Characterization of three propane-inducible oxygenases in Mycobacterium sp. strain ENV421.

AIMS: Mycobacterium sp. strain ENV421 has the ability to cometabolize a variety of chemicals following growth on propane as a sole source of carbon and energy. In this study, we used genetic and biochemical approaches to identify and characterize multiple propane-inducible oxygenase genes in ENV421. METHODS AND RESULTS: Gene clusters encoding a CYP153-type cytochrome P450 oxygenase (P450), an AlkB-type alkane monooxygenase (AlkB) and a soluble diiron monooxygenase were identified and cloned using degenerate PCR primers. Reverse transcriptase PCR showed that all three gene clusters were induced by propane. Substrate specificity studies revealed that despite the fact that ENV421 does not grow on medium length alkanes, cloned versions of both the AlkB and P450 were capable of octane oxidation, forming n-octanol. Additionally, the P450 oxygenase had the ability to oxidize indole, medium-to-long-chain alkylbenzenes and a variety of para-substituted methylalkylbenzenes. Successful cloning and expression of the diiron monooxygenase was not achieved, so its substrate specificity could not be determined. CONCLUSIONS: Three types of short-to-medium-chain alkane oxygenases were induced by propane in ENV421, even though the cloned AlkB and P450 oxygenases did not oxidize propane. Curiously, they both oxidized octane, which is not a growth substrate for ENV421. Furthermore, the P450, typically operating as terminal alkane hydroxylase, exhibited interesting regio- and stereoselectivity, catalysing linear alkanes, alkylbenzenes and indole. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first example of a propane-inducible P450 with a broad substrate specificity, including linear alkanes, alkylbenzenes and a multiring compound. The induction of three distinct oxygenase classes by propane is also an interesting finding because it might explain why propane serves as an effective stimulant that promotes the biodegradation of a various environmental contaminants.

The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation.

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1.

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.

The arsenite oxidase genes (aroAB) in novel chemoautotrophic arsenite oxidizers.

Six novel bacterial strains were isolated from the environment which can oxidize arsenite [As(III)] to the less mobile form arsenate [As(V)] coupled to CO(2) fixation under either aerobic or denitrifying conditions. PCR primers were designed to the conserved molybdopterin domain of the large subunit of arsenite oxidase in order to identify the arsenite oxidase genes from these isolates. The amino acid sequences for the arsenite oxidases reported here were 72-74% identical to that of strain NT-26, the only previously reported autotrophic arsenite oxidizer. Indeed the autotrophic arsenite oxidase genes form a distinct phylogenetic group, separated from previously described heterotrophic arsenite oxidase genes, with the exception of the heterotroph Agrobacterium tumefaciens. The arsenite oxidase primers described here represent a powerful culture-independent tool to assess the diversity of arsenite oxidase genes in environmental bacteria.

Taxonomic positioning of two biological control agents for plant diseases as Lysobacter enzymogenes based on phylogenetic analysis of 16S rDNA, fatty acid composition and phenotypic characteristics.

AIMS: To taxonomically position two bacterial strains conferring biological control activity towards plant diseases. METHODS AND RESULTS: Key phenotypic characteristics, including gliding motility and a high percentage of G + C content, indicated biocontrol strains N4-7 and C3 were essentially identical to those described for Lysobacter enzymogenes. Cellular fatty acid analysis confirmed a close relatedness of strains N4-7 and C3 to L. enzymogenes and a more distant relatedness to L. antibioticus. The 16S rDNA phylogenetic analysis revealed a distinct Lysobacter clade that included both strains within the gamma-proteobacteria. CONCLUSIONS: The combined taxonomic methods provide clear evidence that N4-7 and C3 should be grouped as strains of L. enzymogenes and not Stenotrophomonas maltophilia or a novel taxon. Phylogenetic analysis of 16S rDNA formed a Lysobacter clade that included several other environmentally diverse bacterial strains obtained from databases and confirmed relatedness of strains N4-7 and C3 to L. enzymogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Inclusion of N4-7 and C3 as strains of L. enzymogenes is among the first description of members of this genus as biocontrol agents of plant diseases. These results suggest that members of the Lysobacter group might provide a new source as plant-associated microbes that display biocontrol activity.

