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.

Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2682-2690 ◽  
Author(s):  
O. A. Karlsen ◽  
Ø. Larsen ◽  
H. B. Jensen
Keyword(s):  
Copper Ions ◽  
Sequence Similarity ◽  
Physiological Role ◽  
Growth Conditions ◽  
Inverse Pcr ◽  
Restriction Enzyme Digestion ◽  
Methylococcus Capsulatus ◽  
Gene Encoding ◽  
Reading Frame ◽  
Methylomicrobium Album

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a σ 54-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria.

scholarly journals Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide

1996 ◽  
Vol 316 (2) ◽  
pp. 685-690 ◽  
Author(s):  
Masahiro TAMOI ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Higher Plants ◽  
Sequence Similarity ◽  
Native Enzyme ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
Synechococcus Pcc 7942 ◽  
Pcc 7942

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62±4.5 and 420±10.5 μM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx. molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.

scholarly journals Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis

2010 ◽  
Vol 192 (13) ◽  
pp. 3304-3310 ◽  
Author(s):  
Yuchen Liu ◽  
Robert H. White ◽  
William B. Whitman
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
Direct Conversion ◽  
Maximum Activity ◽  
Lysine Biosynthesis ◽  
Diaminopimelic Acid ◽  
Reading Frame ◽  
Cell Extracts ◽  
Methanothermobacter Thermautotrophicus

ABSTRACT The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Δmmp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and α-ketoglutarate as substrates was 24.3 ± 2.0 nmol min−1 mg of protein−1. The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70°C and pH 8.0 to 9.0. The apparent Km s of MJ1391 for ll-DAP and α-ketoglutarate were 82.8 ± 10 μM and 0.42 ± 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales.

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 Trichloroethene Reductive Dehalogenase fromDehalococcoides ethenogenes: Sequence of tceA and Substrate Range Characterization

2000 ◽  
Vol 66 (12) ◽  
pp. 5141-5147 ◽  
Author(s):  
Jon K. Magnuson ◽  
Margaret F. Romine ◽  
David R. Burris ◽  
Mark T. Kingsley
Keyword(s):  
Signal Sequence ◽  
Sequence Similarity ◽  
Inverse Pcr ◽  
Binding Motif ◽  
Gene Encoding ◽  
Sequence Comparisons ◽  
Reading Frame ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Ethenogenes ◽  
Range Characterization

ABSTRACT The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE,cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase,tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)2. Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds

2002 ◽  
Vol 363 (2) ◽  
pp. 377-386 ◽  
Author(s):  
Ronald P. de VRIES ◽  
Patricia A. vanKUYK ◽  
Harry C.M. KESTER ◽  
Jaap VISSER
Keyword(s):  
Aspergillus Niger ◽  
Sugar Beet ◽  
Aromatic Compounds ◽  
Plant Cell Wall ◽  
Sequence Similarity ◽  
Feruloyl Esterase ◽  
Cell Wall Polysaccharides ◽  
Gene Encoding ◽  
Reading Frame ◽  
Wheat Arabinoxylan

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255–262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. nigerfaeA, encoding feruloyl esterase A (FAEA), and A. nigerbphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.

scholarly journals Tylosin Resistance in Arcanobacterium pyogenes Is Encoded by an Erm X Determinant

2003 ◽  
Vol 47 (11) ◽  
pp. 3519-3524 ◽  
Author(s):  
B. Helen Jost ◽  
Adam C. Field ◽  
Hien T. Trinh ◽  
J. Glenn Songer ◽  
Stephen J. Billington
Keyword(s):  
Antimicrobial Agents ◽  
Growth Promotion ◽  
The United States ◽  
Feedlot Cattle ◽  
Leader Peptide ◽  
Nucleotide Sequencing ◽  
Reading Frame ◽  
Constitutive Resistance ◽  
Genes Encoding ◽  
Arcanobacterium Pyogenes

ABSTRACT Arcanobacterium pyogenes, a commensal on the mucous membranes of many economically important animal species, is also a pathogen, causing abscesses of the skin, joints, and visceral organs as well as mastitis and abortion. In food animals, A. pyogenes is exposed to antimicrobial agents used for growth promotion, prophylaxis, and therapy, notably tylosin, a macrolide antibiotic used extensively for the prevention of liver abscessation in feedlot cattle in the United States. Of 48 A. pyogenes isolates, 11 (22.9%) exhibited inducible or constitutive resistance to tylosin (MIC of ≥128 μg/ml). These isolates also exhibited resistance to other macrolide and lincosamide antibiotics, suggesting a macrolide-lincosamide resistance phenotype. Of the 11 resistant isolates, genomic DNA from nine hybridized to an erm(X)-specific probe. Cloning and nucleotide sequencing of the A. pyogenes erm(X) gene indicated that it was >95% similar to erm(X) genes from Corynebacterium and Propionibacterium spp. Eight of the erm(X)-containing A. pyogenes isolates exhibited inducible tylosin resistance, which was consistent with the presence of a putative leader peptide upstream of the erm(X) open reading frame. For at least one A. pyogenes isolate, 98-4277-2, erm(X) was present on a plasmid, pAP2, and was associated with the insertion sequence IS6100. pAP2 also carried genes encoding the repressor-regulated tetracycline efflux system determinant Tet 33. The repA gene from pAP2 was nonfunctional in Escherichia coli and at least one A. pyogenes isolate, suggesting that there may be host-encoded factors required for replication of this plasmid.

scholarly journals Molecular Cloning and Expression Analysis of the Rhodobacter capsulatus sodB Gene, Encoding an Iron Superoxide Dismutase

1998 ◽  
Vol 180 (20) ◽  
pp. 5413-5420 ◽  
Author(s):  
Néstor Cortez ◽  
Néstor Carrillo ◽  
Cécile Pasternak ◽  
Angelika Balzer ◽  
Gabriele Klug
Keyword(s):  
Superoxide Dismutase ◽  
Rhodobacter Capsulatus ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Selection Procedure ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Species Activity

ABSTRACT Genetic complementation of a sodA sodB Escherichia colimutant strain was used to clone Rhodobacter capsulatusgenes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed inE. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodBmutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodBgene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.

scholarly journals Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product fromBacillus subtilis

1998 ◽  
Vol 180 (14) ◽  
pp. 3697-3703 ◽  
Author(s):  
Takashi Inaoka ◽  
Yoshinobu Matsumura ◽  
Tetsuaki Tsuchido
Keyword(s):  
Superoxide Dismutase ◽  
Stationary Phase ◽  
Specific Activity ◽  
Upstream Region ◽  
Gene Encoding ◽  
Sod Activity ◽  
Vegetative Cells ◽  
Reading Frame ◽  
Superoxide Dismutase Gene

ABSTRACT Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designatedsodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region ofsodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutantEscherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

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