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2.
J Bacteriol ; 183(5): 1595-9, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11160090

ABSTRACT

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/isolation & purification , Muramoylpentapeptide Carboxypeptidase/metabolism , Peptidyl Transferases , Bacillus subtilis/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cloning, Molecular , Enzyme Stability , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/growth & development , Hydrogen-Ion Concentration , Kinetics , Lactams/metabolism , Muramoylpentapeptide Carboxypeptidase/chemistry , Muramoylpentapeptide Carboxypeptidase/genetics , Penicillin-Binding Proteins , Plasmids , Thiolester Hydrolases/metabolism
3.
Nature ; 390(6657): 249-56, 1997 Nov 20.
Article in English | MEDLINE | ID: mdl-9384377

ABSTRACT

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.


Subject(s)
Bacillus subtilis/genetics , Genome, Bacterial , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Cloning, Organism , DNA, Bacterial , Molecular Sequence Data
4.
FEMS Microbiol Lett ; 149(2): 189-94, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9141660

ABSTRACT

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond to a sigma 70-type promoter. This promoter responds to heat shock and involves an inverted repeat different from the CIRCE sequence characteristic of the Gram-positive heat-shock promoters.


Subject(s)
Operon/genetics , Sequence Analysis, DNA/methods , Streptomyces/genetics , Transcription, Genetic/genetics , Base Sequence , Chromosome Mapping , Gene Expression Regulation, Bacterial/physiology , Genes, Bacterial/genetics , Heat-Shock Proteins/genetics , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Recombinant Proteins/genetics
5.
DNA Seq ; 6(3): 179-84, 1996.
Article in English | MEDLINE | ID: mdl-8722574

ABSTRACT

The dnaK operon of Streptomyces coelicolor A3(2) was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two of the most conserved regions in 30 different DnaK proteins (HSP70). The isolated insert-a BamHI 5.6-kb fragment-was sequenced and shown to contain three open-reading frames organized in an operon and coding for proteins analogous to DnaK, GrpE and DnaJ, successively.


Subject(s)
Escherichia coli Proteins , Genes, Bacterial , HSP70 Heat-Shock Proteins/genetics , Operon/genetics , Streptomyces/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Conserved Sequence/genetics , DNA Probes , HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/chemistry , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Molecular Sequence Data , Open Reading Frames/genetics , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Streptomyces/chemistry
6.
FEMS Microbiol Lett ; 78(1): 101-5, 1992 Nov 15.
Article in English | MEDLINE | ID: mdl-1468613

ABSTRACT

Two beta-lactamase genes called blaL and blaU have been cloned independently in Liège and in Umeå, from Streptomyces cacaoi. Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of beta-lactamases (active-site serine penicillinases). DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S. cacaoi strains used in Liège and in Umeå.


Subject(s)
Genes, Bacterial , Streptomyces/genetics , beta-Lactamases/genetics , Amino Acid Sequence , Molecular Sequence Data , Nucleic Acid Hybridization , Polymerase Chain Reaction , Streptomyces/enzymology
7.
Biochim Biophys Acta ; 748(2): 213-9, 1983 Oct 28.
Article in English | MEDLINE | ID: mdl-6626553

ABSTRACT

The bovine beta-crystallin Bp chain is organized into two very similar domains, with short extensions at both N- and C-termini, and two alternative models for the beta Bp dimer have been proposed (Wistow, G., Slingsby, C., Blundell, T., Driessen, H.P.C., De Jong, W.W. and Bloemendal, H. (1981) FEBS Lett. 133, 9-16). By limited proteolysis the C-terminal arms can be cleaved off rapidly from the beta Bp dimer, while the N-terminal arms are more difficult to remove. Trypsin divides the beta Bp chain into two fragments which approximately correspond to the two structural domains. Dissociation and reassociation of the different products of limited proteolysis indicated that: the C-terminal arm extends freely from the surface and is not involved in subunit-contact; at least one N-terminal arm seems required for dimer formation; the N-terminal domains have a greater tendency to associate than the C-terminal domains and, when mixed, the purified domains reassociate partially to a Mr 50 000 structure like native beta Bp. These findings support the more extended dimer model of beta Bp.


Subject(s)
Crystallins/metabolism , Animals , Cattle , Chymotrypsin , Electrophoresis, Polyacrylamide Gel , Kinetics , Macromolecular Substances , Molecular Weight , Peptide Fragments/analysis , Protein Conformation , Trypsin/metabolism
8.
Eur J Biochem ; 135(2): 315-20, 1983 Sep 15.
Article in English | MEDLINE | ID: mdl-6136410

ABSTRACT

Incubation of calf lens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different beta-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamylyltransferase, EC 2.3.2.13). One of these is beta Bp, the predominant subunit of beta-crystallin, of which the amino acid sequence is known. The site of amine-labeling in beta Bp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled beta-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled beta subunits.


Subject(s)
Acyltransferases/metabolism , Crystallins/metabolism , Lens, Crystalline/enzymology , Acylation , Animals , Binding Sites , Cattle , Electrophoresis, Paper , Electrophoresis, Polyacrylamide Gel , Fluorescence , Peptide Fragments/isolation & purification , Substrate Specificity , Transglutaminases
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