Degradation of phenanthrene and naphthalene by a Burkholderia species strain.

Burkholderia sp. TNFYE-5 was isolated from soil for the ability to grow on phenanthrene as sole carbon and energy source. Unlike most other phenanthrene-degrading bacteria, TNFYE-5 was unable to grow on naphthalene. Growth substrate range experiments coupled with the ring-cleavage enzyme assay data suggest that TNFYE-5 initially metabolizes phenanthrene to 1-hydroxy-2-naphthoate with subsequent degradation through the phthalate and protocatechuate and beta-ketoadipate pathway. A metabolite in the degradation of naphthalene by TNFYE-5 was isolated by high-pressure liquid chromatography (HPLC) and was identified as salicylate by UV-visible spectral and gas chromatography-mass spectrometry analyses. Thus, the inability to degrade salicylate is apparently one major reason for the incapability of TNFYE-5 to grow on naphthalene.

Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants.

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group, Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, and Mycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.

Genetic structure and functional implication of the fcb gene cluster for hydrolytic dechlorination of 4-chlorobenzoate from Pseudomonas sp. DJ-12.

The fcb gene cluster responsible for the hydrolytic dechlorination of 4-chlorobenzoate (4CBA) was cloned from the chromosomal DNA of Pseudomonas sp. DJ-12, and its nucleotide sequence analyzed. The gene cluster was organized in the order fcbB-fcbA-fcbT1-fcbT2-cbT3-fcbC, which is different from that reported in other bacteria. A promoter-like sequence (-35 and -10 region) is located upstream of the fcbB gene and putative ribosome-binding sequences were found upstream of the respective orfs. A stem-loop transcription terminator structure is found downstream of fcbC. This suggests that the six orfs are transcribed into a polycistronic mRNA. The FcbA, FcbB, and FcbC enzymes for dechlorination of 4CBA have a relationship in common with the enzymes involved in fatty acid metabolism on the basis of their deduced amino acid sequences. The proteins encoded by fcbT1, fcbT2, and fcbT3 show similarity to those encoded by dctP, dctQ, and dctM of Rhodobacter capsulatus respectively, which encode transporter proteins for C4-dicarboxylate. It is likely, therefore, that these proteins of DJ-12 play a role in transport of 4CBA into the cell.

Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria.

A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 micromol. min(-1). mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.

Characterization of the phthalate permease OphD from Burkholderia cepacia ATCC 17616.

The ophD gene, encoding a permease for phthalate transport, was cloned from Burkholderia cepacia ATCC 17616. Expression of the gene in Escherichia coli results in the ability to transport phthalate rapidly into the cell. Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease. An ophD knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain. This means that 17616 must have a second phthalate-inducible phthalate uptake system.

Role of quinolinate phosphoribosyl transferase in degradation of phthalate by Burkholderia cepacia DBO1.

Two distinct regions of DNA encode the enzymes needed for phthalate degradation by Burkholderia cepacia DBO1. A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD+ was identified between these two regions by sequence analysis and functional assays. Southern hybridization experiments indicate that DBO1 and other phthalate-degrading B. cepacia strains have two dissimilar genes for this enzyme, while non-phthalate-degrading B. cepacia strains have only a single gene. The sequenced gene was labeled ophE, due to the fact that it is specifically induced by phthalate as shown by lacZ gene fusions. Insertional knockout mutants lacking ophE grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates. The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate. The recruitment of this gene for growth on phthalate thus gives B. cepacia an advantage over other phthalate-degrading bacteria in the environment.

Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1.

