Isolation of Pseudomonas oleovorans Carrying Multidrug Resistance Proteins MdtA and MdtB from Wastewater.

The pollution of industrial wastewater has become a global issue in terms of economic development and ecological protection. Pseudomonas oleovorans has been studied as a bacterium involved in the treatment of petroleum pollutants. Our study aimed to investigate the physicochemical properties and drug resistance of Pseudomonas oleovorans isolated from industrial wastewater with a high concentration of sulfate compounds. Firstly, Pseudomonas oleovorans was isolated and then identified using matrix-assisted flight mass spectrometry and 16S rDNA sequencing. Then, biochemical and antibiotic resistance analyses were performed on the Pseudomonas oleovorans, and a microbial high-throughput growth detector was used to assess the growth of the strain. Finally, PCR and proteomics analyses were conducted to determine drug-resistance-related genes/proteins. Based on the results of the spectrum diagram and sequencing, the isolated bacteria were identified as Pseudomonas oleovorans and were positive to reactions of ADH, MTE, CIT, MLT, ONPG, and ACE. Pseudomonas oleovorans was sensitive to most of the tested antibiotics, and its resistance to SXT and CHL and MIN and TIM was intermediate. The growth experiment showed that Pseudomonas oleovorans had a good growth rate in nutrient broth. Additionally, gyrB was the resistance gene, and mdtA2, mdtA3, mdtB2, mdaB, and emrK1 were the proteins that were closely associated with the drug resistance of Pseudomonas oleovorans. Our results show the biochemical properties of Pseudomonas oleovorans from industrial wastewater with a high concentration of sulfate compounds and provide a new perspective for Pseudomonas oleovorans to participate in biological removal of chemical pollutants in industrial wastewater.

Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans.

Bacterial Type IV CRISPR-Cas systems are thought to rely on multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements, but the substrate requirements and potential DNA nuclease activities for many systems within this type are uncharacterized. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in a PAM-dependent manner and elicits interference without showing DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. Deletion of the native Type IV CRISPR array resulted in upregulation of pilN operon transcription in the absence of genome cleavage, indicating that Type IV-A CRISPR-Cas systems can function in host gene regulation. These systems resemble CRISPR interference (CRISPRi) methodology but represent a natural CRISPRi-like system that is found in many Pseudomonas and Klebsiella species and allows for gene silencing using engineered crRNAs.

Complete genome sequence of the Pseudomonas oleovorans strain ODT-83 isolated from oyster.

A bacterial strain ODT-83 is isolated from oysters, which is capable of adsorbing norovirus (NoV) via histo-blood group antigen-like (HBGA-like) substances. To better understand its genetic background associated with the production of HBGA-like substances, the genome of the ODT-83 was completely sequenced and analyzed. The ODT-83 only contains one circular chromosome, with a length of 5,384,159 bp. Both the 16S rRNA gene phylogeny and the average nucleotide identity (ANI) analyses confirm that the ODT-83 is a new Pseudomonas oleovorans strain. The whole genome encodes a total of 5037 predicted open reading frames (ORFs), 66 tRNA genes and 12 rRNA genes. Two gene clusters are detected on the genome, which are involved in the synthesis of polysaccharides of alginate and Pel, respectively. These results lay the foundation for further research on the interaction between the P. oleovorans strain ODT-83 and NoV.

Removal of gaseous tetrahydrofuran via a three-phase airlift bioreactor loaded with immobilized cells of GFP-tagged Pseudomonas oleovorans GDT4.

Tetrahydrofuran (THF) is a common highly toxic cyclic aliphatic ether that frequently exists in waste gases. Removal of gaseous THF is a serious issue with important environmental ramifications. A novel three-phase airlift bioreactor (TPAB) loaded with immobilized cells was developed for efficient THF removal from gas streams. An effective THF-degrading transformant, Pseudomonas oleovorans GDT4, which contains the pTn-Mod-OTc-gfp plasmid and was tagged with a green fluorescent protein (GFP), was constructed. Continuous treatment of THF-containing waste gases was succeeded by the GFP-labelled cells immobilized with calcium alginate and activated carbon fiber in the TPAB for 60 days with >90% removal efficiency. The number of fluorescent cells in the beads reached 1.7 × 1011 cells·g-1 of bead on day 10, accounting for 83.3% of the total number of cells. The amount further increased to 3.0 × 1011 cells·g-1 of bead on day 40. However, it decreased to 2.5 × 1011 cells·g-1 of bead with a substantial increase in biomass in the liquid because of cell leakage and hydraulic shock. PCR-DGGE revealed that P. oleovorans was the dominant microorganism throughout the entire operation. The maximum elimination capacity was affected by empty bed residence time (EBRT). The capacity was only 25.9 g m-3·h-1 at EBRT of 80 s, whereas it reached 37.8 g m-3·h-1 at EBRT of 140 s. This work provides an alternative method for full-scale removal of gaseous THF and presents a useful tool for determining the biomass of a specific degrader in immobilized beads.

Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines.

Amination of bulky ketones, particularly in (R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S) selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R) amines for the production of high-value products and to screen for those converting putrescine.IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Draft genome sequence of Pseudomonas oleovorans strain MGY01 isolated from deep sea water.

Pseudomonas oleovorans MGY01 isolated from the deep-sea water of the South China Sea could effectively degrade malachite green. The draft genome of P. oleovorans MGY01 was sequenced and analyzed to gain insights into its efficient metabolic pathway for degrading malachite green. The data obtained revealed 109 Contigs (N50; 128,269 bp) with whole genome size of 5,201,892 bp. The draft genome sequence of strain MGY01 will be helpful in studying the genetic pathways involved in the degradation of malachite green.

Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp.

OPHC2 is a thermostable organophosphate (OP) hydrolase in the ß-lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962- and 106-fold improvements in catalytic efficiency for methyl-parathion and ethyl-paraoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 × 10(7) -fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 Å resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPHM2 from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg(-1) ) toward methyl-parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in ß-lactamase superfamily and provide efficient and robust enzymes for OP detoxification.

Biodegradation of tetrahydrofuran by Pseudomonas oleovorans DT4 immobilized in calcium alginate beads impregnated with activated carbon fiber: mass transfer effect and continuous treatment.

A novel entrapment matrix, calcium alginate (CA) coupled with activated carbon fiber (ACF), was prepared to immobilize Pseudomonas oleovorans DT4 for degrading tetrahydrofuran (THF). The addition of 1.5% ACF increased the adsorption capacity of the immobilized bead, thus resulting in an enhanced average removal rate of 30.3mg/(Lh). The synergism between adsorption and biodegradation was observed in the hybrid CA-ACF beads instead of in the system comprising CA beads and freely suspended ACF. The effective diffusion coefficient of the CA-ACF bead was not significantly affected by bead size, but the bead's value of 1.14×10(-6)cm(2)/s (for the bead diameter of 0.4 cm) was larger than that of the CA bead by almost one order of magnitude based on the intraparticle diffusion-reaction kinetics analysis. Continuous treatment of the THF-containing wastewater was succeeded by CA-ACF immobilized cells in a packed-bed reactor for 54 d with a >90% removal efficiency.

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization.

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.

Survival of phosphate-solubilizing bacteria against DNA damaging agents.

Phosphate-solubilizing bacteria (PSBs) were isolated from different plant rhizosphere soils of various agroecological regions of India. These isolates showed synthesis of pyrroloquinoline quinone (PQQ), production of gluconic acid, and release of phosphorus from insoluble tricalcium phosphate. The bacterial isolates synthesizing PQQ also showed higher tolerance to ultraviolet C radiation and mitomycin C as compared to Escherichia coli but were less tolerant than Deinococcus radiodurans. Unlike E. coli, PSB isolates showed higher tolerance to DNA damage when grown in the absence of inorganic phosphate. Higher tolerance to ultraviolet C radiation and oxidative stress in these PSBs grown under PQQ synthesis inducible conditions, namely phosphate starvation, might suggest the possible additional role of this redox cofactor in the survival of these isolates under extreme abiotic stress conditions.

A newly isolated strain capable of effectively degrading tetrahydrofuran and its performance in a continuous flow system.

A Gram-negative strain DT4, capable of growing aerobically on tetrahydrofuran (THF) as the sole carbon and energy source was isolated from a pharmaceutical wastewater treatment plant. It was identified as Pseudomonas oleovorans by morphological and physiological characteristics as well as Biolog profiling and 16S rDNA sequence. Cells of P. oleovorans DT4 pre-cultured in THF could degrade 5 mM THF completely without lag phase. The generation time of 2.7 h and the maximum degradation rate of 203.9 mg THF/(h g dry weight) were observed, demonstrating that DT4 bears the highest THF-degrading activity in ever described strains. Furthermore, THF concentration as high as 100 mM was tolerated by the culture. Several important compounds including gamma-butyrrolactone and benzene could be directly metabolized, whereas other pollutants (e.g., tetrahydropyrane) could be cometabolized by DT4. THF removal was achieved in a continuous flow system with the maximum specific growth rate 0.113 h(-1) and half-saturation constant 1.224 mg/L, indicating the great potential of THF bioremediation in future full-scale application.

