Determinants for overproduction of the Pseudomonas oleovorans cytoplasmic membrane protein alkane hydroxylase in alk+ Escherichia coli W3110.

The Pseudomonas oleovorans alkB gene is expressed in alk+ Escherichia coli W3110 to 10 to 15% of the total cell protein, which is exceptional for a (foreign) cytoplasmic membrane protein. In other E. coli recombinants such as alk+ HB101, AlkB constitutes 2 to 3% of the total protein. In this study, we have investigated which factors determine the expression level of alkB in alk+ W3110. In particular, we have investigated the role of AlkB-induced stimulation of phospholipid synthesis. Blocking phospholipid synthesis in alk+ W3110 did not specifically alter the expression of alkB, and we conclude that stimulation of phospholipid synthesis is not a prerequisite for high-level expression of the membrane protein. W3110 is able to produce exceptionally high levels of alkane monooxygenase, because the rate of alkB mRNA synthesis in W3110 is an order of magnitude higher than that in HB101. This may be due in part to the higher copy number of pGEc47 in W3110 in comparison with HB101.     

Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids.

Pseudomonas putida PpG6 is able to utilize purified n-alkanes of six to ten carbon atoms for growth. It can also grow on the primary terminal oxidation products of these alkanes and on 1-dodecanol but not on the corresponding 2-ketones or 1,6-hexanediol, adipic acid, or pimelic acid. Revertible point mutants can be isolated which have simultaneously lost the ability to grow on all five n-alkane growth substrates but which can still grow on octanol or nonanol. An acetate-negative mutant defective in isocitrate lysase activity is unable to grow on even-numbered alkanes and fatty acids. Analysis of double mutants defective in acetate and propionate or in acetate and glutarate metabolism shows that alkane carbon is assimilated only via acetyl-coenzyme A and propionyl-coenzyme A. These results support the following conclusions: (i) The n-alkane growth specificity of P. putida PpG6 is due to the substrate specificity of whole-cell alkane hydroxylation; (ii) there is a single alkane hydroxylase enzyme complex; (iii) the physiological role of this complex is to initiate the monoterminal oxidation of alkane chains; and (iv) straight-chain fatty acids from butyric through nonanoic are degraded exclusively by beta-oxidation from the carboxyl end of the molecule.     

Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonas putida.

In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase proteins in mutants lacking the ability to convert n-alkanes to their primary alcohols. Purified heptane is an effective inducer in a mutant lacking detectable hydroxylase activity.     

Genetics of alkane oxidation byPseudomonas oleovorans

Many Pseudomonads are able to use linear alkanes as sole carbon and energy source. The genetics and enzymology of alkane metabolism have been investigated in depth forPseudomonas oleovorans, which is able to oxidize C5-C12 n-alkanes by virtue of two gene regions, localized on the OCT-plasmid. The so-calledalk-genes have been cloned in pLAFR1, and were subsequent analyzed using minicell expression experiments, DNA sequencing and deletion analysis. This has led to the identification and characterization of thealkBFGHJKL andalkST genes which encode all proteins necessary to convert alkanes to the corresponding acyl-CoA derivatives. These then enter the -oxidation-cycle, and can be utilized as carbon- and energy sources. Medium (C6-C12)- or long-chain (C13-C20) n-alkanes can be utilized by many strains, some of which have been partially characterized. The alkane-oxidizing enzymes used by some of these strains (e.g. twoP. aeruginosa strains, aP. denitrificans strain and a marinePseudomonas sp.) appear to be closely related to those encoded by the OCT-plasmid.     

Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli.

We tested the synthesis and in vivo function of the inducible alkane hydroxylase of Pseudomonas oleovorans GPo1 in several Escherichia coli recombinants. The enzyme components (AlkB, AlkG and AlkT) were synthesized at various rates in different E. coli hosts, which after induction produced between twofold and tenfold more of the Alk components than did P. oleovorans. The enzyme components were less stable in recombinant E. coli hosts than in P. oleovorans. In addition, the specific activity of the alkane mono-oxygenase component AlkB was five or six times lower in E. coli than in P. oleovorans. Evidently, optimal functioning of the hydroxylase system requires factors or a molecular environment that are available in Pseudomonas but not in E. coli. These factors are likely to include correct interactions of AlkB with the membrane and incorporation of iron into the AlkG and AlkB apoproteins.     

Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations.

The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particulate and soluble fractions. Spinach reductase and spinach ferredoxin could replace the soluble hydroxylase component. Two alkane hydroxylase mutants show in vitro complementation (S. Benson and J. Shapiro, J. Bacteriol., 123: 759-760, 1975): one, alk-7, lacks an active soluble component and the other, alk-181, lacks an active particulate component. Together with previous results on a particulate alcohol dehydrogenase enzyme (Benson and Shapiro, J. Bacteriol., 126: 794-798, 1976), these results allowed us to assay three plasmid-determined inducible activities: soluble alkane hydroxylase (alkA+), particulate alkane hydroxylase (alkB+), and particulate alcohol dehydrogenase (alkC+). Growth tests and in vitro complementation assays revealed three groups of plasmid mutations that block expression of alkane hydroxylase activity: alkA, which so far includes only the alk-7 mutation; alkB, which includes alk-181 and 11 other mutations; and a pleiotropic-negative class, which includes nine mutations that lead to loss of alkA+, alkB+, and alkC+ activities. Thus, the alk+ gene cluster found on IncP-2 plasmids contains at least four cistrons. We believe it is significant that two of these determined the presence of membrane proteins. The accompanying paper shows that these loci are part of a single regulon.     

Pyrimidine biosynthesis in Pseudomonas oleovorans

AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate transcarbamoylase activity. METHODS AND RESULTS: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source. Two uracil auxotrophs of P. oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity. Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized. At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps. oleovorans enzyme. CONCLUSIONS: Pyrimidine biosynthesis is regulated in Ps. oleovorans. Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps. oleovorans to increase the synthesis of polyhydroxyalkanoates.     

Methanol metabolism by Pseudomonas oleovorans

A typical facultative methylotroph Pseudomonas oleovorans oxidizes methanol to formaldehyde by a specific dehydrogenase which is active towards phenazine metosulphate. Direct oxidation of formalydehyde to CO2 via formiate is a minor pathway because the activities of dehydrogenases of formaldehyde and formiate are lwo. Most formaldehyde molecules are involved in the hexulose phosphate cycle, which is confirmed by a high activity of hexulose phosphate synthase. Formaldehyde is oxidized to CO2 in the dissimilation branch of the cycle providing energy for biosynthesis; this confirmed by higher levels of dehydrogenases of glucose-6-phosphate and 6-phosphogluconate during the methylotrophous growth of the cells. The acceptor of formaldehyde (ribulose-5-phosphate) is regenerated and pyruvate is synthesized in the assimilation branch of the hexulose phosphate cycle. Aldolase of 2-keto-3-deoxy-6-phosphogluconate plays an important role in this process. Further metabolism of trioses involves reactions of the tricarboxylic acid cycle which performs mainly an anabolic function due to complete repression of alpha-ketoglutarate dehydrogenase during the methylotrophous growth. The carbon of methanol is partially assimilated as CO2 by the carboxylation of pyruvate or phosphoenolpyruvate. NH+4 is assimilated by the reductive amination of alpha-ketoglutarate.     

Pseudomonas oleovorans subsp. lubricantis subsp. nov., and Reclassification of Pseudomonas pseudoalcaligenes ATCC 17440T as Later Synonym of Pseudomonas oleovorans ATCC 8062T

Isolate RS1^T isolated from used metalworking fluid was found to be a Gram-negative, motile, and non-spore forming rod. Based on phylogenetic analyses with 16S rRNA, isolate RS1^T was placed into the mendocina sublineage of Pseudomonas. The major whole cell fatty acids were C_18:1ω7c (32.6%), C_16:0 (25.5%), and C_15:0 ISO 2OH/C_16:1ω7c (14.4%). The sequence similarities of isolate RS1^T based on gyrB and rpoD genes were 98.9 and 98.0% with Pseudomonas pseudoalcaligenes, and 98.5 and 98.1% with Pseudomonas oleovorans, respectively. The ribotyping pattern showed a 0.60 similarity with P. oleovorans ATCC 8062^T and 0.63 with P. pseudoalcaligenes ATCC17440^T. The DNA G + C content of isolate RS1^T was 62.2 mol.%. The DNA–DNA relatedness was 73.0% with P. oleovorans ATCC 8062^T and 79.1% with P. pseudoalcaligenes ATCC 17440^T. On the basis of morphological, biochemical, and molecular studies, isolate RS1^T is considered to represent a new subspecies of P. oleovorans. Furthermore, based on the DNA–DNA relatedness (>70%), chemotaxonomic, and molecular profile, P. pseudoalcaligenes ATCC 17440^T and P. oleovorans ATCC 8062^T should be united under the same name; according to the rules of priority, P. oleovorans, the first described species, is the earlier synonym and P. pseudoalcaligenes is the later synonym. As a consequence, the division of the species P. oleovorans into two novel subspecies is proposed: P. oleovorans subsp. oleovorans subsp. nov. (type strain ATCC 8062^T = DSM 1045^T = NCIB 6576^T), P. oleovorans subsp. lubricantis subsp. nov. (type strain RS1^T = ATCC BAA-1494^T = DSM 21016^T).     

