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Pseudomonas putidais a fast-growing bacterium found mostly in temperate soil and water habitats. The metabolic versatility ofP. putidamakes this organism attractive for biotechnological applications such as biodegradation of environmental pollutants and synthesis of added-value chemicals (biocatalysis). This organism has been extensively studied in respect to various stress responses, mechanisms of genetic plasticity and transcriptional regulation of catabolic genes.P. putidais able to colonize the surface of living organisms, but is generally considered to be of low virulence. A number ofP. putidastrains are able to promote plant growth. The aim of this review is to give historical overview of the discovery of the speciesP. putidaand isolation and characterization ofP. putidastrains displaying potential for biotechnological applications. This review also discusses some major findings inP. putidaresearch encompassing regulation of catabolic operons, stress-tolerance mechanisms and mechanisms affecting evolvability of bacteria under conditions of environmental stress.

Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa. Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency.

The drilling, processing and transportation of oil are the main sources of pollution in water and soil. The current work analyzes the microbial diversity and aromatic compounds degradation potential in the metagenomes of communities in the wastewater treatment plant (WWTP) of a crude oil refinery. By focusing on the degradation of phenol, we observed the involvement of diverse indigenous microbial communities at different steps of the WWTP. The anaerobic bacterial and archaeal genera were replaced by aerobic and facultative anaerobic bacteria through the biological treatment processes. The phyla Proteobacteria, Bacteroidetes and Planctomycetes were dominating at different stages of the treatment. Most of the established protein sequences of the phenol degradation key enzymes belonged to bacteria from the class Alphaproteobacteria. From 35 isolated strains, 14 were able to grow on aromatic compounds, whereas several phenolic compound-degrading strains also degraded aliphatic hydrocarbons. Two strains, Acinetobacter venetianus ICP1 and Pseudomonas oleovorans ICTN13, were able to degrade various aromatic and aliphatic pollutants and were further characterized by whole genome sequencing and cultivation experiments in the presence of phenol to ascertain their metabolic capacity in phenol degradation. When grown alone, the intermediates of catechol degradation, the meta or ortho pathways, accumulated into the growth environment of these strains. In the mixed cultures of the strains ICP1 and ICTN13, phenol was degraded via cooperation, in which the strain ICP1 was responsible for the adherence of cells and ICTN13 diminished the accumulation of toxic intermediates., This study was supported by the ERA-NET project WRANA (Inno-INDIGO/0004/2014), and by the Institutional Research Funding IUT20-19 from Eesti Teadusagentuur (Estonian Research Council) to MK, by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI) to PMS, and by Department of Biotechnology, Ministry of Science and Technology, New Delhi, BT/IN/INNO-INDIGO/29/AK/2015-16 to AK. The isolated strains are deposited in the Collection of Environmental and Laboratory Microbial Strains (CELMS; financed by the Estonian Ministry of Education and Research) (RLOMRCELMS) the public catalogue of which is available on the Estonian Electronic Microbial dataBase (EEMB) website http://eemb.ut.ee.

Bioaugmentation in wastewater treatment plants (WWTPs) is challenging due to low survival and persistence of applied microbes. This study aimed to track the capacity and survival of fluorescent-tagged Pseudomonas oleovoransICTN13 as a model organism applicable in bioaugmentation of phenol-containing wastewater. The isolate was immobilized in alginate biopolymer, and enhanced efficacy and survival for biodegradation of phenol against free cells were studied. Encapsulated cells resulted in enhanced phenol removal efficiency (~94%) compared to free cells (~72%). Encapsulation of cells facilitated an extended storage time of 30 days. Remarkably, phenol and COD removal efficacy of encapsulated cells was sustained up to ~ 92–93% in a reactor after 45 days, while free cells could produce ~ 80–84% removal efficiency. Fluorescence microscopy showed high survival of the encapsulated cells, whereas gradual deterioration of free cells was observed. Thus, the findings highlight the importance of bio augmented strain in WWTPs where encapsulation is a crucial factor.

