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1.
Appl Environ Microbiol ; 82(14): 4299-4308, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27208130

ABSTRACT

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4 Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2 IMPORTANCE: In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer and the network responsible for microoxic metabolism through the interaction between the gene regulators phaR and fixK2 These results contribute to the understanding of the physiological conditions required for polyhydroxybutyrate biosynthesis. The interaction between these two main metabolic pathways is also reflected in the symbiotic phenotypes of soybeans inoculated with phaR mutants, which were more competitive for nodulation and enhanced dry matter production by the plants. Therefore, this knowledge may be applied to the development of superior strains to be used as improved inoculants for soybean crops.


Subject(s)
Bradyrhizobium/genetics , Bradyrhizobium/metabolism , Gene Expression Regulation, Bacterial , Hydroxybutyrates/metabolism , Metabolic Networks and Pathways/genetics , Polyesters/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon/metabolism , Cytoplasmic Granules/metabolism , Gene Deletion , Mannitol/metabolism , Soil Microbiology
2.
J Appl Microbiol ; 109(3): 1067-75, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20408935

ABSTRACT

AIMS: Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant γ-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. METHODS AND RESULTS: A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. CONCLUSIONS AND SIGNIFICANCE AND IMPACT OF THE STUDY: Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA.


Subject(s)
Dioxygenases/genetics , Hemiterpenes/metabolism , Latex/metabolism , Xanthomonas/enzymology , Dioxygenases/metabolism , Escherichia coli/genetics , Mutagenesis, Insertional , Pseudomonas putida/genetics , Xanthomonas/genetics , Xanthomonas/growth & development
3.
Biomacromolecules ; 2(1): 295-303, 2001.
Article in English | MEDLINE | ID: mdl-11749186

ABSTRACT

The degradation of natural rubber (NR), synthetic poly(cis-1,4-isoprene) (SR), and cross-linked NR (latex gloves) by Gram-positive and Gram-negative bacteria was analyzed by weight loss, gel permeation chromatography, and determination of the protein content. Weight losses of 11-18% and an increase in protein up to 850 microg/mL after incubation of Nocardia sp. DSMZ43191, Streptomyces coelicolor, Streptomyces griseus, bacterial isolate 18a, Acinetobacter calcoaceticus, and Xanthomonas sp. with latex gloves as a carbon source indicated degradation of the polymer. An increase of protein up to 1250 microg/mL was obtained upon incubation of the bacteria with SR. No or only little weight losses and no increase in the protein content were found for nondegrading control strains such as Streptomyces lividans and Streptomyces exfoliatus and for mutants of degrading strains of S. coelicolor and S. griseus, which have been identified by their inability to produce clearing zones on opaque latex agar. Measurement of the average molecular weight of synthetic rubber before and after degradation showed a time-dependent shift to lower values for the degrading strains. Diketone derivates of oligo(cis-1,4-isoprene) were identified as metabolites of rubber degradation. An oxidative degradation pathway of poly(cis-1,4-isoprene) to acetyl-coenzymeA and propionyl-coenzymeA by beta-oxidation is suggested for bacterial degradation of isoprene rubber.


Subject(s)
Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Rubber/metabolism , Acrylates/metabolism , Biodegradation, Environmental , Gloves, Protective/microbiology , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/growth & development , Gram-Positive Bacteria/enzymology , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/growth & development , Hemiterpenes , Latex/chemistry , Latex/metabolism , Molecular Structure , Molecular Weight , Mutation , Polyethylenes/chemistry , Polyethylenes/metabolism , Propionates/metabolism , Rubber/chemistry , Time Factors
4.
J Biol Chem ; 276(39): 36215-24, 2001 Sep 28.
Article in English | MEDLINE | ID: mdl-11457823

ABSTRACT

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.


