Functional expression, purification, and characterization of the recombinant Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700

For the investigation of the NADPH-dependent Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700, the encoding gene mekA with a C-terminal strep-tag was cloned and expressed under the control of a l-rhamnose inducible promoter from Escherichia coli. The mekA gene was found by analyzing the methylethylketone (MEK) degradation pathway by Onaca et al. J Bacteriol 189:3759–3767, 2007. Sequence analysis of the corresponding protein, which catalyzes the Baeyer-Villiger oxidation of MEK to ethyl acetate, showed two binding sites (Rossman-fold motifs) for cofactors NAD(P)H and FAD. Although expression of mekA resulted in large amounts of inclusion bodies compared to soluble protein, high amounts of purified and active MekA were obtained by affinity chromatography. The substrate spectrum of MekA was investigated with purified enzyme and whole cells using a variety of aliphatic, aromatic, and cyclic ketones including four chiral substrates. The specific activity of MekA with MEK as substrate was determined to be 1.1 U/mg protein. K _M values were determined for MEK and the cofactors NADPH and NADH to be 6, 11, and 29 μM, respectively.     

Cell surface display of lipase in Pseudomonas putida KT2442 using OprF as an anchoring motif and its biocatalytic applications

We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47 degrees C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47 degrees C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.     

Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51.

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.     

Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Microbial growth on 2-bromobutane

A member of the genus Arthrobacter was isolated which grew at the expense of 2-bromobutane as sole source of carbon and energy. Evidence is presented which suggests that the initial conversion of 2-bromobutane to 2-butanol is a spontaneous chemical hydrolysis and not mediated by the organism. Further evidence from oxygen consumption experiments indicates that 2-bromobutane is oxidized through 2-butanol, methyl ethyl ketone, ethyl acetate to acetate and ethanol. Results of experiments with cells grown on pathway intermediates reveal that the enzymes necessary for the oxidation of 2-butanol, methyl ethyl ketone, ethyl acetate, ethanol and acetaldehyde are not coordinately, but individually induced by their respective substrates.     

Cometabolic oxidation of polychlorinated biphenyls in soil with a surfactant-based field application vector.

Polychlorinated biphenyl (PCB)-degradative genes, under the control of a constitutive promoter, were cloned into a broad-host-range plasmid and a transposon. These constructs were inserted into a surfactant-utilizing strain, Pseudomonas putida IPL5, to create a field application vector (FAV) in which a surfactant-degrading organism cometabolizes PCB. By utilizing a surfactant not readily available to indigenous populations and a constitutive promoter, selective growth and PCB-degradative gene expression are decoupled from biphenyl. Since PCB degradation via the biphenyl degradation pathway is nonadaptive in the absence of biphenyl, there is no selective pressure for PCB gene maintenance. The recombinant strains exhibited degradative activity against 25 of 33 PCB congeners in Aroclor 1248 in the absence of biphenyl. Whole-cell enzyme assays indicated that PCB-degradative activity of a recombinant strain carrying the PCB genes on a plasmid was approximately twice that of the same strain carrying the PCB genes on a transposon. Plasmid loss rates in the absence of antibiotic selection averaged 7.4% per cell division and were highly variable between experiments. Surfactant-amended slurries of PCB-contaminated electric power plant substation soil were inoculated with approximately 10(5) recombinant cells per ml. The populations of the added strains increased to greater than 10(9) cells per ml in 2 days, and cell growth coincided with PCB degradation. By 15 days, 50 to 60% of the indicator congener 2,3,2',5'-tetrachlorobiphenyl was degraded. The effectiveness of PCB degradation by the plasmid-containing strain depended on plasmid stability. The transposon-encoded PCB genes were much more stable, and in surfactant-amended soil slurries, PCB degradation was more consistent between experiments.     

Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil

One rhamnolipid-producing bacterial strain named Pseudomonas aeruginosa BSFD5 was isolated and characterized. Its rhlABRI cassette including necessary genes for rhamnolipid synthesis was cloned and transformed into the chromosome of P. putida KT2440 by a new random transposon vector without introducing antibiotic-resistance marker, generating a genetically engineered microorganism named P. putida KT2440-rhlABRI, which could stably express the rhlABRI cassette and produce rhamnolipid at a yield of 1.68?g?l(-1). In experiments using natural soil, it was shown that P. putida KT2440-rhlABRI could increase the dissolution of pyrene and thus promote its degradation by indigenous microorganisms. P. putida KT2440-rhlABRI thus demonstrated potential for enhancing the remediation of soils contaminated with polycyclic aromatic hydrocarbons.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

Cloning and transposon vectors derived from satellite bacteriophage P4 for genetic manipulation of Pseudomonas and other gram-negative bacteria.

We developed transposon and cloning shuttle vectors for genetic manipulation of Pseudomonas and other gram-negative bacteria, exploiting the unique properties and the broad host range of the satellite bacteriophage P4. P4::Tn5 AP-1 and P4::Tn5 AP-2 are suicide transposon vectors which have been used for efficient Tn5 mutagenesis in Pseudomonas putida. pKGB2 is a phasmid vector with a cloning capacity of about 7.5 kb; useful unique cloning sites are SacI and SacII in the streptomycin resistance determinant and PvuI and XhoI in the kanamycin resistance determinant. pKGB4 is a cosmid derived from pKGB2 and carries the additional cloning site SmaI in the kanamycin resistance determinant; its cloning capacity is about 18 kb. These vectors and their recombined derivatives were transferred from Escherichia coli to P. putida by transduction and may be used for other bacterial species susceptible to P4 infection.     

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.     

Performance and Microbial Diversity of a Trickle‐Bed Air Biofilter under Interchanging Contaminants

A trickle‐bed air biofilter (TBAB) was evaluated under conditions of interchanging the feed volatile organic compounds (VOCs) in the sequence methyl ethyl ketone (MEK), toluene, methyl isobutyl ketone (MIBK), styrene, and then back to MEK. The obtained performance results revealed that the biofilter provided high removal efficiency within the critical loading of each VOC, which was previously defined in the non‐interchanging VOC fed biofilter. The biofilter easily acclimated to the oxygenated compounds (MEK and MIBK), but re‐acclimation was delayed for the aromatic compounds (toluene and styrene). Ratios of the molar mass of CO₂ produced per molar mass of VOC removed were investigated. It has been found that the ratios for the aromatic compounds closely resembled the theoretical complete chemical oxidation based ratios while larger differences were encountered with the oxygenated compounds. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes was used to assess the impact of interchanging VOCs on the bacterial community structure in the biofilter. The results from denaturing gradient gel electrophoresis (DGGE) showed that the structure of the microbial community in the biofilter was different after each interchange of VOCs.     

Genetic organization of sulphur-controlled aryl desulphonation in Pseudomonas putida S-313

Pseudomonas putida S-313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR-type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate-specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.     

Transposition of DEH,a broad-host-range transposon flanked by ISPpu12, in Pseudomonas putida is associated with genomic rearrangements and dehalogenase gene silencing

Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families. The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehR(I), was isolated from strain PP3 by using the TOL plasmid pWW0. DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehR(I) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria. Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P. putida PaW340. Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P. putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing. Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.