Title: Study on the variability of a tandem repeat region in the Escherichia coli tolA gene and its role in stress adaptation
Other Titles: Studie van de variabiliteit en rol in stressadaptatie van een tandem herhaling in het Escherichia coli tolA gen
Authors: Zhou, Kai; S0196456
Issue Date: 15-Jan-2013
Abstract: DNA tandem repeats (TRs), also designated as satellite DNA, are an array of consecutive sequence of nucleotides that are repeated in a head-to-tail manner. DNA sequences harboring TRs exist in both prokaryotic and eukaryotic genomes, and generally are considered to be hypermutable loci in which the TR copy number can increase or decrease as a result of strand-slippage replication or recombination. Variable intergenic/intragenic TRs are considered as an adaptation strategy and believed to facilitate most bacteria in coping with dynamic and complex environmental stresses. Here we report a variable tandem repeat region uncovered in the Escherichia coli (E. coli) tolA gene, and further reveal the role of that region in the stress tolerance of E. coli. Through analyzing the genome sequence of E. coli MG1655 by Tandem Repeat Finder (TRF) program, the tolA gene was selected as the target of this study. Thirteen imperfect repeats were identified in the repetitive region of MG1655 tolA. Moreover, in silico analysis of 123 E. coli tolA sequences from Genbank and PCR analysis of the tolA TR region from 111 additional E. coli strains revealed that this TR region is highly variable. Nine different TR sizes with 8 up to 16 repeat units were found in the in silico analysis, and 6 of these were also found by PCR analysis. The 13-unit TR emerged as the predominant type from both approaches (47.2% and 86.5%, respectively). Remarkably, TRs in pathogenic strains appeared to be more variable than those in non-pathogens. To demonstrate the occurrence of TR variation in a clonal population, a selection system for TR deletion events was constructed by inserting the 13-unit TR region of MG1655 in frame into a plasmid-borne chloramphenicol acetyltransferase (cat) gene. The resulting cat gene no longer confers chloramphenicol resistance, unless the insert size is reduced by TR contraction. Using this system, Cm resistant revertants with a TR contraction were recovered at a frequency of 1.1×10-7, and contraction was shown to be recA dependent and enhanced in a DNA repair deficient mutS background. The biological impact of TR variation was examined by comparing the ability of a set of synthetic chromosomal tolA variants with in frame TRs comprised of 2 to 39 repeat units to rescue the altered susceptibility of an E. coli ΔtolA mutant to deoxycholic acid (DOC), sodium dodecyl sulfate (SDS), hyperosmolarity and infection with filamentous bacteriophage. Interestingly, although each of the TolA variants was able to at least partly rescue the ΔtolA mutant, the extent was clearly dependent on both the repeat number and the type of stress imposed, indicating the existence of opposing selective forces with regard to the optimal TR copy number. More specifically, DOC tolerance increased with an increasing number of TR units from TolA2TR to TolA39TR, and the plating efficiencies of the corresponding strains increased over a range of four orders of magnitude. In contrast to DOC sensitivity, all TolA TR variants complemented sensitivity to SDS and hyperosmolarity equally well and up to wild-type level. However, when hyperosmolarity was combined with low pH, the TolA6TR, TolA8TR, and TolA13TR strains outperformed the other variants carrying either lower or higher TR copy numbers. Moreover, all TolA variants were significantly less susceptible to filamentous phage fd than the strain expressing wild-type TolA (i.e. TolA13TR). These findings underscore the intricate phenotypical changes brought about by TR variation of TolA. Finally, to study the dynamics of TR variation in E. coli tolA, we specifically designed a selection system by introducing two stop codons in one of the TR units of tolA. This allowed us to demonstrate that tolA TR variations occur in a clonal wild-type population at a frequency of at least 6.9 × 10-5, thereby proving that tolA TRs can dynamically change on short evolutionary time scales. It should be noted here that this selection system only detects TR contractions which preserve the reading frame.. Subsequently, we could also demonstrate that TR contractions of tolA are RecA dependent and enhanced in a DNA repair deficient uvrD background. In summary, we revealed a highly variable TR region in the tolA gene of E. coli. Moreover, we demonstrated the pleiotropic phenotypic effects of TR copy number variations in this gene, and the existence of environmental stress factors that constitute a possible selective force able to drive TR rearrangements. Recombination-dependent TR rearrangements in tolA could be detected in clonal populations by both a plasmid- and a chromosome-based selection system, providing further support for a role of TR regions as hypermutable contingency loci that allow rapid and flexible adaptation to complex environmental conditions. Being the first to systematically study an intragenic minisatellite in E. coli, this work has also contributed to a more detailed insight in the role of hypervariable contingency loci in E. coli adaptation.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre for Food and Microbial Technology

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