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Contamination of the environment with crude oil or other fuels is an enormous disaster for all organisms. The microbial communities for bioremediation have been an effective tool for eliminating pollution. This study aimed to determine individual cultures' and a strain mixture's ability to utilize alkanes (single alkanes and crude oil). The proper study of pure cultures is necessary to design synergistically working consortia. The Acinetobacter venetianus ICP1 and Pseudomonas oleovorans ICTN13 strains isolated from a wastewater treatment plant of a crude oil refinery can grow in media containing various aromatic and aliphatic hydrocarbons. The genome of the strain ICP1 contains four genes encoding alkane hydroxylases, whose transcription depended on the length of the alkane in the media. We observed that the hydrophobic cells of the strain ICP1 adhered to hydrophobic substrates, and their biofilm formation increased the bioavailability and biodegradation of the hydrocarbons. Although strain ICTN13 also has one alkane hydroxylase-encoding gene, the growth of the strain in a minimal medium containing alkanes was weak. Importantly, the growth of the mixture of strains in the crude oil-containing medium was enhanced compared with that of the single strains, probably due to the specialization in the degradation of different hydrocarbon classes and co-production of biosurfactants. [ABSTRACT FROM AUTHOR]

RpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of a large number of genes to facilitate survival under starvation conditions and other stresses. The results of our study demonstrate that the frequency of emergence of base substitution mutants is significantly increased in long-term-starved populations of rpoS-deficient Pseudomonas putida cells. The increasing effect of the lack of RpoS on the mutation frequency became apparent in both a plasmid-based test system measuring [Phe.sup.+] reversion and a chromosomal rpoB system detecting rifampin-resistant mutants. The elevated mutation frequency coincided with the death of about 95% of the cells in a population of rpoS-deficient P. putida. Artificial overexpression of superoxide dismutase or catalase in the rpoS-deficient strain restored the survival of cells and resulted in a decline in the mutation frequency. This indicated that, compared to wild-type bacteria, rpoS-deficient cells are less protected against damage caused by reactive oxygen species. 7,8-Dihydro-8-oxoguanine (GO) is known to be one of the most stable and frequent base modifications caused by oxygen radical attack on DNA. However, the spectrum of base substitution mutations characterized in rpoS-deficient P. putida was different from that in bacteria lacking the GO repair system: it was broader and more similar to that identified in the wild-type strain. Interestingly, the formation of large deletions was also accompanied by a lack of RpoS. Thus, the accumulation of DNA damage other than GO elevates the frequency of mutation in these bacteria. It is known that oxidative damage of proteins and membrane components, but not that of DNA, is a major reason for the death of cells. Since the increased mutation frequency was associated with a decline in the viability of bacteria, we suppose that the elevation of the mutation frequency in the surviving population of carbon-starved rpoS-deficient P. putida may be caused both by oxidative damage of DNA and enzymes involved in DNA replication and repair fidelity.

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.

Transcription of the plasmid-borne phenol catabolic operon pheBA in Pseudomonas putida is activated by the LysR-family regulator CatR in the presence of the effector molecule cis, cis-muconate (CCM), which is an intermediate of the phenol degradation pathway. In addition to the positive control of the operon, several factors negatively affect transcription initiation from the pheBA promoter. First, the activation of the pheBA operon depends on the extracellular concentration of phenol. The pheBA promoter is rapidly activated in the presence of micromolar concentrations of phenol in minimal growth medium, but the initiation of transcription from this promoter is severely delayed after sudden exposure of bacteria to 2.5 mM phenol. Second, the transcriptional activation from this promoter is impeded when the growth medium of bacteria contains amino acids. The negative effects of amino acids can be suppressed either by overproducing CatR or by increasing, the intracellular amount of CCM. However, the intracellular amount of CCM is a major limiting factor for the transcriptional activation of the pheBA operon, as accumulation of CCM in a P. putida catB-defective strain, unable to metabolize CCM (but expressing CatR at a natural level), almost completely relieves the negative effects of amino acids. The intracellular amount of CCM is negatively affected by the catabolite repression control protein via downregulating at the post-transcriptional level the expression of the pheBA-encoded catechol 1,2-dioxygenase and the phenol monooxygenase, the enzymes needed for CCM production.

We analyzed the target preferences of a Tn3 family transposon Tn4652. Alignment of 93 different insertion sites revealed a consensus sequence which resembles that of Tn3, indicating that despite a low similarity between Tn4652 and Tn3 transposases, their target site recognition is conserved. doi:10.1128/JB.01863-06

As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene omp[A.sub.PP0773] and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that omp[A.sub.PP0773] and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.

Plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. Here we show that a large catabolic TOL plasmid, pWW0, from Pseudomonas putida carries genes (rulAB genes) encoding an error-prone DNA polymerase Pol V homologue which increase the survival of bacteria under conditions of accumulation of DNA damage. A study of population dynamics in stationary phase revealed that the presence of pWW0-derived rulAB genes in the bacterial genome allows the expression of a strong growth advantage in stationary phase (GASP) phenotype of P. putida. When rulAB-carrying cells from an 8-day-old culture were mixed with Pol V-negative cells from a 1-day-old culture, cells derived from the aged culture out-competed cells from the nonaged culture and overtook the whole culture. At the same time, bacteria from an aged culture lacking the rulAB genes were only partially able to out-compete cells from a fresh overnight culture of the parental P. putida strain. Thus, in addition to conferring resistance to DNA damage, the plasmid-encoded Pol V genes significantly increase the evolutionary fitness of bacteria during prolonged nutritional starvation of a P. putida population. The results of our study indicate that RecA is involved in the control of expression of the pWW0-encoded Pol V.

In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the dinB gene) in stationary-phase mutagenesis in Pseudomonas putida. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately. A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the P. putida wild-type strain and its Pol Iv-defective dinB knockout derivative. Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type P. putida populations starved for more than 1 week. No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving P. putida cells was observed. The occurrence of 1-bp deletions in P. putida cells did not require a functional RecA protein. RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the P. putida dinB gene was not significantly influenced by the DNA damage-inducing agent mitomycin C. Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving P. putida cells.

Simultaneous degradation of phenol in the presence of different N sources including the two s-triazines atrazine and cyanuric acid by Pseudomonas sp. strain ADP, the degradation of atrazine to cyanuric acid, and the effects of the adaptation of cells to phenol are studied. Results indicate that Pseudomonas sp. strain ADP is an ideal bacterium that can be used for investigating the regulatory interactions amongst different catabolic genes and stress response mechanisms during the simultaneous degradation of phenol and atrazine.

Research has been conducted on stationary-phase mutations. The hypothesis that the mutation process changes take place only during bacteria starvation has been tested, and the results are reported.

Research has been conducted on the Pseudomonas putida transposon Tn4652. Results indicate that the Tn4652 transposase promoter's transcription is controlled by the stationary-phase-specific sigma factor sigma (super)S.

Results demonstrate that transcription from the pheBA and catBCA promoters, encoding catechol 1,2-doxygenase and phenol monooxygenase and catechol degradation, respectively, is dependent on the growth medium. Data also show that amino acids in the medium silences both the promoters exponentially.

Research has been conducted on the sigma factor activating gene expression in stationary phase and under stress conditions. Results demonstrate that the transcription level depends on the growth phase of the Pseudomonas putida cells and is higher in the stationary phase.

Research has been conducted on the Tn3 family transposon Tn4652. The creation of the fusion promoters by Tn4652 in activating silent genes is discussed.

Results show the activation binding site of CatR contains two G nucleotides important for promoter activity but not for CatR binding as indicated by the site-directed mutagenesis. Data also demonstrate a catR-mediated regulation mechanism conservation between the pheBA and the catBCA promoters.

Research has been conducted on the transposase regulation in Pseudomonas putida. Results indicate that Tn4652 transposase is controlled by a transposon-encoded protein.

The Tn 4652 transposase gene tnpA was sequenced and the corresponding promoter was mapped to the right end of the element to study the mechanisms behind the regulation of the transposase gene. The positive funtion of the integration host factor (IHF) was verified by the absence of enhancing effect of the IHF in the P. putida ihfA-defective stran A8759. The results suggested that IHF is not only instrumental in facilitating transcription from the tnpA promoter but also absorbs some of the negative effects of terminal sequences of Tn4652 on the activity of the promoters.

The chromosomal catR gene mediates transcription of the phenol monooxygenase- and catechol 1,2-dioxygenase encoding pheBA operon within PEST 1226, in Pseudomonas putida PaW85. Expression analysis of the deletion subclones obtained from PAT1140 indicates that a regulatory protein chromosomally encoded in P. putida mediates the transcription of the PheBA operon. cis,cis-muconate incites the preparation of catechol 1,2-dioxygenase and plasmid encoded phenol monooxygenase.

Abstract Background We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. Results By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC-deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. Conclusions ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.