AlkB homologues in thermophilic bacteria of the genus Geobacillus

Screening of alkane hydroxylase genes (alkB) was performed in the thermophilic aerobic bacteria of the genus Geobacillus. Total DNA was extracted from the biomass of 11 strains grown on the mixture of saturated C10-C20 hydrocarbons, PCR amplification of fragments of alkB genes was performed with degenerate oligonucleotide primers, PCR products were cloned and sequenced. For the first time in the genome of thermophilic bacteria the presence of a set of alkB gene homologues was revealed. The strains each contain three to six homologues among which only two are universal for all of the strains. Comparative phylogenetic analysis of the nucleotide sequences and the inferred amino acid sequences showed close relatedness of six of the revealed variants of geobacilli sequences to the alkB4, alkB3, and alkB2 genes that had previously been revealed by other authors in Rhodococcus erythropolis strains NRRL B-16531 and Q15. The rest two variants of alkB sequences were unique. Analysis of the GC composition of all the Geobacillus alkB homologues revealed closer proximity to the rhodococcal chromosomal DNA than to the chromosomal DNA of geobacilli. This may be an indication of the introduction of the alkB genes into the Geobacillus genome by interspecies horizontal transfer; and rhodococci or other representatives of the Actinobacteria phylum were probably the donors of these genes. Analysis of the codon usage in fragments of alkB genes confirms the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. Formation of a set of several alkB homologues in a genome of a particular microorganism may result from free gene exchange within this pool.     

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.     

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.     

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.     

Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6

Biotransformation using alkane-oxidizing bacteria or their alkane hydroxylase (AH) systems have been little studied at the molecular level. We have cloned and sequenced genes from Gordonia sp. TF6 encoding an AH system, alkB2 (alkane 1-monooxygenase), rubA3 (rubredoxin), rubA4 (rubredoxin), and rubB (rubredoxin reductase). When expressed in Escherichia coli, these genes allowed the construction of biotransformation systems for various alkanes. Normal alkanes with 5 to 13 carbons were good substrates for this biotransformation, and oxidized to their corresponding 1-alkanols. Surprisingly, cycloalkanes with 5 to 8 carbons were oxidized to their corresponding cycloalkanols as well. This is the first study to achieve biotransformation of alkanes using the E. coli expressing the minimum component genes of the AH system. Our biotransformation system has facilitated assays and analysis leading to improvement of AH systems, and has indicated a cycloalkane oxidation pathway in microorganisms for the first time.     

New alk genes detected in Antarctic marine sediments

Alkane monooxygenases (Alk) are the key enzymes for alkane degradation. In order to understand the dispersion and diversity of alk genes in Antarctic marine environments, this study analysed by clone libraries the presence and diversity of alk genes ( alkB and alkM ) in sediments from Admiralty Bay, King George Island, Peninsula Antarctica. The results show a differential distribution of alk genes between the sites, and the predominant presence of new alk genes, mainly in the pristine site. Sequences presented 53.10–69.60% nucleotide identity and 50.90–73.40% amino acid identity to alkB genes described in Silicibacter pomeroyi , Gordonia sp., Prauserella rugosa , Nocardioides sp., Rhodococcus sp., Nocardia farcinica , Pseudomonas putida , Acidisphaera sp., Alcanivorax borkumensis , and alkM described in Acinetobacter sp. This is the first time that the gene alkM was detected and described in Antarctic marine environments. The presence of a range of previously undescribed alk genes indicates the need for further studies in this environment.     

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.     

Alkane hydroxylase homologues in Gram-positive strains

We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.     

alkB homologs in thermophilic bacteria of the genus Geobacillus

Screening for alkane hydroxylase genes (alkB) was performed in thermophilic aerobic bacteria of the genus Geobacillus. Total DNAs were isolated from the biomass of 11 strains grown on a mixture of saturated C₁₀–C₂₀ hydrocarbons. Fragments of alkB genes were amplified by PCR with degenerate oligonucleotide primers, and the PCR products were cloned and sequenced. For the first time, a set of alkB gene homologs was detected in the genomes of thermophilic bacteria. The strains each contained three to six homologs, of which only two were common for all of the strains. Phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences showed that six of the variants revealed in Geobacillus were closely related to alkB4, alkB3, and alkB2, found in Rhodococcus erythropolis strains NRRL B-16531 and Q15. All variants of alkB sequences were unique. Analysis of the GC composition showed that the Geobacillus alkB homologs are closer to Rhodococcus than to Geobacillus chromosomal DNA. It was assumed that the alkB genes were introduced in the Geobacillus genome via interspecific horizontal transfer and that Rhodococcus or other representatives of Actinobacteria served as donors. Analysis of the codon usage in the fragments of alkB genes confirmed the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. The formation of a set of several alkB homologs in a genome of a particular microorganism may result from free gene exchange within this pool.     

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.     

Physiological, numerical and molecular characterization of alkyl ether-utilizing rhodococci

Twenty-seven Gram-positive strains were characterized physiologically and numerically and classified them into four groups according to their specific activities for utilization of linear alkyl ethers (AEs), cyclic AEs, monoalkoxybenzenes and 1,4-diethoxybenzene. The comparative analysis of the 16S ribosomal RNA gene and 16S-23S intergenic spacer region showed that they belonged to the genera Rhodococcus and Gordonia. Alkyl ether-utilizing rhodococci appeared to involve various and diverse cytochromes P450 of the families CYP116 (25 positive strains from 27), CYP153 (5/27), CYP249 (1/27) and a new family P450RR1 (27/27). The presence of P450RR1 was strongly related to the specific activity for utilization of 2-methoxyphenol and 2-ethoxyphenol. In addition, 26 of 27 strains contained multiple alkB genes coding for probable non-haem iron containing alkane monooxygenases and hydroxylases. Similar DNA fragments coding for a tetrahydrofuran monooxygenase A subunit (ThmA) were found in all cyclic AE-utilizing strains and nearly identical DNA fragments coding for likely orthologues of a propane monooxygenase A subunit (PrmA) in all linear AE-utilizing strains. The substrate availability in the degradation of aryl AEs, cyclic AEs and linear AEs agreed with the molecular probing of the respective genes encoding cytochrome P450RR1, ThmA and PrmA.     

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.     

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   

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.     

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.     

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.     

Regulatory mutations of the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains with plasmids carrying pleiotropic alk mutations gave rise to alkane-positive "revertants," which differ from wild type. Some had restricted ability to utilize alkane and primary alcohol growth substrates, and others could grow on undecane and dodecanol, which are not utilized by alk+ strains. These revertants showed altered responses to normal inducers of alkA+, alkB+, and alkC+ activities. Some revertants were constitutive for these activities. Constitutive mutants could also be isolated directly from wild type, but they appeared spontaneously at a frequency of less than 2 X 10(-8). Regulatory mutations of all three types, pleiotropic negative, altered inducer specificity, and constitutive, were tightly linked in transduction crosses with a polar alkB mutation. These results demonstrate that the IncP-2 plasmid alk gene cluster constitutes a regulon. They also permit the identification of at least one cistron whose gene product participates in inducer recognition and suggest that the alkABC regulon is not under simple repressor control.