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

Highlights

• 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.

Abstract

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.

Introduction

Genetic changes in microbial populations can occur fast through the acquisition and incorporation of foreign DNA or through mutation. Point mutations, in general, occur as a result of DNA damage and via errors introduced during DNA replication. Cells have multiple mechanisms for coping with DNA damage – DNA repair and DNA damage tolerance by translesion DNA synthesis (TLS) carried out by specialized DNA polymerases, e.g., Pol IV and Pol V [[1], [2], [3]]. DNA synthesis occurring during DNA repair may contribute to mutagenesis [4]. For instance, in E. coli the process of double-strand break repair (DSBR) by homologous recombination is switched from a high-fidelity pathway to an error-prone pathway due to the involvement of DNA damage-induced error-prone TLS DNA polymerases, thereby facilitating occurrence of mutations under stressful conditions [[5], [6], [7], [8]]. Several studies have indicated that the network of factors affecting mutation frequency in bacteria could be much more sophisticated than initially presumed. For example, Al Mamun et al. [9] found that stress-induced mutagenesis in E. coli is affected by at least 93 genes, including several upstream activators of RpoS, RpoE and SOS stress responses. Thus, defining new factors affecting mutation rates is highly important for understanding the mechanisms of evolutionary processes in nature.

Besides maintaining genome integrity, homologous recombination (HR) can induce genetic rearrangements in bacteria. In addition, HR is an essential process for the integration of horizontally transferred genes into their new bacterial host genome [10]. There are several factors affecting the frequency of HR either positively or negatively. HR is less efficient when the recombining DNAs are not identical. Recombination between DNA molecules that are not entirely identical generates mismatches within the heteroduplex region of the strand exchange products. These mismatches can be detected by DNA mismatch repair (MMR) proteins such as MutS and MutL which, according to the initial studies by [11] and [12], block and/or abort mismatched recombination intermediates. In addition, HR is decreased in bacteria by the action of UvrD helicase, an enzyme participating both in MMR and nucleotide excision repair (NER) pathways [13,14], due to its ability to remove RecA filaments from DNA [[15], [16], [17]].

It has been demonstrated in a variety of experimental systems that HR is stimulated by DNA-damaging treatments [18]. There are two major HR pathways in bacteria, the RecBCD and the RecFOR pathways, both of which depend upon the RecA protein [[19], [20], [21]]. The RecBCD pathway operates at DNA double-strand breaks (DSBs) and utilizes RecBCD helicase-nuclease responsible for generating ssDNA and loading RecA [22]. The RecFOR pathway operates at single-stranded DNA (ssDNA) regions not associated with DSBs, and in this case the RecF, RecO and RecR proteins facilitate the loading of RecA protein onto single-stranded DNA (ssDNA) via the displacement of single-strand-binding protein SSB [23]. RecA promotes the central step of HR, aligning and pairing of two DNA molecules, and promoting a strand-exchange in which the complementary strand of the DNA duplex is transferred to the originally bound ssDNA [16,24]. The search for homology occurs by combining one-dimensional sliding along dsDNA with hopping between sites that are physically close within a genome, and jumping between sites that are distant within the genome but physically close, whereas a sufficient threshold (8 bases) stabilizes the interaction and permits DNA strand exchange process to be started [24,25].

Nucleoid-associated proteins (NAPs) such as HU, IHF, H-NS, Fis contribute to the dynamic nature of nucleoid structure by bending, wrapping or bridging the genomic DNA [26,27]. Integration Host Factor (IHF) was initially identified as the protein essential for the site-specific integrative recombination of phage lambda in E. coli [28,29]. Subsequently, it was found that IHF affects many cellular functions including a variety of site-specific recombination events, transposition, chromosome replication initiation, and gene expression [27]. Recently, it was demonstrated that IHF promotes site-specific integration of foreign DNA at the CRISPR locus by inducing sharp bend at the leader sequence, allowing the Cas1-Cas2 integrase to catalyse the integration reaction [30].

We have previously shown that HR is elevated during the prolonged starvation of soil bacterium Pseudomonas putida in the presence of phenol which is a potential carbon source and stressor for soil bacteria. This was demonstrated by employing an assay which enables detection of HR events between a broad-host-range RK2-derived plasmid and the bacterial chromosome, which results in restoring the expression of the phenol monooxygenase gene pheA [31]. HR between chromosomal loci, on the contrary, took place mainly in growing cells of P. putida; however, it occurred at high frequency under starvation conditions when the NER enzymes UvrA or UvrB were missing [32]. Our previous studies also demonstrated that the chromosomal location of the HR target influences the frequency and dynamics of HR events in P. putida [31,32]. A bioinformatic screen for sequence determinants affecting recombination through genome revealed that chromosomal DNA regions of P. putida which flanked the test system in the strains exhibiting lower HR frequency were strongly enriched in binding sites for at least three NAPs (FIS, IHF and MvaT/MvaU) if compared to those which expressed higher frequency of HR [31]. Based on the results of the in silico analysis we hypothesized 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 influence the frequency of HR. In order to examine the effects of individual NAPs on the frequency of HR, we focused on one particular NAP, IHF, and measured whether the absence or overexpression of IHF could affect the frequency of HR. As we have previously observed that the frequency of point mutations can also vary at different chromosomal positions of P. putida [33], the effect of IHF on the occurrence of point mutations was also investigated. Our results suggest that IHF facilitates both the HR processes and the occurrence of point mutations in P. putida.