Subject(s)
Bacterial Proteins , Carboxylic Ester Hydrolases/chemistry , Gram-Negative Anaerobic Straight, Curved, and Helical Rods/enzymology , Hydrolases/chemistry , Polyesters/chemistry , Polymers/chemistry , Amino Acid Sequence , Carboxylic Ester Hydrolases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Hydrogen-Ion Concentration , Lipase/chemistry , Molecular Sequence Data , Protein Binding , Substrate Specificity , Temperature , Time Factors
5.
Int J Syst Evol Microbiol ; 51(Pt 3): 905-908, 2001 May.
Article in English | MEDLINE | ID: mdl-11411713

ABSTRACT

The phylogenetic positions of the type strain and strain A62 of [Pseudomonas] lemoignei, as well as those of nine bacterial isolates closely related to [Pseudomonas] lemoignei, were re-evaluated by analysing morphological, physiological and molecular-biological characteristics. 16S rDNA analysis confirmed that the type strain of [Pseudomonas] lemoignei and strain A62 belong to the order 'Burkholderiales' of the beta-Proteobacteria and represent a new cluster, for which the name Paucimonas gen. nov. is proposed. Paucimonas contains a single species, Paucimonas lemoignei comb. nov.


Subject(s)
Betaproteobacteria/classification , Gram-Negative Bacteria/classification , Phylogeny , Betaproteobacteria/genetics , Betaproteobacteria/physiology , DNA, Ribosomal/genetics , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/physiology , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics
6.
Adv Biochem Eng Biotechnol ; 71: 293-325, 2001.
Article in English | MEDLINE | ID: mdl-11217416

ABSTRACT

Polyesters, such as microbially produced poly[(R)-3-hydroxybutyric acid] [poly(3HB)], other poly[(R)-hydroxyalkanoic acids] [poly(HA)] and related biosynthetic or chemosynthetic polyesters are a class of polymers that have potential applications as thermoplastic elastomers. In contrast to poly(ethylene) and similar polymers with saturated, non-functionalized carbon backbones, poly(HA) can be biodegraded to water, methane, and/or carbon dioxide. This review provides an overview of the microbiology, biochemistry and molecular biology of poly(HA) biodegradation. In particular, the properties of extracellular and intracellular poly(HA) hydrolyzing enzymes [poly(HA) depolymerases] are described.


Subject(s)
Bacteria/metabolism , Biodegradation, Environmental , Polyesters/chemistry , Polyesters/metabolism , Air , Amino Acid Sequence , Carbon Dioxide , Hydrolysis , Methane , Molecular Sequence Data , Sewage , Time Factors , Water
7.
J Org Chem ; 65(23): 7800-6, 2000 Nov 17.
Article in English | MEDLINE | ID: mdl-11073584

ABSTRACT

Lipase catalysis in nonaqueous media is recognized as a powerful tool in organic and more recently polymer synthesis. Even though none of the currently known polyhydroxyalkanoate (PHA) depolymerases have lipase activity, they do have a catalytic center that resembles that of lipases. Motivated by the above, the potential of using the poly(3-hydroxybutyrate), PHB, depolymerase from Psuedomonas lemoignei in organic media to catalyze ester-forming reactions was investigated. The effect of different organic solvents (benzene-d(6), cyclohexane-d(12), and acetonitrile-d(3)) on the activity of the PHB-depolymerase toward propylation of L-lactide was studied. A significant difference in the catalytic rate was observed as a function of solvent polarity. The selectivity of the PHB-depolymerase (P. lemoignei) to catalyze the propylation of a series of different lactones including epsilon-caprolactone, delta-butyrolactone, gamma-butyrolactone, and D, L, meso, and racemic lactides has been studied with the PHB-depolymerase (P. lemoignei) in organic solvents. Important differences in the reactivity of these lactones, as well as selective hydrolysis of stereochemically different linear lactic acid dimers, were observed. Moreover, the ability of the PHB-depolymerase to catalyze the solventless polymerization of epsilon-caprolactone and trimethylene carbonate was investigated.


Subject(s)
Carboxylic Ester Hydrolases/chemistry , Pseudomonas/enzymology , Catalysis
8.
J Bacteriol ; 182(20): 5916-8, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11004196

ABSTRACT

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.