Pseudomonas oleovorans subsp. lubricantis subsp. nov., and reclassification of Pseudomonas pseudoalcaligenes ATCC 17440T as later synonym of Pseudomonas oleovorans ATCC 8062 T.

Isolate RS1(T) isolated from used metalworking fluid was found to be a Gram-negative, motile, and non-spore forming rod. Based on phylogenetic analyses with 16S rRNA, isolate RS1(T) was placed into the mendocina sublineage of Pseudomonas. The major whole cell fatty acids were C(18:1)omega7c (32.6%), C(16:0) (25.5%), and C(15:0) ISO 2OH/C(16:1)omega7c (14.4%). The sequence similarities of isolate RS1(T) based on gyrB and rpoD genes were 98.9 and 98.0% with Pseudomonas pseudoalcaligenes, and 98.5 and 98.1% with Pseudomonas oleovorans, respectively. The ribotyping pattern showed a 0.60 similarity with P. oleovorans ATCC 8062(T) and 0.63 with P. pseudoalcaligenes ATCC17440(T). The DNA G + C content of isolate RS1(T) was 62.2 mol.%. The DNA-DNA relatedness was 73.0% with P. oleovorans ATCC 8062(T) and 79.1% with P. pseudoalcaligenes ATCC 17440(T). On the basis of morphological, biochemical, and molecular studies, isolate RS1(T) is considered to represent a new subspecies of P. oleovorans. Furthermore, based on the DNA-DNA relatedness (>70%), chemotaxonomic, and molecular profile, P. pseudoalcaligenes ATCC 17440(T) and P. oleovorans ATCC 8062(T) should be united under the same name; according to the rules of priority, P. oleovorans, the first described species, is the earlier synonym and P. pseudoalcaligenes is the later synonym. As a consequence, the division of the species P. oleovorans into two novel subspecies is proposed: P. oleovorans subsp. oleovorans subsp. nov. (type strain ATCC 8062(T) = DSM 1045(T) = NCIB 6576(T)), P. oleovorans subsp. lubricantis subsp. nov. (type strain RS1(T) = ATCC BAA-1494(T) = DSM 21016(T)).

Engineering the monomer composition of polyhydroxyalkanoates synthesized in Saccharomyces cerevisiae.

Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical beta-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C6 to C14) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as beta-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.

Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase.

We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3 HB-co-4 HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3 HB-co-4 HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures.

Genetic characterization of the poly(hydroxyalkanoate) synthases of various Pseudomonas oleovorans strains.

We identified the poly(hydroxyalkanoate) synthase (PHAS) genes of three strains of Pseudomonas oleovorans by using polymerase chain reaction (PCR)-based detection methods. P. oleovorans NRRL B-14682 contains Class I PHA synthase gene (phaC), NRRL B-14683 harbors Class II phaC1 and phaC2 genes, and NRRL B-778 contain both the Class I and II PHA synthase genes. Inverse-PCR and chromosomal walking techniques were employed to obtain the complete sequences of the Class I phaCs of NRRL B-778 (phbC778; 1698 bps) and B-14682 (phbC14682; 1899 bps). BLAST search indicated that these genes are new and had not been previously cloned. The gene product of phbC778 (i.e., PhbC778; 566 amino acid residues) is homologous to the Class I PHA synthases of Pseudomonas sp. HJ-2 and Pseudomonas sp. strain 61-3, and that of phbC14682 (PhbC14682; 632 amino acids) is homologous to PHAS of Delftia acidovorans. The PhbC14682 contains an extra sequence of 33 amino acids in its conserved alpha/beta-hydrolase domain, making it only the second Class I PHA synthase found to contain this cellular proteolytic sequence. Consistent with their Pseudomonas origin, the codon-usage profiles of PhbC778 and PhbC14682 are similar to those of Pseudomonas Class II PHASs. These new Pseudomonas Class I phbC genes provide valuable addition to the gene pool for the construction of novel PHASs through gene shuffling.

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs.

Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.

Engineering of chimeric class II polyhydroxyalkanoate synthases.

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

rpoS gene expression in carbon-starved cultures of the Polyhydroxyalkanoate-accumulating species Pseudomonas oleovorans.

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H2O2 and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.