Formation of new PHE plasmids in pseudomonads in a phenol-polluted environment.

Several years ago, a laboratory-constructed plasmid with a single-component phenol monooxygenase gene (pheBA operon) flanked by two IS elements was released to a phenol-polluted area. During the following years, we found in the test area widely distributed pheBA operon-containing bacteria. The new pheBA+ strains belong predominantly to the Pseudomonas fluorescens group, and they did not arise via selection of the released PHE plasmid. On the contrary, the formation of several different types of PHE plasmids occurred, namely pPHE101 (60,958 bp) from the IncP-9 group, non-transferable plasmid pPHE69 (44,717 bp), mobilizable plasmid pPHE20 (39,609 bp) and the IncP-7 type plasmid pPHE24ΔpheBA (120,754 bp), in which the pheBA operon was translocated from the plasmid to the chromosome. In two cases, PHE plasmid-bearing strains exist in a multi-plasmid state, also containing the non-catabolic plasmids pG20 (133,709 bp) and pG69 (144,433 bp) with backbones sharing 97% DNA identity and with redundant genes for the initiation of replication, repA1and repA2, of which only one was active. Seemingly, several other plasmids and bacterial features besides the pheBA operon were involved in selective distribution of catabolic operons in the natural environment. The comparison of the genetic structure of plasmids and IS elements' functions, as well as resistance to heavy metals of seven completely sequenced plasmids, are discussed.

Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids.

In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

Strategy of Pseudomonas pseudoalcaligenes C70 for effective degradation of phenol and salicylate.

Phenol- and naphthalene-degrading indigenous Pseudomonas pseudoalcaligenes strain C70 has great potential for the bioremediation of polluted areas. It harbours two chromosomally located catechol meta pathways, one of which is structurally and phylogenetically very similar to the Pseudomonas sp. CF600 dmp operon and the other to the P. stutzeri AN10 nah lower operon. The key enzymes of the catechol meta pathway, catechol 2,3-dioxygenase (C23O) from strain C70, PheB and NahH, have an amino acid identity of 85%. The metabolic and regulatory phenotypes of the wild-type and the mutant strain C70ΔpheB lacking pheB were evaluated. qRT-PCR data showed that in C70, the expression of pheB- and nahH-encoded C23O was induced by phenol and salicylate, respectively. We demonstrate that strain C70 is more effective in the degradation of phenol and salicylate, especially at higher substrate concentrations, when these compounds are present as a mixture; i.e., when both pathways are expressed. Moreover, NahH is able to substitute for the deleted PheB in phenol degradation when salicylate is also present in the growth medium. The appearance of a yellow intermediate 2-hydroxymuconic semialdehyde was followed by the accumulation of catechol in salicylate-containing growth medium, and lower expression levels and specific activities of the C23O of the sal operon were detected. However, the excretion of the toxic intermediate catechol to the growth medium was avoided when the growth medium was supplemented with phenol, seemingly due to the contribution of the second meta pathway encoded by the phe genes.

Functional redundancy in phenol and toluene degradation in Pseudomonas stutzeri strains isolated from the Baltic Sea.

In the present study we describe functional redundancy of bacterial multicomponent monooxygenases (toluene monooxygenase (TMO) and toluene/xylene monooxygenase (XylAM) of TOL pathway) and cooperative genetic regulation at the expression of the respective catabolic operons by touR and xylR encoded regulatory circuits in five phenol- and toluene-degrading Pseudomonas stutzeri strains. In these strains both toluene degradation pathways (TMO and Xyl) are active and induced by toluene and phenol. The whole genome sequence of the representative strain 2A20 revealed the presence of complete TMO- and Xyl-upper pathway operons together with two sets of lower catechol meta pathway operons, as well as phenol-degrading operon in a single 292,430bp contig. The much lower GC content and analysis of the predicted ORFs refer to the plasmid origin of the approximately 130kb region of this contig, containing the xyl, phe and tou genes. The deduced amino acid sequences of the TMO, XylA and the large subunit of phenol monooxygenase (LmPH) show 98-100% identity with the respective gene products of the strain Pseudomonas sp. OX1. In both strains 2A20 and OX1 the meta-cleavage pathways for catechol degradation are coded by two redundant operons (phe and xyl). We show that in the strain 2A20 TouR and XylR are activated by different effector molecules, phenol and toluene, respectively, and they both control transcription of the xyl upper, tou (TMO) and phe catabolic operons. Although the growth parameters of redundant strains did not show advantage at toluene biodegradation, the functional redundancy could provide better flexibility to the bacteria in environmental conditions.