Subject(s)
Acyltransferases/metabolism , Cupriavidus necator/metabolism , Hydroxybutyrates/metabolism , Polyesters/metabolism , Acyltransferases/genetics , Cupriavidus necator/genetics , Cupriavidus necator/growth & development , Cytoplasmic Granules/metabolism , Gene Deletion , Kinetics , Mutagenesis , Polymerase Chain Reaction
9.
Appl Environ Microbiol ; 66(9): 3680-5, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10966376

ABSTRACT

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6, 10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of beta-oxidation, (iii) oxidation of the conjugated double bond resulting in a beta-keto acid, and (iv) decarboxylation.


Subject(s)
Butadienes/metabolism , Hemiterpenes , Pentanes , Pseudomonas/metabolism , Streptomyces/metabolism , Biodegradation, Environmental , Butadienes/chemical synthesis , Culture Media , Mass Spectrometry , Pseudomonas/growth & development , Rubber/metabolism , Streptomyces/growth & development
10.
Appl Environ Microbiol ; 66(4): 1385-92, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10742216

ABSTRACT

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.


Subject(s)
Bacterial Proteins , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Polyesters/metabolism , Pseudomonas/enzymology , Valerates/metabolism , Acyltransferases/metabolism , Amino Acid Sequence , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/isolation & purification , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Hydroxybutyrates/metabolism , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA
11.
Appl Environ Microbiol ; 65(4): 1703-9, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10103271

ABSTRACT

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.


Subject(s)
Carboxylic Ester Hydrolases/biosynthesis , Pseudomonas/growth & development , Pseudomonas/metabolism , Succinates/metabolism , Biological Transport , Culture Media , Hydrogen-Ion Concentration , Hydroxybutyrates/metabolism , Substrate Specificity
12.
FEMS Microbiol Lett ; 167(1): 69-73, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9785454

ABSTRACT

A fragment of genomic DNA from Sulfolobus acidocaldarius DSM 639 encoding a lipolytic enzyme was cloned and sequenced. The 314-amino acid polypeptide displays a maximum sequence similarity (43%) to a putative polyhydroxyalkanoate depolymerase from Pseudomonas oleovorans and contains the pentapeptide G-X1-S-X2-G which is typical of serine hydrolases. The protein is highly thermostable and is able to hydrolyse a variety of lipid substrates thus providing a promising tool for potential biotechnological applications.


Subject(s)
Carboxylic Ester Hydrolases/genetics , Esterases/genetics , Lipolysis , Sulfolobus acidocaldarius/enzymology , Sulfolobus acidocaldarius/genetics , Amino Acid Sequence , Carboxylic Ester Hydrolases/metabolism , Cloning, Molecular , DNA, Archaeal/genetics , Enzyme Stability , Esterases/metabolism , Genes, Archaeal , Hot Temperature , Molecular Sequence Data , Pseudomonas/enzymology , Pseudomonas/genetics , Sequence Homology, Amino Acid , Substrate Specificity
13.
J Bacteriol ; 179(12): 4061-5, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9190828

ABSTRACT

A gene, mgt, encoding a protein homologous to the N-terminal module of class A high-molecular-mass penicillin-binding proteins was identified in Ralstonia eutropha. By using specific antibodies, the corresponding Mgt protein was detected in association with the membrane, confirming that the N-terminal hydrophobic segment functioned as a membrane anchor. A derivative in which the hydrophobic sequence was deleted was overexpressed as a maltose-binding fusion protein in Escherichia coli. Cleavage of the product resulted in substantial amounts of soluble Mgt derivative, indicating that folding occurs independently on other proteins or on homologous domains of penicillin-binding proteins.