The biological treatment of oil refinery effluents in wastewater treatment plants (WWTPs) relies on specialized bacteria contributing to remove organic load, nitrogen, sulfur, and phosphorus compounds. Knowledge about bacterial dynamics in WWTPs and how they affect the performance of the wastewater treatment is limited, particularly in tropical countries. The bacterial communities from three compartments of an oil refinery WWTP in Uran, India, were assessed using 16S-metabarcoding, in winter and monsoon seasons, upstream (from the surge pond) and downstream the biotower (clarifier and guard pond), to understand the effects of seasonal variations in WWTP's efficiency. The organic load and ammonia levels of the treated wastewater increased by 3- and 9-fold in the monsoon time-point. A decreased abundance and diversity of 47 genera (325 OTUs) comprising ammonia and nitrite oxidizing bacteria (AOB, NOB, denitrifiers) was observed in the monsoon season downstream the biotower, whereas 23 OTUs of Sulfurospirillum, Desulfovibrio, and Bacillus, putatively performing dissimilatory nitrate reduction to ammonia (DNRA), were 3-fold more abundant in the same compartments (DNRA/denitrifiers winter ratio, This work was supported by the ERA-NET project WRANA (Inn-INDIGO/0004/2014), which included the postdoctoral grant of P.S.-C (grant BPD1/wrana/2017), and by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI).

Nucleoid-associated proteins (NAPs) such as IHF, HU, Fis, and H-NS alter the topology of bound DNA and may thereby affect accessibility of DNA to repair and recombination processes. To examine this possibility, we investigated the effect of IHF on the frequency of homologous recombination (HR) and point mutations in soil bacterium Pseudomonas putida by using plasmidial and chromosomal assays. We observed positive effect of IHF on the frequency of HR, whereas this effect varied depending both on the chromosomal location of the HR target and the type of plasmid used in the assay. The occurrence of point mutations in plasmid was also facilitated by IHF, whereas in the chromosome the positive effect of IHF appeared only at certain DNA sequences and/or chromosomal positions. We did not observe any significant effects of IHF on the spectrum of mutations. However, despite of the presence or absence of IHF, different mutational hot spots appeared both in plasmid and in chromosome. Additionally, the frequency of frameshift mutations in the chromosome was also strongly affected by the location of the mutational target sequence. Taking together, our results indicate that IHF facilitates the occurrence of genetic changes in P. putida, whereas the location of the target sequence affects both the IHF-dependent and IHF-independent mechanisms.

Bacteria evolve as a result of mutations and acquisition of foreign DNA by recombination processes. A growing body of evidence suggests that mutation and recombination rates are not constant across the bacterial chromosome. Bacterial chromosomal DNA is organized into a compact nucleoid structure which is established by binding of the nucleoid-associated proteins (NAPs) and other proteins. This review gives an overview of recent findings indicating that the mutagenic and recombination processes in bacteria vary at different chromosomal positions. Involvement of NAPs and other possible mechanisms in these regional differences are discussed. Variations in mutation and recombination rates across the bacterial chromosome may have implications in the evolution of bacteria.

Stalled RNA polymerases (RNAPs) pose an obstacle for the replicating complexes, which could lead to transcription-replication conflicts and result in genetic instability. Stalled RNAPs and DNA lesions blocking RNAP elongation are removed by transcription-coupled repair (TCR), the process which in bacteria is mediated by TCR factor Mfd and helicase UvrD. Although the mechanism of TCR has been extensively studied, its role in mutagenesis is still obscure. In the current study we have investigated the role of Mfd and UvrD in mutational processes in soil bacterium Pseudomonas putida. Our results revealed that UvrD helicase is essential to prevent the emergence of mutations, as the loss of uvrD resulted in elevated mutant frequency both in exponential- and stationary-phase bacterial cultures. UvrD was also found to be necessary to survive DNA damage, but NER or MMR pathways are not completely abolished in UvrD-deficient P. putida. Mfd-deficiency had a moderate impact on surviving DNA damage and did not influence the frequency of mutations occurred in exponentially growing bacteria. However, the absence of Mfd caused approximately a two-fold decline in stationary-phase mutant frequency compared to the P. putida wild-type strain and suppressed the elevated mutant frequency observed in the ΔuvrD strain. Remarkably, the Mfd-deficient strain also formed less UV-induced mutants. These results suggest that in P. putida the Mfd-mediated TCR could be associated with UV- and stationary-phase mutagenesis.

In addition to its prominence in producing genetic diversity in bacterial species, homologous recombination (HR) plays a key role in DNA repair and damage tolerance. The frequency of HR depends on several factors, including the efficiency of DNA repair systems as HR is involved in recovery of replication forks perturbed by DNA damage. Nucleotide excision repair (NER) is one of the major DNA repair pathways involved in repair of a broad range of DNA lesions generally induced by exogenous chemicals or UV-irradiation and its functions in the cells not exposed to DNA-damaging agents have attracted less attention. In this study we have developed an assay that enables to investigate HR between chromosomal loci of the soil bacterium Pseudomonas putida both in growing and stationary-phase cells. The present assay detects HR events between two non-functional alleles of phenol degrading genes that produce a functional allele and allow the growth of bacteria on phenol as a sole carbon source. Our results indicate that HR between chromosomal loci takes place mainly in the growing cells and the frequency of HR is reduced during the following starvation in NER-proficient P. putida but not in the case when bacteria lack UvrA or UvrB enzymes. The absence of UvrA or UvrB resulted in a hyper-recombination phenotype in P. putida, the cells were filamented and their growth was impaired even in the absence of exogenous DNA damage. However, NER-deficient derivatives that overcame growth defects emerged rapidly. Such adaptation resulted in the decline of the frequency of HR. Although HR in actively replicating P. putida was still elevated in the adapted variants of the UvrA- and UvrB-deficient strains, the dynamics of emergence of the recombinants in these strains turned similar to NER-proficient bacteria. Additionally, we observed that HR was enhanced in the absence of the transcription repair coupling factor Mfd in growing cells but not during starvation. The frequency of HR was not affected by the UvrA homologue UvrA2 neither in NER-proficient bacteria nor in the absence of UvrA, suggesting a minor role of UvrA2 in NER. Thus, we conclude that NER functions are important also without exogenously induced DNA damage in P. putida and both transcription-coupled and global genome NER act to suppress HR in growing cells, whereas UvrA and UvrB are involved in the maintenance of the genome integrity also in stationary-phase cells.

Bacteria form biofilm as a response to a number of environmental signals that are mediated by global transcription regulators and alarmones. Here we report the involvement of the global transcription regulator Fis inPseudomonas putidabiofilm formation through regulation oflapAandlapFgenes. The major component ofP. putidabiofilm is proteinaceous and two large adhesive proteins, LapA and LapF, are known to play a key role in its formation. We have previously shown that Fis overexpression enhancesP. putidabiofilm formation. In this study, we used mini-Tn5transposon mutagenesis to select potential Fis-regulated genes involved in biofilm formation. A total of 90 % of the studied transposon mutants carried insertions in thelapgenes. Since our experiments showed that Fis-enhanced biofilm is mostly proteinaceous, the amounts of LapA and LapF fromP. putidacells lysates were quantified using SDS-PAGE. Fis overexpression increases the quantity of LapA 1.6 times and decreases the amount of LapF at least 4 times compared to the wild-type cells. The increased LapA expression caused by Fis overexpression was confirmed by FACS analysis measuring the amount of LapA-GFP fusion protein. Our results suggest that the profusion of LapA in the Fis-overexpressed cells causes enhanced biofilm formation in mature stages ofP. putidabiofilm and LapF has a minor role inP. putidabiofilm formation.

LapA is the biggest protein in Pseudomonas putida and a key factor for biofilm formation. Its importance and posttranslational regulation is rather thoroughly studied but less is known about the transcriptional regulation. Here we give evidence that transcription of lapA in LB-grown bacteria is initiated from six promoters, three of which display moderate RpoS-dependence. The global transcription regulator Fis binds to the lapA promoter area at six positions in vitro, and Fis activates the transcription of lapA while overexpressed in cells. Two of the six Fis binding sites, Fis-A7 and Fis-A5, are necessary for the positive effect of Fis on the transcription of lapA in vivo. Our results indicate that Fis binding to the Fis-A7 site increases the level of transcription from the most distal promoter of lapA, whereas Fis binding to the Fis-A5 site could be important for modifying the promoter area topology.

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.

This study was supported by the BIO Program of the Spanish Ministry of Science and Innovation, the ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAM. The work in MK Laboratory is supported by Estonian Science Foundation, grant number 9114 to MK, by Estonian Ministry of Research Targeted Financing Project SF0180031s08.