Biodegradation efficiency of functionally important populations selected for bioaugmentation in phenol- and oil-polluted area.

Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was used to monitor the behaviour and relative abundance of mixed phenol-degrading bacterial populations (Pseudomonas mendocina PC1, P. fluorescens strains PC18, PC20 and PC24) during degradation of phenolic compounds in phenolic leachate- and oil-amended microcosms. The analysis indicated that specific bacterial populations were selected in each microcosm. The naphthalene-degrading strain PC20 was the dominant degrader in oil-amended microcosms and strain PC1 in phenolic leachate microcosms. Strain PC20 was not detectable after cultivation in phenolic leachate microcosms. Mixed bacterial populations in oil-amended microcosms aggregated and formed clumps, whereas the same bacteria had a planktonic mode of growth in phenolic leachate microcosms. Colony hybridisation data with catabolic gene specific probes indicated that, in leachate microcosms, the relative proportions of bacteria having meta (PC1) and ortho (PC24) pathways for degradation of phenol and p-cresol changed alternately. The shifts in the composition of mixed population indicated that different pathways of metabolism of aromatic compounds dominated and that this process is an optimised response to the contaminants present in microcosms.

Analysis of river pollution data from low-flow period by means of multivariate techniques: a case study from the oil-shale industry region, northeastern Estonia.

The oil-shale industry has created serious pollution problems in northeastern Estonia. Untreated, phenol-rich leachate from semi-coke mounds formed as a by-product of oil-shale processing is discharged into the Baltic Sea via channels and rivers. An exploratory analysis of water chemical and microbiological data sets from the low-flow period was carried out using different multivariate analysis techniques. Principal component analysis allowed us to distinguish different locations in the river system. The riverine microbial community response to water chemical parameters was assessed by co-inertia analysis. Water pH, COD and total nitrogen were negatively related to the number of biodegradative bacteria, while oxygen concentration promoted the abundance of these bacteria. The results demonstrate the utility of multivariate statistical techniques as tools for estimating the magnitude and extent of pollution based on river water chemical and microbiological parameters. An evaluation of river chemical and microbiological data suggests that the ambient natural attenuation mechanisms only partly eliminate pollutants from river water, and that a sufficient reduction of more recalcitrant compounds could be achieved through the reduction of wastewater discharge from the oil-shale chemical industry into the rivers.

Biodegradation of dimethylphenols by bacteria with different ring-cleavage pathways of phenolic compounds.

The biodegradation of 3,4, 2,4, 2,3, 2,6 and 3,5-dimethylphenol in combination with phenol and p-cresol by axenic and mixed cultures of bacteria was investigated. The strains, which degrade phenol and p-cresol through different catabolic pathways, were isolated from river water continuously polluted with phenolic compounds of leachate of oil shale semicoke ash heaps. The proper research of degradation of 2,4 and 3,4-dimethylphenol in multinutrient environments was performed. The degradation of phenolic compounds from mixtures indicated a flux of substrates into different catabolic pathways. Catechol 2,3-dioxygenase activity was induced by dimethylphenols in Pseudomonas mendocina PC1, where meta cleavage pathway was functional during the degradation of p-cresol. In the case of strains PC18 and PC24 of P. fluorescens, the degradation of p-cresol occurred via the protocatechuate ortho pathway and the key enzyme of this pathway, p-cresol methylhydroxylase, was also induced by dimethylphenols. 2,4 and 3,4-dimethylphenols were converted into the dead-end products 4-hydroxy-3-methylbenzoic acid and 4-hydroxy-2-methylbenzoic acid. In the degradation of 3,4-dimethylphenol, the transient accumulation of 4-hydroxy-2-methylbenzaldehyde repressed the consumption of phenol from substrate mixtures. A mixed culture of strains with different catabolic types made it possible to overcome the incompatibilities at degradation of studied substrate mixtures.

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH(+) phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees.

Occurrence of plasmids in the aromatic degrading bacterioplankton of the baltic sea.

