in which HurP functionally modifies HurR, a sigma factor regulator that is essential for heme-dependent induction of bhuR.

scholarly journals Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

1998 ◽  
Vol 64 (12) ◽  
pp. 4883-4890 ◽  
Author(s):  
Belén Floriano ◽  
José L. Ruiz-Barba ◽  
Rufino Jiménez-Díaz
Keyword(s):  
Enterococcus Faecium ◽  
Pathogenic Bacteria ◽  
Specific Activity ◽  
Sequence Similarity ◽  
Fold Increase ◽  
Leader Peptide ◽  
Nucleotide Sequencing ◽  
Gene Encoding ◽  
Reading Frame ◽  
Fast Flow

ABSTRACT Enterocin I (ENTI) is a novel bacteriocin produced byEnterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI.entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.

Cloning and Nucleotide Sequencing of aStaphylococcus aureus Gene Encoding a Branched-Chain-Amino-Acid Transporter

1998 ◽  
Vol 64 (2) ◽  
pp. 763-767 ◽  
Author(s):  
Uriwan Vijaranakul ◽  
Anming Xiong ◽  
Katherine Lockwood ◽  
R. K. Jayaswal
Keyword(s):  
Amino Acid ◽  
Insertion Site ◽  
Amino Acid Sequences ◽  
Nucleotide Sequencing ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACT We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889–1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureuschromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated α-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.

scholarly journals The Brucella abortus Cyclic β-1,2-Glucan Virulence Factor Is Substituted with O-Ester-Linked Succinyl Residues

2006 ◽  
Vol 188 (14) ◽  
pp. 5003-5013 ◽  
Author(s):  
Mara S. Roset ◽  
Andrés E. Ciocchini ◽  
Rodolfo A. Ugalde ◽  
Nora Iñón de Iannino
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Brucella Abortus ◽  
Brucella Melitensis ◽  
Open Reading Frame ◽  
Nucleotide Sequencing ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Host Interaction

ABSTRACT Brucella periplasmic cyclic β-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic β-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic β-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic β-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-β-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.

scholarly journals Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Δ1-Dehydrogenase Isoenzyme ofRhodococcus erythropolis Strain SQ1

2000 ◽  
Vol 66 (5) ◽  
pp. 2029-2036 ◽  
Author(s):  
R. van der Geize ◽  
G. I. Hessels ◽  
R. van Gerwen ◽  
J. W. Vrijbloed ◽  
P. van der Meijden ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rhodococcus Erythropolis ◽  
Ring Structure ◽  
Structure Characterization ◽  
Targeted Disruption ◽  
Gene Encoding ◽  
Reading Frame ◽  
Cell Extracts ◽  
Steroid Pathway

ABSTRACT Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Δ1-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment ofRhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Δ1-dehydrogenase genes. Heterologous expression ofkstD yielded 3-ketosteroid Δ1-dehydrogenase (KSTD) activity under the control of the lac promoter inEscherichia coli. Targeted disruption of thekstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9α-hydroxy-4-androstene-3,17-dione was not abolished by thekstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ1-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation ofkstD is not sufficient for inactivation of the Δ1-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed thatR. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

scholarly journals The Archaeoglobus fulgidusd-Lactate Dehydrogenase Is a Zn2+Flavoprotein

1999 ◽  
Vol 181 (24) ◽  
pp. 7580-7587 ◽  
Author(s):  
David W. Reed ◽  
Patricia L. Hartzell
Keyword(s):  
Lactate Dehydrogenase ◽  
Fusion Protein ◽  
Factor Xa ◽  
Sole Source ◽  
Open Reading Frame ◽  
Archaeoglobus Fulgidus ◽  
Sulfate Reducer ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilized-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a d-(−)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed inE. coli when coexpressed with the E. coli dnaYgene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for d-lactate, are stable at 80°C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 999-1007
Author(s):  
R G Gregerson ◽  
L Cameron ◽  
M McLean ◽  
P Dennis ◽  
J Strommer
Keyword(s):  
Chromosomal Location ◽  
Higher Plants ◽  
Gene Encoding ◽  
Genomic Libraries ◽  
Regulatory Strategy ◽  
Adh1 Gene ◽  
Adh Genes ◽  
Genes Encoding ◽  
Adh Gene ◽  
Polymerase Chain

Abstract In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

scholarly journals GIT1, a Gene Encoding a Novel Transporter for Glycerophosphoinositol in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1707-1715 ◽  
Author(s):  
J L Patton-Vogt ◽  
S A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Gene ◽  
Sugar Transport ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Uptake And Metabolism ◽  
Cells Cultured

Abstract Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git−) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git− phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

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