Tetrachloroethene Dehalogenase from Dehalospirillum multivorans: Cloning, Sequencing of the Encoding Genes, and Expression of the pceA Gene in Escherichia coli

ABSTRACT The genes encoding tetrachloroethene reductive dehalogenase, a corrinoid-Fe/S protein, of Dehalospirillum multivorans were cloned and sequenced. The pceA gene is upstream ofpceB and overlaps it by 4 bp. The presence of a ς70-like promoter sequence upstream of pceA and of a ρ-independent terminator downstream of pceB indicated that both genes are cotranscribed. This assumption is supported by reverse transcriptase PCR data. The pceA and pceB genes encode putative 501- and 74-amino-acid proteins, respectively, with calculated molecular masses of 55,887 and 8,354 Da, respectively. Four peptides obtained after trypsin treatment of tetrachloroethene (PCE) dehalogenase were found in the deduced amino acid sequence of pceA. The N-terminal amino acid sequence of the PCE dehalogenase isolated from D. multivorans was found 30 amino acids downstream of the N terminus of the deduced pceA product. The pceAgene contained a nucleotide stretch highly similar to binding motifs for two Fe4S4 clusters or for one Fe4S4 cluster and one Fe3S4 cluster. A consensus sequence for the binding of a corrinoid was not found in pceA. No significant similarities to genes in the databases were detected in sequence comparisons. The pceB gene contained two membrane-spanning helices as indicated by two hydrophobic stretches in the hydropathic plot. Sequence comparisons of pceBrevealed no sequence similarities to genes present in the databases. Only in the presence of pUBS 520 supplying the recombinant bacteria with high levels of the rare Escherichia colitRNA4 Arg was pceA expressed, albeit nonfunctionally, in recombinant E. coli BL21 (DE3).

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Protein engineering with synthetic Escherichia coli amber suppressor genes

Genome ◽

10.1139/g89-161 ◽

1989 ◽

Vol 31 (2) ◽

pp. 905-908 ◽

Cited By ~ 3

Author(s):

Jeffrey H. Miller ◽

Lynn G. Kleina ◽

Jean-Michel Masson ◽

Jennifer Normanly ◽

John Abelson

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Protein Engineering ◽

Promoter Sequence ◽

Lac Repressor ◽

Suppressor Genes ◽

Protein Coding ◽

E Coli ◽

Amber Suppressor ◽

Genes Encoding

We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the genes for different tRNAs with the anticodon 5′ CTA 3′. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.Key words: synthetic tRNA, amber suppressor, protein engineering, lac repressor, Escherichia coli.

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Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens

Microbiology ◽

10.1099/mic.0.26455-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2357-2371 ◽

Cited By ~ 93

Author(s):

Simone Laska ◽

Friedrich Lottspeich ◽

Arnulf Kletzin

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

G Protein ◽

Specific Activity ◽

Small Subunit ◽

Sequence Comparisons ◽

Genes Encoding ◽

Molecular Masses ◽

Membrane Bound ◽

Sulfur Reductase

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U (mg protein)−1] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U (mg protein)−1 with methyl viologen and 833 U (mg protein)−1 with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 μmol Fe and 2 μmol Ni (g protein)−1; in addition, 2·5 μmol Mo (g protein)−1 was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.

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Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-240→Gly

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.8.2269-2275.2001 ◽

2001 ◽

Vol 45 (8) ◽

pp. 2269-2275 ◽

Cited By ~ 92

Author(s):

R. Bonnet ◽

C. Dutour ◽

J. L. M. Sampaio ◽

C. Chanal ◽

D. Sirot ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Catalytic Efficiency ◽

Enterobacter Cloacae ◽

E Coli ◽

Genes Encoding ◽

Encoding Gene ◽

Synergy Test ◽

Encoding Genes

ABSTRACT Three clinical strains (Escherichia coli Rio-6,E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla CTX-M genes encoding β-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla CTX-M-9 gene observed inE. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla CTX-M-16, observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240→Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 μg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 μg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher kcat for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla CTX-M-9 genes and the bla CTX-M-16 gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M β-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum β-lactamases in Brazil.

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