Sublines of the major P. aeruginosa reference strain PAO1 are derivatives of the original PAO1 isolate, which are maintained in laboratories worldwide. These sublines display substantial genomic and phenotypic variation due to ongoing microevolution. Here, we examined four sublines, MPAO1, PAO1-L, PAO1-DSM and PAO1-UT, originated from different laboratories, and six DNA polymerase-deficient mutants from the P. aeruginosa MPAO1 transposon library for their employment in elucidation of DNA damage repair and tolerance mechanisms in P. aeruginosa. We found that PAO1 subline PAO1-UT carries a large deletion encompassing the DNA damage inducible imuA-imuB-imuC cassette (PA0669-PA0671), which is implied in mutagenesis in several species. Furthermore, the genetic changes leading to variation in the functionality of the MexEF-OprN efflux system contributed largely to the phenotypic discordance between P. aeruginosa PAO1 sublines. Specifically, we identified multiple mutations in the mexT gene, which encodes a transcriptional regulator of the mexEF-oprN genes, mutations in the mexF, and complete absence of these genes. Of the four tested sublines, MPAO1 was the only subline with the functional MexEF-OprN multidrug efflux system. Active efflux through MexEF-OprN rendered MPAO1 highly resistant to chloramphenicol and ciprofloxacin. Moreover, the functions of specialized DNA polymerase IV and nucleotide excision repair (NER) in 4-NQO-induced DNA damage tolerance appeared to be masked in MPAO1, while were easily detectable in other sublines. Finally, the frequencies of spontaneous and MMS-induced Rifr mutations were also significantly lower in MPAO1 in comparison to the PAO1 sublines with impaired MexEF-OprN efflux system. The MexEF-OprN-attributed differences were also observed between MPAO1 and MPAO1-derived transposon mutants from the two-allele transposon mutant collection. Thus, the accumulating mutations and discordant phenotypes of the PAO1 derivatives challenge the reproducibility and comparability of the results obtained with different PAO1 sublines and also limit the usage of the MPAO1 transposon library in DNA damage tolerance and mutagenesis studies.

Formation of microcolonies (papillae) permits easy visual screening of mutational events occurring in single colonies of bacteria. In this study, we have established a novel papillation assay employable in a wide range of pseudomonads including Pseudomonas aeruginosa and Pseudomonas putida for monitoring mutation frequency in distinct colonies. With the aid of this assay, we conducted a genome-wide search for the factors affecting mutation frequency in P. putida. Screening ∼27,000 transposon mutants for increased mutation frequency allowed us to identify 34 repeatedly targeted genes. In addition to genes involved in DNA replication and repair, we identified genes participating in metabolism and transport of secondary metabolites, cell motility, and cell wall synthesis. The highest effect on mutant frequency was observed when truA (tRNA pseudouridine synthase), mpl (UDP-N-acetylmuramate-alanine ligase) or gacS (multi-sensor hybrid histidine kinase) were inactivated. Inactivation of truA elevated the mutant frequency only in growing cells, while the deficiency of gacS affected mainly stationary-phase mutagenesis. Thus, our results demonstrate the feasibility of the assay for isolating mutants with elevated mutagenesis in growing as well as stationary-phase bacteria.

Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells. We have constructed a test system to investigate HR between a plasmid and a chromosome in carbon-starved populations of the soil bacterium Pseudomonas putida restoring the expression of phenol monooxygenase gene pheA. Our results show that prolonged starvation of P. putida in the presence of phenol stimulates HR. The emergence of recombinants on selective plates containing phenol as an only carbon source for the growth of recombinants is facilitated by reactive oxygen species and suppressed by DNA mismatch repair enzymes. Importantly, the chromosomal location of the HR target influences the frequency and dynamics of HR events. In silico analysis of binding sites of nucleoid-associated proteins (NAPs) revealed that chromosomal DNA regions which flank the test system in bacteria exhibiting a lower HR frequency are enriched in binding sites for a subset of NAPs compared to those which express a higher frequency of HR. We hypothesize that the binding of these proteins imposes differences in local structural organization of the genome that could affect the accessibility of the chromosomal DNA to HR processes and thereby the frequency of HR.

