Kok RG

References (5)

Title : The genes rubA and rubB for alkane degradation in Acinetobacter sp. strain ADP1 are in an operon with estB, encoding an esterase, and oxyR - Geissdorfer_1999_J.Bacteriol_181_4292
Author(s) : Geissdorfer W , Kok RG , Ratajczak A , Hellingwerf KJ , Hillen W
Ref : Journal of Bacteriology , 181 :4292 , 1999
Abstract : Alkanes are oxidized in Acinetobacter sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. rubA and rubB encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in rubA or rubB lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes, estB and oxyR, are located downstream of rubB, but are not necessary for alkane degradation. estB encodes a functional esterase. oxyR encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide. rubA, rubB, estB, and oxyR constitute an operon, which is constitutively transcribed from a sigma70 promoter, and an estB-oxyR containing message is also transcribed from an internal promoter.
ESTHER : Geissdorfer_1999_J.Bacteriol_181_4292
PubMedSearch : Geissdorfer_1999_J.Bacteriol_181_4292
PubMedID: 10400587
Gene_locus related to this paper: acica-estB , acisp-O24848

Title : Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene - Kok_1995_Mol.Microbiol_15_803
Author(s) : Kok RG , van Thor JJ , Nugteren-Roodzant IM , Brouwer MB , Egmond MR , Nudel CB , Vosman B , Hellingwerf KJ
Ref : Molecular Microbiology , 15 :803 , 1995
Abstract : The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signal sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disulphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.
ESTHER : Kok_1995_Mol.Microbiol_15_803
PubMedSearch : Kok_1995_Mol.Microbiol_15_803
PubMedID: 7596283
Gene_locus related to this paper: acica-este3

Title : Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase - Kok_1995_J.Bacteriol_177_3295
Author(s) : Kok RG , van Thor JJ , Nugteren-Roodzant IM , Vosman B , Hellingwerf KJ
Ref : Journal of Bacteriology , 177 :3295 , 1995
Abstract : Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A. calcoaceticus BD413 is proposed.
ESTHER : Kok_1995_J.Bacteriol_177_3295
PubMedSearch : Kok_1995_J.Bacteriol_177_3295
PubMedID: 7768830
Gene_locus related to this paper: acica-este3

Title : A gene of Acinetobacter calcoaceticus BD413 encodes a periplasmic peptidyl-prolyl cis-trans isomerase of the cyclophilin sub-class that is not essential for growth - Kok_1994_Biochim.Biophys.Acta_1219_601
Author(s) : Kok RG , Christoffels VM , Vosman B , Hellingwerf KJ
Ref : Biochimica & Biophysica Acta , 1219 :601 , 1994
Abstract : Downstream of the Acinetobacter calcoaceticus estA gene, encoding a cell-bound esterase, an open reading frame (orf) was identified, which may encode a protein with a mass of 20.4 kDa. This protein shows extensive similarity to both prokaryotic and eukaryotic peptidyl-prolyl cis-trans isomerases (PPIases) of the cyclophilin sub-class, especially to the periplasmic rotamase (RotA) of Escherichia coli. A putative signal sequence suggests that the product of the Acinetobacter gene, we termed rotA, is located outside the cytoplasm. Transcription of the gene is initiated from a promoter, just upstream of the rotA orf. The observation that two A. calcoaceticus rotA deletion mutants display no apparent mutant phenotype, suggests that this PPIase is not essential for growth of the organism. These mutants, to our knowledge, are the first prokaryotic PPIase mutants reported.
ESTHER : Kok_1994_Biochim.Biophys.Acta_1219_601
PubMedSearch : Kok_1994_Biochim.Biophys.Acta_1219_601
PubMedID: 7948017
Gene_locus related to this paper: acica-este2

Title : Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases - Kok_1993_J.Gen.Microbiol_139_2329
Author(s) : Kok RG , Christoffels VM , Vosman B , Hellingwerf KJ
Ref : J Gen Microbiol , 139 :2329 , 1993
Abstract : Acinetobacter calcoaceticus BD413, when grown in batch culture in nutrient broth, produces both extracellular lipase activity and cell-bound esterase activity during and after the transition between exponential growth and the stationary phase. From a library of A. calcoaceticus DNA in Escherichia coli, plasmids were isolated that enabled E. coli to grow on media with tributyrin as the sole carbon source. Assays with model substrates classified the product of the cloned gene as an esterase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to contain one open reading frame, termed estA, which encodes a protein of 40.0 kDa. The amino acid sequence of this protein shows homology to a number of lipolytic enzymes, most notably to esterases. Deletion of estA only partially abolished cell-bound esterase activity in A. calcoaceticus, indicating that BD413 forms at least two esterases. Both esterases show the same temporal regulation of expression. beta-Galactosidase activity was measured in strains in which a promoterless lacZ gene was inserted into estA. Induction of lacZ expression in these strains also occurred at the end of exponential growth in batch cultures, indicating that production of the esterase is regulated at the genetic level.
ESTHER : Kok_1993_J.Gen.Microbiol_139_2329
PubMedSearch : Kok_1993_J.Gen.Microbiol_139_2329
PubMedID: 8254303
Gene_locus related to this paper: acica-este1 , acica-este2