Effect of transcription of mutational target gene on mutation frequency.

<p>Influence of transcription was studied in growing bacteria. Average number of Phe⁺ mutants accumulated per 1×10⁷ viable cells is shown with 95%-confidence intervals. To compare the frequency of Phe⁺ mutants in growing <i>P. putida</i>, bacteria were grown in the presence or absence of 1 mM IPTG and the number of mutants emerged onto phenol minimal plates on day 8 were counted. In total, at least 30 independent cultures were examined in three parallel experiments for each strain.</p

Spectrum of Phe⁺ mutations in <i>P. putida</i> strains carrying the phe-lacI test system at various chromosomal positions.

a<p>Frequency of mutation per site calculated per total number of Phe⁺ mutants accumulated per 1×10⁷cells for day 7 is shown in parentheses.</p>b<p>Positions of nucleotides are given in respect to the <i>lacI</i> coding sequence so that the first translated codon GTG is at position 1–3. Mutations at positions −345 to −354 alter the LacI operator sequence.</p

Mutation Frequency and Spectrum of Mutations Vary at Different Chromosomal Positions of <em>Pseudomonas putida</em>

<div><p>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 <em>Pseudomonas putida</em> 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 IS<em>1411</em> 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 <em>vice versa</em>, 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 IS<em>1411</em> 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 <em>P. putida</em> 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.</p> </div

Time-dependent appearance of mutational hot spots in <i>P. putida</i> strains carrying the phe-lacI test system.

<p>Time-dependent appearance of mutational hot spots in <i>P. putida</i> strains carrying the phe-lacI test system.</p

The frequency of accumulation of Phe⁺ mutants in <i>P. putida</i> strains carrying the pheA+C test system at different chromosomal locations^a.

a<p>Average numbers of Phe⁺ mutants per day and total number of mutants for day 9 calculated per 1×10⁷ cells with 95% confidence intervals are shown. The results with at least 6 independent populations of each strain are presented.</p>b<p>Strains carrying the mutational target gene opposite to the direction of the movement of replisome in the chromosome are indicated in bold.</p>c<p>Based on the comparison of the frequency of Phe⁺ mutations accumulated for day 9 statistically significantly different (<i>P</i><0.05) homogeneity groups (a–d) appeared.</p

The location of the P<i>_tac</i> promoter and the LacI operator sequence in the phe-lacI test system.

<p>−10 and −35 hexamers of the P<i>_tac</i> promoter are boxed and the operator sequence is underlined. The nucleotide positions of the operator sequence are given from translational initiator codon GTG of the <i>lacI</i> gene.</p

Chromosomal location of the randomly inserted test system in various <i>P. putida</i> strains.

<p>The locations of the phe-lacI test system detecting mutations which inactivate LacI repressor are shown in panel A (designated as lacI) and the locations of the pheA+C test system detecting only frameshift mutations are shown in panel B. The black arrows demonstrate the direction of transcription of the <i>P. putida</i> chromosomal genes containing the insertions of the test system. When the transcribed strand is the leading strand template for replication, the RNA polymerase and the replisome move in the same direction (co-directional orientation); when the transcribed strand is the lagging strand template, the RNA polymerase and the replisome converge (head-on orientations). The direction of transcription of the mutational target genes (the <i>lacI</i> gene in the phe-lacI test system and the <i>pheA</i> gene in the pheA+C test system) in different <i>P. putida</i> strains is indicated by green or red arrows. The red arrows designate head-on orientations of the transcription of the mutational target gene and the movement of the replisome in the chromosome and the green arrows point to co-directional transcription and replication. The replication of the chromosome starts at <i>oriC</i> region (indicated by two-directional arrow) and terminates at <i>dif</i> sites. Location of <i>P. putida dif</i> sequence is according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048511#pone.0048511-Carnoy1" target="_blank">[87]</a>.</p