Summary Evolution of catabolic pathways for the degradation of synthetic nitroaromatic compounds is currently ongoing process because these compounds have been in nature only for a short time. Bacteria isolated from contaminated areas contain pathways for the degradation of nitroaromatic compounds at different stages of progression. Therefore, the emergence of pathways for the degradation of such chemicals provides a good opportunity to investigate evolutionary processes leading to the emergence of new metabolic routes and their regulatory systems. In Burkholderia sp. strain DNT the regulatory gene encoding the LysR-type transcriptional regulator DntR is placed divergently of the dinitrotoluene (DNT) dioxygenase genes. This regulator still recognizes salicylate, an effector of its NagR-like ancestor but not DNT. In this issue of Molecular Microbiology, de las Heras et al. demonstrate that the DntR does not respond to any metabolic intermediates of the DNT catabolic pathway. The results of this study suggest that the catabolic pathway for the degradation of DNT has reached to an early stage of evolution when novel specificities of the catabolic enzymes have already acquired but the cognate regulatory system is still missing. This research addresses some fundamental questions about bottlenecks to be solved during evolution of new catabolic operons.

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.

Root colonization of plant growth-promoting bacteria is a complex multistep process that is influenced by several factors. For example, during adherence to plant roots, bacteria have to endure reactive oxygen species (ROS) produced by plants. In this study, we report that the global transcriptional regulator Fis is involved in the regulation of ROS-tolerance of Pseudomonas putida and thereby affects barley root colonization. Fis overexpression reduced both ROS-tolerance and adherence to barley roots and activated the transcription of the nuoA-N operon encoding NADH dehydrogenase I, the first enzyme of a membrane-bound electron-transport chain. The nuoA-N knockout mutation in the fis-overexpression background increased the ROS-tolerance and adherence to barley roots. We show that nuoA has two transcriptional start sites located 104 and 377 nucleotides upstream of the coding sequence, indicating the presence of two promoters. The DNase I footprint analysis revealed four Fis binding sites: Fis-nuo1 to Fis-nuo4, situated between these two promoters. Site-directed mutagenesis in a promoter-lacZ reporter and β-galactosidase assay further confirmed direct binding of Fis to Fis-nuo2 and probably to Fis-nuo4 but not to Fis-nuo1 and Fis-nuo3. Additionally, the results implied that Fis binding to Fis-nuo4 could affect transcription of the nuoA-N operon by modification of upstream DNA topology. Moreover, our transposon mutagenesis results indicated that Fis might be involved in the regulation of several alternative ROS detoxification processes utilizing NADH.

In a growth-restricting environment, mutants arise that are able to take over bacterial populations by a process known as adaptive mutation or stationary-phase mutation. This process is best studied in Escherichia coli. The genus Pseudomonas represents one of the largest groups of bacteria able to colonize multiple habitats and to adapt rapidly to new environments. The majority of bacteria including pseudomonads contain a different set of DNA polymerases and DNA repair enzymes than those identified in E. coli. The aim of this review is to provide an overview of the results of studies of mutagenic processes in pseudomonads and to discuss these results in the light of the mechanisms of stationary-phase mutagenesis discovered in E. coli.

The rpoB gene encoding for beta subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif(r)) phenotype of bacteria. Here we have characterized rpoB/Rif(r) system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif(r) clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif(r) mutations characterized for P. aeruginosa grown at 37 degrees C and that characterized for P. putida grown at 30 degrees C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 degrees C. The strong Rif(r) phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 degrees C and expressed weak Rif(r) phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 degrees C and 37 degrees C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif(r) mutants from selective plates are critical when the rpoB/Rif(r) test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.

Although many nitroaromatic compounds have been in nature for only a few decades, bacteria have already evolved the ability to metabolize them. Both horizontal transfer of genes and mutagenesis induced under stressful conditions might facilitate evolution of new catabolic pathways. Nitrotoluene degradation pathways are supposedly derived from an ancestral naphthalene degradation pathway. The 2-nitrotoluene degradation genes in Acidovorax sp. strain JS42 are controlled by the transcriptional activator NtdR, which differs from NagR, the activator of the naphthalene degradation operon in Ralstonia sp. strain U2, by only five amino acids. Both regulators respond to salicylate, an intermediate of naphthalene degradation, but NtdR also recognizes a wide range of nitroaromatic compounds. In this issue of Molecular Microbiology, Ju et al. present results of site-directed mutagenesis of NtdR and NagR and show how the nitrotoluene-responsive regulator NtdR can be generated from a NagR-like ancestor by only a few mutations. The reconstructed hypothetical pathway for the evolution of NtdR from NagR demonstrates stepwise broadening of the effector range of the evolving protein without loss of the original activity. These results provide strong evidence for the idea that promiscuity of proteins is an important step in the evolution of new functions.

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.

ColRS two-component system is well conserved in pseudomonads, but its exact role has remained obscure. Here, we report that Pseudomonas putida deficient in ColR experiences serious carbon source-specific stress that leads to the lysis of a subpopulation of bacteria growing on solid glucose medium. We observed that on glucose medium colR-deficient bacteria aggregated, produced a Congo Red-binding substance and had enhanced membrane permeability. Detection of a large amount of cytoplasmic beta-galactosidase and other proteins as well as chromosomal DNA in the growth medium of a colR mutant indicated that cell lysis took place if ColR was absent. Investigation of colony morphology revealed concavities in the centre of the colonies of colR mutant suggesting that cell lysis occurred mainly in the areas of the highest cell density. Analysis of bacteria at a single cell level by flow cytometry showed that population of glucose-grown colR-deficient cells was heterogeneous. In addition to the wild type-like population, we detected a subpopulation of cells with damaged membrane permeable to propidium iodide. Interestingly, inactivation of oprB1 encoding a glucose porin eliminated the cell lysis as well as autoaggregation and membrane leakiness of a colR mutant indicating that glucose influx could be responsible for membrane stress in the absence of ColRS system.

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida . The genome of this organism contains two uvrA genes, uvrA and uvrA2 . Genetic studies on the effects of uvrA , uvrA2 , uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida . However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB , uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2 , uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida —besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.

Several bacterial species carry in their genomes a so-called "mutagenesis" gene cluster encoding ImuB which is similar to Y-family DNA polymerases, and DnaE2 related to the catalytic subunit DnaE of Pol III. Y-family DNA polymerases are known to be involved in stationary-phase mutagenesis and DnaE2 homologues characterized so far have expressed a mutator phenotype. In this study, we raised a question about the involvement of ImuB and DnaE2 in stationary-phase mutagenesis. Here, we show that Pseudomonas putida ImuB and DnaE2 have antagonistic effects on stationary-phase mutagenesis. ImuB facilitated accumulation of stationary-phase mutants up to two-fold. In contrast to that, DnaE2 had no significant effect on emergence of 1-bp deletion mutants and moreover, it acted as an anti-mutator in accumulation of base substitution mutants in starving bacteria. Similar antagonistic effects of DnaE2 and ImuB on mutagenesis appeared also in UV-mutagenesis study. This data distinguishes the DnaE2 of P. putida from its homologues studied in other organisms.

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.

Summary Bacteria use two-component signal transduction pathways to sense both extracellular and intracellular environment and to coordinate cellular events according to changing conditions. Adaptation can be either physiological or genetical. Here, we present evidence that a genome reorganization process such as transposition can be controlled by certain environmental cues sensed by a two-component signal transduction system. We demonstrate that transposition-dependent accumulation of phenol-utilizing mutants is severely decreased in Pseudomonas putida defective in a two-component system colRS. Translocation of Tn4652 is decreased both in colR- and colS-defective strains, indicating that signal transduction from a histidine kinase ColS to a response regulator ColR is necessary for the activation of Tn4652 in bacteria starving on phenol. However, overexpression of ColR in a colS-defective strain restores Tn4652 transposition, suggesting that absence of the signal from ColS can be compensated by an elevated amount of ColR. In vitro analysis of purified ColR and ColS proteins evidenced that they constitute a functional phosphorelay. Site-directed mutagenesis revealed that a conserved H221 can be the phosphoryl-accepting residue in ColS and that aspartate residues D8 and D51 of ColR are necessary for the phosphotransfer from ColS to ColR. To our knowledge, Tn4652 is the first bacterial transposon regulated by a two-component system. This finding indicates that transpositional activity can respond to signals sensed and processed by the host.

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas , Na 2 -succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to ∼35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas . Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.

Summary Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn 4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al ., 2001, J Bacteriol 183: 5445‐5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida , might contribute to the transposition of Tn 4652 as well. Here, we demonstrate that transposition of Tn 4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn 4652 occurs in a P. putida ihfA -defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain. This indicates that the amount of IHF is a bottleneck in Tn 4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro , transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase.

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 .

Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source.