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Highlights • TCR factor Mfd and helicase UvrD help to tolerate DNA damage. • Mfd-mediated TCR contributes to UV- and stationary-phase mutagenesis in P. putida. • The absence of UvrD does not entirely disable NER and MMR in P. putida. • UvrD is essential to maintain low mutant frequency in P. putida. Abstract 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 Δ uvrD 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. [ABSTRACT FROM AUTHOR]

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 Rif r 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 Rifr 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. [Copyright &y& Elsevier]

Abstract: The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rifr) phenotype of bacteria. Here we have characterized rpoB/Rifr 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 48h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rifr 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 Rifr mutations characterized for P. aeruginosa grown at 37°C and that characterized for P. putida grown at 30°C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30°C. The strong Rifr 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°C and expressed weak Rifr phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30°C and 37°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 Rifr mutants from selective plates are critical when the rpoB/Rifr test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures. [Copyright &y& Elsevier]

[Display omitted] • Survival and degradation efficacy of Ofp tagged ICTN13 was tested against phenol. • Bacterial species was encapsulated in alginate beads that enhanced longevity. • Encapsulated strain showed higher degradation efficacy in comparison to free cells. • Fluorescence helped tracking the survival of ofp tagged ICTN13 bacteria. • The obtained information allows evaluation of future remediation strategies. 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 oleovorans ICTN13 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. [ABSTRACT FROM AUTHOR]

• The frequency of HR and point mutations are facilitated by IHF. • The effect of IHF on HR depends on the chromosomal position of the target DNA. • Mutational hot spots of the same DNA sequence vary at different locations. 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. [ABSTRACT FROM AUTHOR]