Convergent evolution toward an improved growth rate and a reduced resistance range inProchlorococcusstrains resistant to phage

Prochlorococcusis an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently inProchlorococcusand is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistantProchlorococcusstrains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations inProchlorococcuspopulations, further augmenting the complexity of the interaction network betweenProchlorococcusand its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistantProchlorococcuscells in nature that are growing close to their maximal intrinsic growth rates.

Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC

ABSTRACTWe compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ΔmutS)P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 μg/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 μg/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showedampCoverexpression, which was caused bydacBorampRmutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-typeampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in aP. aeruginosamutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.

Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

A novel mutagenesis technique using error-prone DNA polymerase δ (polδ), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.

Rate and effects of spontaneous mutations that affect fitness in mutator Escherichia coli

Knowledge of the mutational parameters that affect the evolution of organisms is of key importance in understanding the evolution of several characteristics of many natural populations, including recombination and mutation rates. In this study, we estimated the rate and mean effect of spontaneous mutations that affect fitness in a mutator strain of Escherichia coli and review some of the estimation methods associated with mutation accumulation (MA) experiments. We performed an MA experiment where we followed the evolution of 50 independent mutator lines that were subjected to repeated bottlenecks of a single individual for approximately 1150 generations. From the decline in mean fitness and the increase in variance between lines, we estimated a minimum mutation rate to deleterious mutations of 0.005 (±0.001 with 95% confidence) and a maximum mean fitness effect per deleterious mutation of 0.03 (±0.01 with 95% confidence). We also show that any beneficial mutations that occur during the MA experiment have a small effect on the estimate of the rate and effect of deleterious mutations, unless their rate is extremely large. Extrapolating our results to the wild-type mutation rate, we find that our estimate of the mutational effects is slightly larger and the inferred deleterious mutation rate slightly lower than previous estimates obtained for non-mutator E. coli .

Shotgun Cloning of Transposon Insertions in the Genome ofCaenorhabditis elegans

We present a strategy to identify and map large numbers of transposon insertions in the genome ofCaenorhabditis elegans. Our approach makes use of the mutator strainmut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence ofC. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predictedC. elegansgenes. The strategy presented provides an approach to isolate insertions of natural transposable elements in manyC. elegansgenes and to create a large-scale collection ofC. elegansmutants.