Sphingomonas yanoikuyae B1 is able to utilize toluene, m-xylene, p-xylene, biphenyl, naphthalene, phenanthrene, and anthracene as sole sources of carbon and energy for growth. A forty kilobase region of DNA containing most of the genes for the degradation of these aromatic compounds was previously cloned and sequenced. Insertional inactivation of bphC results in the inability of B1 to grow on both polycyclic and monocyclic compounds. Complementation experiments indicate that the metabolic block is actually due to a polar effect on the expression of bphA3, coding for a ferredoxin component of a dioxygenase. Lack of the ferredoxin results in a nonfunctional polycyclic aromatic hydrocarbon dioxygenase and a nonfunctional toluate dioxygenase indicating that the electron transfer components are capable of interacting with multiple oxygenase components. Insertional inactivation of a gene for a dioxygenase oxygenase component downstream of bphA3 had no apparent effect on growth besides a polar effect on nahD which is only needed for growth of B1 on naphthalene. Insertional inactivation of either xylE or xylG in the meta-cleavage operon results in a polar effect on bphB, the last gene in the operon. However, insertional inactivation of xylX at the beginning of this cluster of genes does not result in a polar effect suggesting that the genes for the meta-cleavage pathway, although colinear, are organized in at least two operons. These experiments confirm the biological role of several genes involved in metabolism of aromatic compounds by S. yanoikuyae B1 and demonstrate the interdependency of the metabolic pathways for polycyclic and monocyclic aromatic hydrocarbon degradation.

Novel organization of the genes for phthalate degradation from Burkholderia cepacia DBO1.

Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthalate degradation to two separate regions on the cosmid clones. Analysis of the nucleotide sequence of these two regions showed that the genes for phthalate degradation are arranged in at least three transcriptional units. The gene for phthalate dioxygenase reductase (ophA1) is present by itself, while the genes for an inactive transporter (ophD) and 4,5-dihydroxyphthalate decarboxylase (ophC) are linked and the genes for phthalate dioxygenase oxygenase (ophA2) and cis-phthalate dihydrodiol dehydrogenase (ophB) are linked. ophA1 and ophDC are adjacent to each other but are transcribed in opposite directions, while ophA2B is located 4 kb away. The genes for the oxygenase and reductase components of phthalate dioxygenase are located approximately 7 kb away from each other. The gene for the putative phthalate permease contains a frameshift mutation in contrast to genes for other permeases. Strains deleted for ophD are able to transport phthalate into the cell at rates equivalent to that of the wild-type organism, showing that this gene is not required for growth on phthalate.

Active efflux of organic solvents by Pseudomonas putida S12 is induced by solvents.

Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the srpABC genes include aromatic and aliphatic solvents and alcohols. General stress conditions such as pH, temperature, NaCl, or the presence of organic acids did not induce srp transcription. Although the solvent efflux pump in P. putida S12 is a member of the resistance-nodulation-cell division family of transporters, the srpABC genes were not induced by antibiotics or heavy metals.

Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes.

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.

Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance.

Bacteria able to grow in aqueous:organic two-phase systems have evolved resistance mechanisms to the toxic effects of solvents. One such mechanism is the active efflux of solvents from the cell, preserving the integrity of the cell interior. Pseudomonas putida S12 is resistant to a wide variety of normally detrimental solvents due to the action of such an efflux pump. The genes for this solvent efflux pump were cloned from P. putida S12 and their nucleotide sequence determined. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins known to be involved in proton-dependent multidrug efflux systems. Transfer of the genes for the solvent efflux pump to solvent-sensitive P. putida strains results in the acquisition of solvent resistance. This opens up the possibilities of using the solvent efflux system to construct bacterial strains capable of performing biocatalytic transformations of insoluble substrates in two-phase aqueous:organic medium.

Evidence for the role of 2-hydroxychromene-2-carboxylate isomerase in the degradation of anthracene by Sphingomonas yanoikuyae B1.

Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1.

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni.

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences.