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B Witholt

Publications and source records attributed to B Witholt.

At least 91 records · Page 5Linked to original sources
PubMed1989

The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression.

We have identified and sequenced the Pseudomonas OCT plasmid-encoded alkane hydroxylase gene (alkB) and its promoter. The transcription initiation site of the alkBAC mRNA was determined by nuclease S1 mapping. A putative interaction site with RNA-polymerase was identified based on homology of the alk promoter with other Pseudomonas promoters. The alkB gene encodes a 401-amino acid polypeptide which, despite an unusual codon composition, can be expressed at high levels in Escherichia coli and Pseudomonas. The amino-terminal sequence of the purified cytoplasmic membrane alkane hydroxylase was determined and was found to be in agreement with the nucleotide sequence. The translation product of the alkB gene contains nine hydrophobic sequences of which eight are sufficiently long to be membrane-spanning segments. The amino-terminal sequence resembles that of several bacterial integral membrane proteins and is not cleaved off following translation.

Amino Acid Sequence↗
PubMed1989

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase.

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.

Aldehyde Dehydrogenase↗
PubMed1989

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels.

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M(r) = 79,000) shares a high degree of similarity with maize branching enzyme I (M(r) = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.

Journal Article↗
PubMed1989

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.

Biodegradation, Environmental↗
PubMed1989

Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads.

The fluorescent pseudomonads are classified as a group, one characteristic of which is that they do not accumulate poly-3-hydroxybutyrate (PHB) during nutrient starvation in the presence of excess carbon source. In this paper we show that prototype strains from this subclass, such as Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, do accumulate poly-3-hydroxyalkanoates (PHA) when grown on fatty acids. These PHAs are composed of medium-chain-length (C6 to C12) 3-hydroxy fatty acids. The ability to form these polyesters does not depend on the presence of plasmids. A specificity profile of the enzymes involved in the biosynthesis of PHA was determined by growing Pseudomonas oleovorans on fatty acids ranging from C4 to C18. In all cases, PHAs were formed which contained C6 to C12 3-hydroxy fatty acids, with a strong preference for 3-hydroxyoctanoate when Ceven fatty acids were supplied and 3-hydroxynonanoate when Codd fatty acids were the substrate. These results indicate that the formation of PHAs depends on a specific enzyme system which is distinct from that responsible for the synthesis of PHB. While the fluorescent pseudomonads are characterized by their inability to make PHB, they appear to share the capacity to produce PHAs. This characteristic may be helpful in classifying pseudomonads. It may also be useful in the optimization of PHA production for biopolymer applications.

Fatty Acids↗
PubMed1989

Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments.

EDTA-induced outer membrane losses from whole cells of wild-type Escherichia coli (O111:B4) and several lipopolysaccharide (LPS) mutants derived from E. coli K-12 D21 were analyzed. EDTA treatment induced losses of LPS (up to 40%), outer membrane proteins OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine. The extent of these releases was strain specific. Successively more EDTA was necessary to induce these losses from strains containing LPS with increasing polysaccharide chain length. An additional heat shock immediately following the EDTA treatment had no effect on LPS release, but it decreased the release of outer membrane proteins and reduced the leakage of periplasmic proteins, suggesting that the temporary increase in outer membrane "permeability" caused by Ca2+-EDTA treatment was rapidly reversed by the redistribution of outer membrane components, a process which is favored by a mild heat shock. The fact that the material released from E. coli C600 showed a constant ratio of lipoprotein, OmpA, and phosphatidylethanolamine at all EDTA concentrations tested suggests that the material is lost as specific outer membrane patches. The envelope alterations caused by EDTA did not result in cell lysis.

Bacterial Outer Membrane Proteins↗
PubMed1989

Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells.

Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes.

Bacterial Outer Membrane Proteins↗
PubMed1989

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.

Amino Acid Sequence↗
PubMed1988

Alkane utilization in Pseudomonas oleovorans. Structure and function of the regulatory locus alkR.

The OCT plasmid-localized alkBAC operon encodes enzymes for alkane hydroxylation and alkanol dehydrogenation. The positively controlled expression of the operon is very efficient in both Pseudomonas putida and Escherichia coli. Two regulatory functions have been ascribed to the regulatory locus alkR: inducer recognition and transcriptional activation of the operon. We have cloned and localized the alkR locus on a 4.9-kilobase pair SalI fragment. The alkR region was analyzed for translation productions in E. coli minicells. Two proteins were identified: a 99- and a 48-kDa peptide. The positions of the cistrons encoding these proteins were established. Both cistrons were shown to be essential for an Alk phenotype. The first cistron (alkS), which encodes the 99-kDa protein, complemented alkR mutations affecting inducer specificity. Furthermore, we found that alkS is responsible for activation of expression of the alkBAC operon since it is required for the induction of the alkB gene product alkane hydroxylase. The second cistron (alkT), which encodes the 48-kDa protein, is required for reconstitution of an Alk phenotype but has no function in regulation of alkBAC expression. Thus, the expression of the alkBAC operon is regulated by a 99-kDa protein, whereas the 48-kDa protein is probably a component of the alkane hydroxylase complex.

Bacterial Proteins↗
PubMed1988

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers.

An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6-dichlorohexane-degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28,000. The affinity for its substrates was relatively low with Km values for short-chain haloalkanes in the range 0.1-0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5-C9 1-halo-n-alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e.g. methylbromide, dibromomethane, 1,2-dibromoethane, 1,3-dichloropropene, and bis(2-chloroethyl)ether. The degradation of chiral 2-bromoalkanes appeared to proceed without stereochemical preference. Optically active 2-bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.

Actinomycetales↗
PubMed1988

Formation of Polyesters by Pseudomonas oleovorans: Effect of Substrates on Formation and Composition of Poly-(R)-3-Hydroxyalkanoates and Poly-(R)-3-Hydroxyalkenoates.

Pseudomonas oleovorans grows on C(6) to C(12)n-alkanes and 1-alkenes. These substrates are oxidized to the corresponding fatty acids, which are oxidized further via the beta-oxidation pathway, yielding shorter fatty acids which have lost one or more C(2) units. P. oleovorans normally utilizes beta-oxidation pathway intermediates for growth, but in this paper we show that the intermediate 3-hydroxy fatty acids can also be polymerized to intracellular poly-(R)-3-hydroxyalkanoates (PHAs) when the medium contains limiting amounts of essential elements, such as nitrogen. The monomer composition of these polyesters is a reflection of the substrates used for growth of P. oleovorans. The largest monomer found in PHAs always contained as many C atoms as did the n-alkane used as a substrate. Monomers which were shorter by one or more C(2) units were also observed. Thus, for C-even substrates, only C-even monomers were found, the smallest being (R)-3-hydroxyhexanoate. For C-odd substrates, only C-odd monomers were found, with (R)-3-hydroxyheptanoate as the smallest monomer. 1-Alkenes were also incorporated into PHAs, albeit less efficiently and with lower yields than n-alkanes. These PHAs contained both saturated and unsaturated monomers, apparently because the 1-alkene substrates could be oxidized to carboxylic acids at either the saturated or the unsaturated ends. Up to 55% of the PHA monomers contained terminal double bonds when P. oleovorans was grown on 1-alkenes. The degree of unsaturation of PHAs could be modulated by varying the ratio of alkenes to alkanes in the growth medium. Since 1-alkenes were also shortened before being polymerized, as was the case for n-alkanes, copolymers which varied with respect to both monomer chain length and the percentage of terminal double bonds were formed during nitrogen-limited growth of P. oleovorans on 1-alkenes. Such polymers are expected to be useful for future chemical modifications.

Journal Article↗
PubMed1987

Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli.

