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.     

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.     

Regulation of membrane peptides by the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains carrying the plasmid alk genes will grow on n-alkanes. Induced alk+ strains contain membrane activities for alkane hydroxylation and dehydrogenation of aliphatic primary alcohols. P. putida cytoplasmic and outer membranes can be separated by sucrose gradient centrifugation after disruption of cells by either mild detergent lysis or passage through a French press. Both the membrane component of alkane hydroxylase and membrane alcohol dehydrogenase fractionated with the cytoplasmic membrane. Induction of the alk regulon resulted in the appearance of at least three new plasmid-determined cytoplasmic membrane peptides of about 59,000 (59K), 47,000 (47K), and 40,000 (40K) daltons as well as the disappearance of a pair of chromosomally encoded outer membrane peptides of about 43,000 daltons. The 40K peptide is the membrane component of alkane hydroxylase and the product of the plasmid alkB gene because the alkB1029 mutation altered the properties of alkane hydroxylase in whole cells, reduced its thermal stability in cell extracts, and led to increased electrophoretic mobility of the inducible 40K peptide. These results are consistent with a model for vectorial oxidation of n-alkanes in the cytoplasmic membrane of P. putida.     

Biosynthesis of synthons in two-liquid-phase media

The Pseudomonas oleovorans alkane hydroxylase and xylene oxygenase from Pseudomonas putida are versatile mono-oxygenases for stereo- and regioselective oxidation of aliphatic and aromatic hydrocarbons. Pseudomonas oleovorans and alkanol dehydrogenase deficient mutants of Pseudomonas have previously been used to produce alkanols from various alkanes and optically active epoxides from alkenes. Similarly, P. putida strains have been used to produce aromatic alcohols, aromatic acids, and optically active styrene oxides. A limitation in the use of Pseudomonas strains for bioconversions is that these strains can degrade some of the products formed. To counter this problem, we have constructed Escherichia coli recombinants, which contain the alk genes from the OCT plasmid of P. oleovorans [E. coli HB101 (pGEc47)] and the xylMA genes from the TOL plasmid of P. putida mt-2 [E. coli HB101 (pGB63)], encoding alkane hydroxylase and xylene oxygenase, respectively. Escherichia coli HB101 (pGEc47) was used to produce octanoic acid from n-octane and E. coli HB101 (pBG63) was put to use for the oxidation of styrene to styrene oxide in two-liquid phase biocatalysis at high cell densities. The alk(+) recombinant strain E. coli HB101 (pGEc47) was grown to 40 g/L cell dry mass in the presence of n-octane, which was converted to octanoic acid by the alkane oxidation system, the product accumulating in the aqueous phase. The xyl(+) recombinant E. coli HB101 (pBG63) was grown to a cell density of 26 g/L cell dry mass in the presence of around 7% (v/v) n-dodecane, which contained 2% (v/v) styrene. The recombinant E. coli (xyl(+)) converted styrene to (S)-(+)-styrene oxide at high enantiomeric excess (94% ee) and this compound partitioned almost exclusively into the organic phase. Using these high-cell-density two-liquid-phase cultures, the products accumulated rapidly, yielding high concentrations of products (50 mM octanoic acid and 90 mM styrene oxide) in the respective phases. (c) 1996 John Wiley & Sons, Inc.     

Synthesis of alkane hydroxylase of Pseudomonas oleovorans increases the iron requirement of alk+ bacterial strains

The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. Expression of the alk genes in P. oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures. Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E. coli W3110 (pGEc47). AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E. coli W3110 (pGEc47). Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium. Our results show that a glucose-grown E. coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes.     

In vivo formation of gene fusions in Pseudomonas putida and construction of versatile broad-host-range vectors for direct subcloning of Mu d1 and Mu d2 fusions

The Mu d1 and Mu d2 prophages were integrated into the conjugative broad-host-range plasmid R751. The two plasmids were then transferred into Pseudomonas putida, and derivatives carrying intact Mu prophages were recovered. After induction of Mu at 42 degrees C, both operon and gene fusions were observed on 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) plates. Broad-host-range vectors were constructed which allow direct cloning of both operon or gene fusions and their analysis in Escherichia coli and P. putida. By using one of these vectors, two operon fusions were isolated from the P. putida chromosome and comparatively analyzed in E. coli and P. putida.     

In vivo formation of gene fusions in Pseudomonas putida and construction of versatile broad-host-range vectors for direct subcloning of Mu d1 and Mu d2 fusions.

The Mu d1 and Mu d2 prophages were integrated into the conjugative broad-host-range plasmid R751. The two plasmids were then transferred into Pseudomonas putida, and derivatives carrying intact Mu prophages were recovered. After induction of Mu at 42 degrees C, both operon and gene fusions were observed on 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) plates. Broad-host-range vectors were constructed which allow direct cloning of both operon or gene fusions and their analysis in Escherichia coli and P. putida. By using one of these vectors, two operon fusions were isolated from the P. putida chromosome and comparatively analyzed in E. coli and P. putida.     

Local anesthetics block induction of the Pseudomonas alk regulon.

The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzymatic activities involved in the metabolism of n-alkanes in Pseudomonas putida. Procaine reversibly inhibited existing alkane hydroxylase activity. Induction of a soluble aliphatic amidase activity was not affected. These results support the hypothesis that induction of the plasmid-determined alkane metabolic system in P. putida involves a membrane component(s).     

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.     

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.