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 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.

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 Cytochrome P460 Genes from the MethanotrophMethylococcus capsulatus Bath

1998 ◽  
Vol 180 (24) ◽  
pp. 6440-6445 ◽  
Author(s):  
David J. Bergmann ◽  
James A. Zahn ◽  
Alan B. Hooper ◽  
Alan A. DiSpirito
Keyword(s):  
Sequence Similarity ◽  
Immunoblot Analysis ◽  
Open Reading Frames ◽  
Binding Motif ◽  
Type I ◽  
Methylococcus Capsulatus ◽  
Gene Encoding ◽  
Degenerate Oligonucleotide ◽  
Apparent Similarity ◽  
Ammonia Oxidizing Bacterium

ABSTRACT P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacteriumNitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that containscyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing usingcyp indicated that a cytochrome P460 similar to that fromM. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b andMethylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45,Methylomicrobium albus BG8, and Methylomonassp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.

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 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 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.

scholarly journals Rhizobium daejeonense sp. nov. isolated from a cyanide treatment bioreactor

2005 ◽  
Vol 55 (6) ◽  
pp. 2543-2549 ◽  
Author(s):  
Zhe-Xue Quan ◽  
Hee-Sung Bae ◽  
Jong-Hwan Baek ◽  
Wen-Feng Chen ◽  
Wan-Taek Im ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Growth Conditions ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
Phenotypic Properties ◽  
Type Strains

A polyphasic study was carried out to determine the taxonomic position of two aerobic, cyanide-degrading bacterial strains, designated L61T and L22, which had been isolated from a bioreactor for the treatment of nickel-complexed cyanide. The two isolates exhibited almost identical taxonomic characteristics. Phylogenetic analysis inferred from comparative 16S rRNA gene sequences indicated that the isolates fall in a sublineage of the genus Rhizobium comprising the type strains of Rhizobium giardinii, Rhizobium radiobacter, Rhizobium rubi, Rhizobium larrymoorei, Rhizobium vitis, Rhizobium undicola, Rhizobium loessense, Rhizobium galegae and Rhizobium huautlense. Cells of the two isolates are Gram-negative, aerobic, motile and non-spore-forming rods (0·6–0·7×1·1–1·3 μm), with peritrichous flagella. The DNA G+C content is 60·1–60·9 mol%. Cellular fatty acids are C16 : 0 (2·2–3·3 %), C18 : 0 (2·1–3·2 %), C19 : 0 cyclo ω8c (9·9–16·8 %), C20 : 3 ω6,9,12c (2·7–3·3 %), summed feature 3 (7·2–7·7 %) and summed feature 7 (67·8–73·7 %). The strains formed nodules on a legume plant, Medicago sativa. A nifH gene encoding denitrogenase reductase, the key component of the nitrogenase enzyme complex, was detected in L61T by PCR amplification by using a nifH-specific primer system. Strains L61T and L22 were distinguished from the type strains of recognized Rhizobium species in the same sublineage based on low DNA–DNA hybridization values (2–4 %) and/or a 16S rRNA gene sequence similarity value of less than 96 %. Moreover, some phenotypic properties with respect to substrate utilization as a carbon or nitrogen source, antibiotic resistance and growth conditions could be used to discriminate L61T and L22 from Rhizobium species in the same sublineage. Based on the results obtained in this study, L61T and L22 are considered to be representatives of a novel species of Rhizobium, for which the name Rhizobium daejeonense sp. nov. is proposed. The type strain is L61T (=KCTC 12121T=IAM 15042T=CCBAU 10050T).

scholarly journals The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

2003 ◽  
Vol 71 (4) ◽  
pp. 1938-1943 ◽  
Author(s):  
Maciej Lis ◽  
Howard K. Kuramitsu
Keyword(s):  
Streptococcus Mutans ◽  
Kinase Activity ◽  
Sequence Similarity ◽  
Physiological Role ◽  
Amino Acid Sequence Similarity ◽  
Gene Encoding ◽  
Lipid Kinase ◽  
Intact Gene ◽  
Wild Type Phenotype

ABSTRACT We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.

scholarly journals Shewanella oneidensis MR-1 Sensory Box Protein Involved in Aerobic and Anoxic Growth

2011 ◽  
Vol 77 (13) ◽  
pp. 4647-4656 ◽  
Author(s):  
A. Sundararajan ◽  
J. Kurowski ◽  
T. Yan ◽  
D. M. Klingeman ◽  
M. P. Joachimiak ◽  
...  
Keyword(s):  
Reductase Activity ◽  
Shewanella Oneidensis ◽  
Physiological Role ◽  
Growth Conditions ◽  
Electron Acceptors ◽  
Wild Type ◽  
Lower Growth ◽  
Content Type ◽  
Reading Frame ◽  
Consumption Rates

ABSTRACTAlthough little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) inShewanella oneidensisMR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of differentc-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, includingc-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia inS. oneidensisMR-1.

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

2008 ◽  
Vol 410 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Fui-Ching Tan ◽  
Qi Cheng ◽  
Kaushik Saha ◽  
Ilka U. Heinemann ◽  
Martina Jahn ◽  
...  
Keyword(s):  
Expression Vector ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Homology Search ◽  
Protein Database ◽  
Gene Encoding ◽  
Reading Frame ◽  
Green Fluorescent

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

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