Plasmids are mobile genetic elements that provide their hosts with many beneficial traits including in some cases the ability to degrade different aromatic compounds. To fulfill the knowledge gap regarding catabolic plasmids of the Baltic Sea water, a total of 209 biodegrading bacterial strains were isolated and screened for the presence of these mobile genetic elements. We found that both large and small plasmids are common in the cultivable Baltic Sea bacterioplankton and are particularly prevalent among bacterial genera Pseudomonas and Acinetobacter. Out of 61 plasmid-containing strains (29% of all isolates), 34 strains were found to carry large plasmids, which could be associated with the biodegradative capabilities of the host bacterial strains. Focusing on the diversity of IncP-9 plasmids, self-transmissible m-toluate (TOL) and salicylate (SAL) plasmids were detected. Sequencing the repA gene of IncP-9 carrying isolates revealed a high diversity within IncP-9 plasmid family, as well as extended the assumed bacterial host species range of the IncP-9 representatives. This study is the first insight into the genetic pool of the IncP-9 catabolic plasmids in the Baltic Sea bacterioplankton.

Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens.

p-Cresol methylhydroxylase (PCMH), a key enzyme responsible for the catabolism of p-cresol via the protocatechuate ortho pathway, was used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluore-scens strains PC18 and PC24. Although both strains catabolize p-cresol using PCMH, different whole-cell kinetic parameters for this compound were revealed. Affinity for the substrate and the specific growth rate were higher in PC18, whereas maximum p-cresol tolerance was higher in PC24. In addition, PCMH of strain PC18 was induced during growth on phenol. In both strains, the pchACXF operon, which encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, was sequenced. Transcriptional regulation of these operons by PchR, a putative sigma(54)-dependent regulator, was shown. Although the promoters of these operons resembled sigma(54)-controlled promoters, they differed from the consensus sequence by having T instead of C at position -12. Complementation assays confirmed that the amino acid sequence differences of the PchR regulators between the two strains studied led to different effector-binding capabilities of these proteins: (1) phenol was a more efficient effector for PchR of PC18 than p-cresol, (2) phenol did not activate the regulator of PC24, and (3) both regulators responded similarly to p-cresol.

Survival and catabolic performance of introduced Pseudomonas strains during phytoremediation and bioaugmentation field experiment.

A long-term field experiment was carried out to estimate the efficiency of bioaugmentation in combination with phytoremediation for oil shale chemical industry solid waste dump area remediation. Soil samples for microbiological and chemical analysis were collected during 3 years after bacterial biomass application. Microbial communities in soil samples were analysed using both culture-based and molecular methods. The survival of the introduced bacterial strains was confirmed by cultivation-based Box-PCR genomic fingerprints and denaturing gradient gel electrophoresis fingerprinting of the 16S rRNA and lmPH genes. The introduced bacterial strains as well as corresponding catabolic genes were recovered several years after biomass application, predominantly from the rhizosphere of birches. Soil samples from bioaugmented plots showed an elevated potential for degradation of phenolic compounds even 40 months after treatment. Based on our results we can conclude that the introduced Pseudomonas strains both survived, and their metabolic traits have persisted at the contaminated site over a long period of time.

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20.

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 ISPre1-like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 ISPre2-like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer - we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of ISPre2-like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.

Evaluation of different phenol hydroxylase-possessing phenol-degrading pseudomonads by kinetic parameters.

Phenol-degrading pseudomonads possessing different phenol hydroxylases (PH) were evaluated by the values of apparent half-saturation constant for phenol-oxygenating activity (K ( S )), maximum specific growth rate (mu (max)), lag-time length (lambda), inhibition constant (K ( I )) and growth yield factor (Y ( X/S )). Strains of the same PH type showed similar kinetic parameters: single-component PH (sPH) harbouring strains had higher values of K ( S ) and lower values of mu (max) than the strains having multicomponent PH (mPH). However, the values of K ( I ) and the dependencies of the lag-time length on initial phenol concentration were strain-specific. The elevated ratio between specific activities of catechol 1,2-dioxygenase (C12O) and muconate cycloisomerase in sPH-strains caused irreversible accumulation of a high amount of exogenous cis,cis-muconate (CCM) which resulted in decreased Y ( X/S ) values. Co-presence of sPH and mPH genes did not give the strains PC16 and P69 any extra advantage and according to determined kinetic parameters only one PH was active during phenol degradation. At the same time simultaneous functioning of catechol ortho and meta cleavage pathways (strain PC20) resulted in higher mu (max) and Y ( X/S ) values. Evaluation of strains showed that the type of PH determined the efficiency of phenol degradation, whereas the tolerance to elevated phenol concentrations was strain-specific.

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes.

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.