Two novel alkane hydroxylase-rubredoxin fusion genes isolated from a Dietzia bacterium and the functions of fused rubredoxin domains in long-chain n-alkane degradation

Two alkane hydroxylase-rubredoxin fusion gene homologs (alkW1 and alkW2) were cloned from a Dietzia strain, designated DQ12-45-1b, which can grow on crude oil and n-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1- and AlkG2-type Rds. Expression of the alkW1 gene in DQ12-45-1b was induced when cells were grown on C(8) to C(32) n-alkanes as sole carbon sources, but expression of the alkW2 gene was not detected. Functional heterologous expression in an alkB deletion mutant of Pseudomonas fluorescens KOB2Δ1 suggested the alkW1 could restore the growth of KOB2Δ1 on C(14) and C(16) n-alkanes and induce faster growth on C(18) to C(32) n-alkanes than alkW1ΔRd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2Δ1 itself. In addition, the artificial fusion of AlkB from the Gram-negative P. fluorescens CHA0 and the Rds from both Gram-negative P. fluorescens CHA0 and Gram-positive Dietzia sp. DQ12-45-1b significantly increased the degradation of C(32) alkane compared to that seen with AlkB itself. In conclusion, the alkW1 gene cloned from Dietzia species encoded an alkane hydroxylase which increased growth on and degradation of n-alkanes up to C(32) in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chain n-alkanes (such as C(32)) in the Gram-negative bacterium.     

Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains

We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C₆–C₁₁) or long-chain n -alkanes (C₁₂–C₁₆), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2–93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n -alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB , respectively, to grow on n -alkanes, showing that the cloned genes do indeed encode alkane hydroxylases.     

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.     

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.     

Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR

AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (相似文献   

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.     

Rubredoxin reductase from Alcanivorax borkumensis: expression and characterization

Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems.     

Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa

Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P. aeruginosa isolates often grow on medium- as well as long-chain n-alkanes. Heterologous expression showed that the two alkane hydroxylase homologs of P. aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C₁₂-C₁₆ n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P. putida GPo1 counterparts in n-octane oxidation. The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P. aeruginosa strains tested in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.     

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.     

Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.     

Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5

Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.     

Propane and n-butane oxidation by Pseudomonas putida GPo1

Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of n-octane.     

A conserved amino acid motif (R-X-G-R-R) in the Glut1 glucose transporter is an important determinant of membrane topology.

The Glut1 glucose transporter is one of over 300 members of the major facilitator superfamily of membrane transporters. These proteins are extremely diverse in substrate specificity and differ in their transport mechanisms. The two most common features shared by many members of this superfamily are the presence of 12 predicted transmembrane segments and an amino acid motif, R-X-G-R-R, present at equivalent positions within the cytoplasmic loops joining transmembrane segments 2-3 and 8-9. The structural and functional roles of the arginine residues within these motifs in Glut1 were investigated by expression of site-directed mutant transporters in Xenopus oocytes followed by analyses of intrinsic transport activity and the membrane topology of mutant glycosylation-scanning reporter Glut1 molecules. Substitution of lysine residues for the cluster of 3 arginine residues in each of the 2 cytoplasmic pentameric motifs of Glut1 revealed no absolute requirement for arginine side chains at any of the 6 positions for transport of 2-deoxyglucose. However, removal of the 3 positive charges at either site by substitution of glycines for the arginines completely abolished transport activity as the result of a local perturbation in the membrane topology in which the cytoplasmic loop was aberrantly translocated into the exoplasm along with the two flanking transmembrane segments. Substitution of lysines for the arginines had no affect on membrane topology. We conclude that the positive charges in the R-X-G-R-R motif form critical local cytoplasmic anchor points involved in determining the membrane topology of Glut1. These data provide a simple explanation for the presence of this conserved amino acid motif in hundreds of functionally diverse membrane transporters that share a common predicted membrane topology.     

Differential expression of the components of the two alkane hydroxylases from Pseudomonas aeruginosa

Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C(10) to C(22)/C(24) alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes.     

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.