Subject(s)
Alcaligenes/chemistry , Bacterial Proteins , Carrier Proteins/analysis , Glycosyltransferases/analysis , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/analysis , Peptidyl Transferases , Animals , Base Sequence , Carrier Proteins/biosynthesis , Escherichia coli/genetics , Glycosyltransferases/biosynthesis , Molecular Sequence Data , Muramoylpentapeptide Carboxypeptidase/biosynthesis , Penicillin-Binding Proteins , Rabbits
14.
FEMS Microbiol Lett ; 150(2): 179-88, 1997 May 15.
Article in English | MEDLINE | ID: mdl-9170260

ABSTRACT

Using natural rubber latex as the sole source of carbon and energy 50 rubber-degrading bacteria were isolated. Out of those 50 isolates, 33 were identified as Streptomyces species and 8 as Micromonospora species. Screening of 1220 bacteria obtained from different culture collections revealed 46 additional rubber-degrading bacteria (Streptomyces 31 strains, Micromonospora 5, Actinoplanes 3, Nocardia 2, Dactylosporangium 1, Actinomadura 1, unidentified 3). All rubber-degrading isolates were identified as members of the actinomycetes, a large group of mycelium-forming Gram-positive bacteria. Interestingly no Gram-negative bacterium could be isolated. In most strains expression of extracellular rubber-degrading enzymes was repressed by glucose and/or succinate. The reduction of the average molecular mass of solution-cast films of natural rubber from 640000 to 25000 in liquid culture upon bacterial growth indicates the participation of an endo-cleavage mechanism of degradation.


Subject(s)
Actinomycetales/metabolism , Rubber/metabolism , Biodegradation, Environmental , Carbohydrate Metabolism , Energy Metabolism , Environmental Microbiology , Enzyme Induction/drug effects , Europe , Asia, Eastern , Gene Expression Regulation, Bacterial/drug effects , Glucose/pharmacology , Phenotype , Species Specificity , Succinates/pharmacology , Succinic Acid
15.
Appl Microbiol Biotechnol ; 46(5-6): 451-63, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9008883

ABSTRACT

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases.


Subject(s)
Bacterial Proteins/metabolism , Carboxylic Ester Hydrolases/metabolism , Gram-Negative Aerobic Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Hydroxy Acids/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Bacteriological Techniques , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/genetics , Gram-Negative Aerobic Bacteria/classification , Gram-Positive Bacteria/classification , Hydrolysis , Molecular Biology , Molecular Sequence Data
16.
FEMS Microbiol Lett ; 143(2-3): 191-4, 1996 10 01.
Article in English | MEDLINE | ID: mdl-8837471

ABSTRACT

Binding of (i) purified wild-type poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ4 of Pseudomonas lemoignei, (ii) a purified truncated form of PhaZ4, which lacked 55 C-terminal amino acids and (iii) commercial lactate dehydrogenase to aqueous suspensions of PHB, chitin or cellulose was studied. Only the wild-type PHB depolymerase was specifically able to bind to PHB granules. No other combination of protein and polymeric substrate resulted in polymer-bound protein. Similar results were obtained for other PHB depolymerases. We concluded that the C-terminal amino acids of PHB depolymerases represent a PHB-specific binding domain or at least an essential part of it.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Amino Acid Sequence , Binding Sites , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/isolation & purification , Escherichia coli , Molecular Sequence Data , Mutagenesis , Pseudomonas/enzymology , Pseudomonas/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Substrate Specificity
17.
FEMS Microbiol Lett ; 142(2-3): 215-21, 1996 Sep 01.
Article in English | MEDLINE | ID: mdl-8810505

ABSTRACT

The poly(3-hydroxyalkanoate) (PHA) degrading isolate K10 was identified as Streptomyces exfoliatus. This bacterium is distinguished from other PHA-degrading strains by its ability to utilize both poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyoctanoate) (PHO). A PHA depolymerase structural gene of S. exfoliatus (phaZ(Sex) was cloned, expressed and partially purified from recombinant Escherichia coli. The depolymerase was specific for PHB and did not hydrolyze PHO. This indicated the presence of at least one additional gene in S. exfoliatus which encodes a PHO depolymerase. 3-Hydroxybutyrate was identified as the only product of PHB hydrolysis. Comparison of the DNA-deduced amino acid sequence revealed high homology to the PHB depolymerase of Comamonas sp. and low to medium homologies to other PHA depolymerases. The PHB depolymerases of S. exfoliatus and Comamonas sp. represent a subgroup within the family of PHA(SCL) depolymerases. To our knowledge, the S. exfoliatus PHB depolymerase is the first briefly characterized PHA depolymerase of a Gram-positive.


