Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation.

An important link between the environment and the physiological state of bacteria is the regulation of the transcription of a large number of genes by global transcription factors. One of the global regulators, Fis (factor for inversion stimulation), is well studied in Escherichia coli, but the role of this protein in pseudomonads has only been examined briefly. According to studies in Enterobacteriaceae, Fis regulates positively the flagellar movement of bacteria. In pseudomonads, flagellar movement is an important trait for the colonization of plant roots. Therefore we were interested in the role of the Fis protein in Pseudomonas putida, especially the possible regulation of the colonization of plant roots. We observed that Fis reduced the migration of P. putida onto the apices of barley roots and thereby the competitiveness of bacteria on the roots. Moreover, we observed that overexpression of Fis drastically reduced swimming motility and facilitated P. putida biofilm formation, which could be the reason for the decreased migration of bacteria onto the root apices. It is possible that the elevated expression of Fis is important in the adaptation of P. putida during colonization of plant roots by promoting biofilm formation when the migration of bacteria is no longer favoured.

Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida.

The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif(r) mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

Elevated mutation frequency in surviving populations of carbon-starved rpoS-deficient Pseudomonas putida is caused by reduced expression of superoxide dismutase and catalase.

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(+) 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.

Involvement of DNA mismatch repair in stationary-phase mutagenesis during prolonged starvation of Pseudomonas putida.

One of the popular ideas is that decline in methyl-directed mismatch repair (MMR) in carbon-starved bacteria might facilitate occurrence of stationary-phase mutations. We compared the frequency of accumulation of stationary-phase mutations in carbon-starved Pseudomonas putida wild-type and MMR-defective strains and found that knockout of MMR system increased significantly emergence of base substitutions in starving P. putida. At the same time, the appearance of 1-bp deletion mutations was less affected by MMR in this bacterium. The spectrum of base substitution mutations which occurred in starving populations of P. putida wild-type strain was distinct from mutation spectrum identified in MMR-defective strains. The spectrum of base substitutions differed also in this case when mutants emerged in starved populations of MutS or MutL-defective strains were comparatively analyzed. Based on our results we suppose that other mechanisms than malfunctioning of MMR system in resting cells might be considered to explain the accumulation of stationary-phase mutations in P. putida. To further characterize populations of P. putida starved on selective plates, we stained bacteria with LIVE/DEAD kit in situ on agar plates. We found that although the overall number of colony forming units (CFU) did not decline in long-term-starved populations, these populations were very heterogeneous on the plates and contained many dead cells. Our results imply that slow growth of subpopulation of cells at the expenses of dead cells on selective plates might be important for the generation of stationary-phase mutations in P. putida. Additionally, the different survival patterns of P. putida on the same selective plates hint that competitive interactions taking place under conditions of prolonged starvation of microbial populations on semi-solid surfaces might be more complicated than previously assumed.

DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.

NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida.

Under growth-restricting conditions bacterial populations can rapidly evolve by a process known as stationary-phase mutagenesis. Bacterial nonhomologous end-joining (NHEJ) system which consists of the DNA-end-binding enzyme Ku and the multifunctional DNA ligase LigD has been shown to be important for survival of bacteria especially during quiescent states, such as late stationary-phase populations or sporulation. In this study we provide genetic evidence that NHEJ enzymes participate in stationary-phase mutagenesis in a population of carbon-starved Pseudomonas putida. Both the absence of LigD or Ku resulted in characteristic spectra of stationary-phase mutations that differed from each other and also from the wild-type spectrum. This indicates that LigD and Ku may participate also in mutagenic pathways that are independent from each other. Our results also imply that both phosphoesterase (PE) and polymerase (POL) domains of the LigD protein are involved in the occurrence of mutations in starving P. putida. The participation of both Ku and LigD in the occurrence of stationary-phase mutations was further supported by the results of the analysis of mutation spectra in stationary-phase sigma factor RpoS-minus background. The spectra of mutations identified in the RpoS-minus background were also distinct if LigD or Ku was absent. Interestingly, the effects of the presence of these enzymes on the frequency of occurrence of certain types of mutations were different or even opposite in the RpoS-proficient and deficient backgrounds. These results imply that RpoS affects performance of mutagenic pathways in starving P. putida that utilize LigD and/or Ku.

Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA - in vivo, in vitro, and in silico studies.

Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin.

Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida.

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.

Different spectra of stationary-phase mutations in early-arising versus late-arising mutants of Pseudomonas putida: involvement of the DNA repair enzyme MutY and the stationary-phase sigma factor RpoS.

Stationary-phase mutations occur in populations of stressed, nongrowing, and slowly growing cells and allow mutant bacteria to overcome growth barriers. Mutational processes in starving cells are different from those occurring in growing bacteria. Here, we present evidence that changes in mutational processes also take place during starvation of bacteria. Our test system for selection of mutants based on creation of functional promoters for the transcriptional activation of the phenol degradation genes pheBA in starving Pseudomonas putida enables us to study base substitutions (C-to-A or G-to-T transversions), deletions, and insertions. We observed changes in the spectrum of promoter-creating mutations during prolonged starvation of Pseudomonas putida on phenol minimal plates. One particular C-to-A transversion was the prevailing mutation in starving cells. However, with increasing time of starvation, the importance of this mutation decreased but the percentage of other types of mutations, such as 2- to 3-bp deletions, increased. The rate of transversions was markedly elevated in the P. putida MutY-defective strain. The occurrence of 2- to 3-bp deletions required the stationary-phase sigma factor RpoS, which indicates that some mutagenic pathway is positively controlled by RpoS in P. putida.