Mechanisms of adaptive mutations in bacteria

University dissertation from Stockholm : Karolinska Institutet, Microbiology and Tumor Biology Center (MTC)

Abstract: Bacteria have the ability to adapt to various challenging environments. The rate, extent and consequences of bacterial adaptation depend on both microbial and environmental factors. This thesis analyses some of the mechanisms bacteria can employ to adapt to novel environments such as the presence of antibiotics, during infection of a host, and when exposed to growth-limiting conditions. Bacterial adaptation to antibiotics is conferred by mutations that usually modify essential cellular functions. In the absence of the drug, the adaptive mutations often confer a fitness cost measured as a decrease in growth rate and/or virulence. Bacteria can in many cases compensate for such fitness costs by acquiring second-site mutations. In Pseudomonas aeruginosa we showed that high levels of quinolone resistance are associated with high fitness costs. However, no-cost and compensatory mutations are common in strains resistant to low levels of quinolones. Decreased fitness, conferred by single resistance mutations in gyrA, was associated with decreased DNA supercoiling. Fitness and supercoiling could be restored to wild-type level without loss of resistance. The supply of mutations might be limiting during adaptation to novel selections. We showed that the rate of adaptation of Salmonella enterica serovar Typhimurium to mice could be accelerated by increases in the mutation supply, mediated by increased mutation rate or population size. An increased mutation rate was however associated with accumulation of deleterious mutations that decreased fitness in secondary environments. Bacterial adaptation to limiting growth conditions was studied in a genetic system that employs a strain with a mutant lac allele on plasmid F'lac128. This strain can adapt to growth on lactose by amplification of the mutant lac allele followed by mutation to Lac+. To further investigate the mechanisms behind this adaptation we identified and compared duplication join points detected before and after selection for gene amplification. We showed that large pre-existing lac duplications, with long sequence homologies, are common in the unselected population. In contrast, after selection for growth on lactose, most duplications were smaller with short sequence homologies at the join points. This discrepancy can be explained by the inefficiency of large duplications to amplify and form Lac+ revertants compared to shorter amplification units. Unique short homology join points could be detected in the unselected population although they were rare. Some short homology duplications may arise from remodeling of large duplications to yield a short amplicon that can amplify more efficiently. A subset of Lac+ revertants was associated with mutation accumulation in unselected genes. We showed that the unselected mutations were only detected in cells where the error-prone polymerase DinB was co-amplified with lac. Thus, the associated mutagenesis is a side effect when dinB and lac are within the same amplified unit. Taken together, these results further confirm that Lac+ revertants do not arise from a stress-induced hypermutable state as has been suggested. Instead Lac+ revertants arise by standard genetic events and stress functions only as an agent of natural selection, favoring growth of cells with a lac amplification.

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