Mutation Frequency and Spectrum of Mutations Vary at Different Chromosomal Positions of Pseudomonas putida

Abstract

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.

Adaptive Divergence in Experimental Populations ofPseudomonas fluorescens. V. Insight into the Niche Specialist Fuzzy Spreader Compels Revision of the ModelPseudomonasRadiation

“…A:T substitution) but also idiosyncrasies. Indeed it can be difficult to compare mutational spectra between species, and even between genes within the same species, since mutation rates and classes are affected by gene-specific parameters such as distance of the gene from the replication origin, the direction of transcription relative to the passage of a replication fork, and the level of transcription (reviewed by Ochman 2003;Juurik et al 2012). Various studies have reported mutational spectra for single genes encoding proteins constrained by functions essential for viability (Garibyan et al 2003;Wolff et al 2004), making it difficult to draw conclusions about the relative occurrence of SNPs and indels and the six classes of substitution.…”

“…A:T substitution) but also idiosyncrasies. Indeed it can be difficult to compare mutational spectra between species, and even between genes within the same species, since mutation rates and classes are affected by gene-specific parameters such as distance of the gene from the replication origin, the direction of transcription relative to the passage of a replication fork, and the level of transcription (reviewed by Ochman 2003;Juurik et al 2012).…”

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

“…First, the NotI-cleaved lacI q -Ptac fragment from the pUC18NotlacI [65] plasmid was cloned into the NotI site of the plasmid pBAM1, resulting in plasmid pBAMlacItac. Then, the genomic DNA of P. putida PaW85 was amplified with primers ligDHindIII and ligD2HindIII to clone the ligD gene.…”

“…The chromosomal location of the lacI q -P tac -ligD expression cassette was determined twice. In the first round of PCR, primer BamI and arbitrary primer ARB6 [65] were used. Second-round PCR was performed with primer ME-I-uus2 and arbitrary primer ARB2 [65].…”

“…In the first round of PCR, primer BamI and arbitrary primer ARB6 [65] were used. Second-round PCR was performed with primer ME-I-uus2 and arbitrary primer ARB2 [65]. The DNA sequencing reactions were analyzed with the Applied Biosystems 3730xl DNA sequencer.…”