The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110(pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subtraction

The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxyiase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5–2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the Induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10–15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild-type W3110. Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low-density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS-PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low-density membrane fraction, alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C_16:1 and C_18:1 increased at the expense of C_17:0cyc and C_19:0cyc*     

Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase,an integral membrane diiron enzyme,and the fatty acid desaturase family

Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site.     

Production of unsaturated polyesters by Pseudomonas oleovorans

Pseudomonas oleovorans was grown separately on 3-hydroxy-6-octenoic acid and 3-hydroxy-7-octenoic acid as the only carbon source and under ammonium nutrient-limiting conditions to produce storage polyesters. The polyesters produced contained mainly unsaturated C8 units. Small amounts of both the saturated and the unsaturated C6 units were also present, but only about 1% of the saturated 3-hydroxyoctanoate units was detected. The polyester obtained from 3-hydroxy-6-octenoic acid, which was a mixture of the cis and trans isomers, also contained units with cis and trans double bonds. The weight average molecular weights of the polymers produced were in the range of 339,000-383,000 as determined by g.p.c. relative to polystyrene, with Mw/Mn ratios of 1.8-2.1. The mechanism of PHA formation from n-octene previously reported is discussed in relation to the present results, and the two were found to be in good agreement.     

Genetic transformation of Pseudomonas oleovorans by electroporation

An electroporation procedure for the transformation of Pseudomonas oleovorans was developed using a model plasmid, pCN51. The optimal electrotransformation was achieved with cells harvested at 45 to 60 min of growth and concentrated to a cell density of 5 OD600nm, plasmid concentration of 6 g per 100 l of cell suspension, and a 0.1-cm gap-width cuvette. Electroporation was performed at the settings of 250 , 25F and 2.5 kV. Transformation yields in the order of 103 colony-forming-unit per electroporation sample were obtained. This is a first report of the electroporation of the commercially valuable bacterium Ps. oleovorans. © Rapid Science Ltd. 1998     

Regulation of alkane oxidation in Pseudomonas putida.

We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.     

Oxidation of methyl tert-butyl ether by alkane hydroxylase in dicyclopropylketone-induced and n-octane-grown Pseudomonas putida GPo1

The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K(s) value (20 to 40 mM) for MTBE.     

The supramolecular architecture of the polyhydroxyalkanoate inclusions in Pseudomonas oleovorans

Abstract A study has been initiated to further clarify the role of H₂O and protein in maintaining a functional amorphous state of poly-β-hydroxyalkanoates (PHAs) in the living cell. It has been clearly demonstrated that the ordered hydrophobic hydrocarbon chains of these materials crystallize at varying rates, depending on repeating unit heterogeneity, subsequent to the removal of the polymer granule from the cell and after solvent extraction. Both isolation and extraction procedures remove water and some protein from the polymer granule, and the polymer crystallizes. We have developed a procedure for the isolation of poly-β-hydroxyoctanoate (PHO) containing granules that maintain their in vivo fluid, amorphous and functional state with water and proteins intact. These purified granules have been observed and their purity determined by 'in vivo' Nomarski computer-enhanced imaging light microscopy. Both this technique and low temperature rapid freeze-fracture electron microscopy have demonstrated an organized para-crystalline network of proteins on the surface of the granule. The structural role of these proteins in maintaining granule integrity and function both in the cell and after granule isolation is discussed. It is suggested that these procedures may permit in vitro studies of the mechanisms of synthesis and degradation of PHAs in a variety of polyester producing microbial systems.