The OCT plasmid encodes enzymes for alkane hydroxylation and alkanol dehydrogenation. Structural components are encoded on the 7.5-kilobase pair alkBAC operon, whereas positive regulatory components are encoded by alkR. We have constructed plasmids containing fusions of cloned alkBAC and alkR DNA and used these fusion plasmids to study the functional expression of the alkBAC operon and the regulatory locus alkR in Pseudomonas putida and in Escherichia coli. Growth on alkanes requires a functional chromosomally encoded fatty acid degradation system in addition to the plasmid-borne alk system. While such a system is active in P. putida, it is active in E. coli only in fadR mutants in which fatty acid degradation enzymes are expressed constitutively. Using such mutants, we found that E. coli as well as P. putida grew on octane as the sole source of carbon and energy when they were supplied with the cloned complete alk system. The alkR locus was strictly necessary in E. coli as well as in P. putida for expression of the alkBAC operon. The alkBAC operon could, however, be further reduced to a 5-kilobase pair operon without affecting the Alk phenotype in either species to a significant extent. Although with this reduction the plasmid-encoded alkanol dehydrogenase activity was lost, chromosomally encoded alkanol dehydrogenases in P. putida and E. coli compensated for this loss. The induction kinetics of the alk system was studied in detail in P. putida and E. coli. We used specific antibodies raised against alkane hydroxylase to follow the appearance of this protein following induction with octane. We found the induction kinetics of alkane hydroxylase to be similar in both species. A steady-state level was reached after about 2 h of induction in which time the alkane hydroxylase accounted for about 1.5% of total newly synthesized protein. Thus, alkBAC expression is very efficient and strictly regulated to both P. putida and E. coli.

Alkanes↗
PubMed1987

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from Escherichia coli.

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca2+ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (greater than 98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cells, and in investigating envelope-related synthesis and assembly processes.

Bacteriological Techniques↗
PubMed1987

Structure of the Pseudomonas putida alkBAC operon. Identification of transcription and translation products.

The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment. We have measured the length and determined the position of the alkBAC operon on this fragment by electron microscopy of R-loops. Furthermore, the 7.3-kilobase pair long alkBAC operon was analyzed for translation products in Escherichia coli minicells. Using a spectrum of overlapping subclones, six different proteins were identified. Starting from the alkBAC promotor, these polypeptides had molecular masses of 41, 15, 49, 58, 59, and 20 kDa, respectively. The 41-kDa protein was identified as alkane hydroxylase by reaction with a specific antibody. The 15- and 49-kDa peptides are soluble components of the alkane hydroxylase complex. The 58-kDa protein is most likely involved in alkanol dehydrogenase activity.

Alkanes↗
PubMed1987

Degradation of n-haloalkanes and alpha, omega-dihaloalkanes by wild-type and mutants of Acinetobacter sp. strain GJ70.

A 1,6-dichlorohexane-degrading strain of Acinetobacter sp. was isolated from activated sludge. The organism could grow with and quantitatively release halide from 1,6-dichlorohexane, 1,9-dichlorononane, 1-chloropentane, 1-chlorobutane, 1-bromopentane, ethylbromide, and 1-iodopropane. Crude extracts contained an inducible novel dehalogenase that liberated halide from the above compounds and also from 1,3-dichloropropane, 1,2-dibromoethane, and 2-bromoethanol. The latter two compounds were toxic suicide substrates for the organism at concentrations of 10 and 5 microM, respectively. Mutants resistant to 1,2-dibromoethane (3 mM) lacked dehalogenase activity and did not utilize haloalkanes for growth. Mutants resistant to both 1,2-dibromoethane (3 mM) and 2-bromoethanol (30 mM) could no longer oxidize or utilize alcohols and were capable of hydrolytic dehalogenation of 1,2-dibromoethane to ethylene glycol.

Acinetobacter↗
PubMed1987

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.

Bacterial Outer Membrane Proteins↗
PubMed1986

Identification of granule-bound starch synthase in potato tubers.

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.

Journal Article↗