ABSTRACT
Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.
Subject(s)
Genetic Vectors , Gram-Negative Bacteria/genetics , Plasmids , Base Sequence , Cloning, Molecular , Conjugation, Genetic , Escherichia coli/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Species SpecificityABSTRACT
An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.
Subject(s)
Aminohydrolases/genetics , Gram-Negative Aerobic Bacteria/genetics , Amino Acid Sequence , Aminohydrolases/chemistry , Aminohydrolases/isolation & purification , Bacterial Proteins/genetics , Base Sequence , Chaperonins/genetics , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genes, Bacterial , Gram-Negative Aerobic Bacteria/enzymology , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Soil MicrobiologyABSTRACT
A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram+ and Gram- bacteria.
Subject(s)
Amidohydrolases/genetics , Brevibacterium/enzymology , Escherichia coli/genetics , Pseudomonas aeruginosa/enzymology , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amino Acid Sequence , Base Sequence , Brevibacterium/genetics , Cloning, Molecular , Escherichia coli/enzymology , Molecular Sequence Data , Pseudomonas aeruginosa/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Restriction MappingABSTRACT
A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertion-mediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.
Subject(s)
Bacterial Proteins/genetics , DNA, Bacterial/isolation & purification , Genes, Bacterial , Pseudomonas/genetics , Vitamin B 12/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Base Sequence , DNA, Bacterial/chemistry , Deoxyribonuclease HindIII , Genetic Complementation Test , Molecular Sequence Data , Molecular Weight , Multigene Family , Mutagenesis, Insertional , Vitamin B 12/biosynthesisABSTRACT
A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contained five different cob genes named cobN to cobQ and cobW. Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cob proteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibiting both cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified as cob(I)alamin adenosyltransferase. CobN is proposed to play a role in cobalt insertion reactions. Four other open reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not lead to a cobalamin-minus phenotype.