Subject(s)
Carboxylic Ester Hydrolases/genetics , Streptomyces/genetics , Amino Acid Sequence , Carboxylic Ester Hydrolases/metabolism , Cloning, Molecular , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Hydroxybutyrates/metabolism , Molecular Sequence Data , Polyesters/metabolism , Recombination, Genetic , Sequence Homology, Amino Acid , Streptomyces/classification , Streptomyces/enzymology
18.
Int J Syst Bacteriol ; 46(3): 769-73, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8782688

ABSTRACT

By using selective enrichment of polyhydroxyalkanoate-degrading bacteria and poly(3-hydroxyvalerate)-containing granules from Chromobacterium violaceum as the carbon source, 10 new Pseudomonas lemoignei strains were isolated; these strains were able to degrade poly(3-hydroxyvalerate), as well as poly(3-hydroxybutyrate), in vitro. The new isolates were characterized and identified by comparing them with P. lemoignei LMG 2207(T) (T = type strain). Like P. lemoignei LMG 2207(T) cells, the cells of the 10 new isolates contained mainly hexadecenoic, hexadecanoic, octadecenoic, and dodecanoic acids, as well as hydroxylated fatty acids, and exhibited respiration in the presence of methylpyruvate, 3-hydroxybutyrate, and 4-hydroxybutyrate, but not in the presence of the 92 other carbon sources included in Biolog GN microplates. The protein patterns of the new isolates were almost identical to each other and very similar to the protein pattern of P. lemoignei LMG 2207(T). Some of the new isolates, but not P. lemoignei LMG 2207(T), contained megaplasmids that were about 200 kbp long. The 16S ribosomal DNA genes of strain A62, a representative of the 10 new isolates, and of P. lemoignei LMG 2207(T) exhibited more than 0.99 sequence similarity. The DNA-DNA reassociation value for two representative strains was 100%, and the levels of DNA-DNA reassociation between these strains and the type strain were 60 and 61%. The taxonomy of P. lemoignei is briefly discussed.


Subject(s)
Polyesters/metabolism , Pseudomonas/classification , Valerates/metabolism , Bacterial Proteins/analysis , Base Composition , Base Sequence , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Fatty Acids/analysis , Molecular Sequence Data , Nucleic Acid Hybridization , Plasmids , Pseudomonas/genetics , Pseudomonas/isolation & purification , Pseudomonas/metabolism , RNA, Bacterial , RNA, Ribosomal, 16S , Soil Microbiology
19.
FEMS Microbiol Lett ; 141(1): 103-9, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8764515

ABSTRACT

Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignei) and S139 (A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 (P. lemoignei) and S196 (A. faecalis), resulted in mutant proteins with almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.


Subject(s)
Alcaligenes/enzymology , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Pseudomonas/enzymology , Serine/physiology , Amino Acid Sequence , Base Sequence , Binding Sites , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/isolation & purification , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Isoflurophate/pharmacology , Molecular Sequence Data , Molecular Weight , Mutation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Alignment
20.
Appl Environ Microbiol ; 61(8): 3113-8, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7487042

ABSTRACT

The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.


Subject(s)
Bacillus subtilis/enzymology , Carboxylic Ester Hydrolases/metabolism , Gram-Negative Aerobic Bacteria/enzymology , Lipase/metabolism , Pseudomonas/enzymology , Amino Acid Sequence , Bacillus subtilis/genetics , Binding Sites , Carboxylic Ester Hydrolases/genetics , Consensus Sequence , Conserved Sequence , Gram-Negative Aerobic Bacteria/genetics , Hydrolysis , Hydroxy Acids/metabolism , Lipase/genetics , Molecular Sequence Data , Polyesters/metabolism , Pseudomonas/genetics , Substrate Specificity
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