Integration Host Factor IHF facilitates homologous recombination and mutagenic processes in Pseudomonas putida.

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.

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.

Mutation frequency and spectrum of mutations vary at different chromosomal positions of Pseudomonas putida.

It is still an open question whether mutation rate can vary across the bacterial chromosome. In this study, the occurrence of mutations within the same mutational target sequences at different chromosomal locations of Pseudomonas putida was monitored. For that purpose we constructed two mutation detection systems, one for monitoring the occurrence of a broad spectrum of mutations and transposition of IS element IS1411 inactivating LacI repressor, and another for detecting 1-bp deletions. Our results revealed that both the mutation frequency and the spectrum of mutations vary at different chromosomal positions. We observed higher mutation frequencies when the direction of transcription of the mutational target gene was opposite to the direction of replisome movement in the chromosome and vice versa, lower mutation frequency was accompanied with co-directional transcription and replication. Additionally, asymmetry of frameshift mutagenesis at homopolymeric and repetitive sequences during the leading and lagging-strand replication was found. The transposition frequency of IS1411 was also affected by the chromosomal location of the target site, which implies that regional differences in chromosomal topology may influence transposition of this mobile element. The occurrence of mutations in the P. putida chromosome was investigated both in growing and in stationary-phase bacteria. We found that the appearance of certain mutational hot spots is strongly affected by the chromosomal location of the mutational target sequence especially in growing bacteria. Also, artificial increasing transcription of the mutational target gene elevated the frequency of mutations in growing bacteria.

Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target.

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.