Escape from growth restriction in small colony variants of Salmonella typhimurium by gene amplification and mutation

Abstract

SummaryAntibiotic resistance in bacteria is generally associated with fitness costs that often can be reduced by second-site compensatory mutations. Here, we examined how a protamine-resistant small colony variant of Salmonella typhimurium adapts to the growth reduction conferred by a resistance mutation in hemC (encoding a haem-biosynthesis enzyme). We show that adaptation occurs in a multi-step process where fitness is successively increased. Thus, the initial adaptive response was selection for an unstable gene amplification of the mutant hemC gene that provided a small fitness increase. Fitness was increased further by a mutation that restored HemC function in one gene copy, relaxing selection for the amplification. Subsequently, the amplification segregated back to the haploid state and even higher fitness. The end result was in most cases mutant strains with a hemC sequence different from that of the wild-type strain. These findings suggest that gene amplification facilitates adaptive evolution. A higher gene dosage increases the target size for compensatory mutations and improves fitness of the cell, thereby allowing an increase in the population size, further increasing the probability of a subsequent stable mutation. Our results provide a novel genetic basis for growth compensation in small colony variants.

Hypermutation and stress adaptation in bacteria

“…Gene amplification preceding the generation of stable adaptive mutations has also been shown in the emergence of antibiotic resistance mutations (Sun et al 2009; see also the reviews by Lindgren and Andersson 2009). Recently, Pranting and Andersson (2011) reported that a protamine resistant, growth restricted mutant (small colony variant) of S. typhimurium escaped from growth restriction by amplifying the mutant gene copy number (partial escape) and acquiring a compensatory mutation, either replacing the original lesion or adjacent to it, in one of the amplified gene copies, followed by segregation to the single gene copy state (full escape) (see also the commentary by Roth (2011)). It will not be surprising if the many mechanisms discovered so far as well as other, yet unidentified ones, may eventually turn out to be not mutually exclusive.…”

Antimicrobials as promoters of genetic variation

“…Once these mutations are acquired, selection favours the elimination of the extra copies. Several studies have provided evidence of this process in a variety of genetic systems [88][89][90][91]. In addition, genome-wide overexpression screening for resistance determinants on E. coli revealed that bacterial genomes present a disturbing potential for adaptive amplification [92,93].…”

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

“…There is considerable evidence that this amplification-under-selection model operates in many situations (Pranting and Andersson 2011; Roth 2011; Elde et al 2012; Näsvall et al 2012; Quinones-Soto et al 2012), including the Salmonella version of the Cairns system. Amplification under selection can also explain the selective evolution of new genes (Bergthorsson et al 2007), a process that has been experimentally demonstrated (Näsvall et al 2012).…”