EVOLUTIONARY REVERSALS OF ANTIBIOTIC RESISTANCE IN EXPERIMENTAL POPULATIONS OF PSEUDOMONAS AERUGINOSA

Antibiotic resistance mutations are accompanied by a fitness cost, and two mechanisms allow bacteria to adapt to this cost once antibiotic use is halted. First, it is possible for resistance to revert; second, it is possible for bacteria to adapt to the cost of resistance by compensatory mutations. Unfortunately, reversion to antibiotic sensitivity is rare, but the underlying factors that prevent reversion remain obscure. Here, we directly study the evolutionary dynamics of reversion by experimentally mimicking reversion mutations—sensitives—in populations of rifampicin‐resistant Pseudomonas aeruginosa. We show that, in our populations, most sensitives are lost due to genetic drift when they are rare. However, clonal interference from lineages carrying compensatory mutations causes a dramatic increase in the time to fixation of sensitives that escape genetic drift, and mutations surpassing the sensitives’ fitness are capable of driving transiently common sensitive lineages to extinction. Crucially, we show that the constraints on reversion arising from clonal interference are determined by the potential for compensatory adaptation of the resistant population. Although the cost of resistance provides the incentive for reversion, our study demonstrates that both the cost of resistance and the intrinsic evolvability of resistant populations interact to determine the rate and likelihood of reversion.     

Positive Epistasis Drives the Acquisition of Multidrug Resistance

The evolution of multiple antibiotic resistance is an increasing global problem. Resistance mutations are known to impair fitness, and the evolution of resistance to multiple drugs depends both on their costs individually and on how they interact—epistasis. Information on the level of epistasis between antibiotic resistance mutations is of key importance to understanding epistasis amongst deleterious alleles, a key theoretical question, and to improving public health measures. Here we show that in an antibiotic-free environment the cost of multiple resistance is smaller than expected, a signature of pervasive positive epistasis among alleles that confer resistance to antibiotics. Competition assays reveal that the cost of resistance to a given antibiotic is dependent on the presence of resistance alleles for other antibiotics. Surprisingly we find that a significant fraction of resistant mutations can be beneficial in certain resistant genetic backgrounds, that some double resistances entail no measurable cost, and that some allelic combinations are hotspots for rapid compensation. These results provide additional insight as to why multi-resistant bacteria are so prevalent and reveal an extra layer of complexity on epistatic patterns previously unrecognized, since it is hidden in genome-wide studies of genetic interactions using gene knockouts.     

R-loop-dependent replication and genomic instability in bacteria

DNA replication, the faithful copying of genetic material, must be tightly regulated to produce daughter cells with intact copies of the chromosome(s). This regulated replication is initiated by binding of specific proteins at replication origins, such as DnaA to oriC in bacteria. However, unregulated replication can sometimes be initiated at other sites, which can threaten genomic stability. One of the first systems of unregulated replication to be described is the one activated in Escherichia coli mutants lacking RNase HI (rnhA). In fact, rnhA mutants can replicate their chromosomes in a DnaA- and oriC-independent process. Because this replication occurs in cells lacking RNase HI, it is proposed that RNA from R-loops is used as a DNA polymerase primer. Replication from R-loops has recently attracted increased attention due to the advent of DNA:RNA hybrid immunoprecipitation coupled with high-throughput DNA sequencing that revealed the high prevalence of R-loop formation in many organisms, and the demonstration that R-loops can severely threaten genomic stability. Although R-loops have been linked to genomic instability mostly via replication stress, evidence of their toxic effects via unregulated replication has also been presented. Replication from R-loops may also beneficially trigger stress-induced mutagenesis (SIM) that assists bacterial adaptation to stress. Here, we describe the cis- and trans-acting elements involved in R-loop-dependent replication in bacteria, with an emphasis on new data obtained with type 1A topoisomerase mutants and new available technologies. Furthermore, we discuss about the mechanism(s) by which R-loops can reshape the genome with both negative and positive outcomes.     

Decreased thermal niche breadth as a trade-off of antibiotic resistance

Evolutionary theory predicts that adaptations, including antibiotic resistance, should come with associated fitness costs; yet, many resistance mutations seemingly contradict this prediction by inducing no growth rate deficit. However, most growth assays comparing sensitive and resistant strains have been performed under a narrow range of environmental conditions, which do not reflect the variety of contexts that a pathogenic bacterium might encounter when causing infection. We hypothesized that reduced niche breadth, defined as diminished growth across a diversity of environments, can be a cost of antibiotic resistance. Specifically, we test whether chloramphenicol-resistant Escherichia coli incur disproportionate growth deficits in novel thermal conditions. Here we show that chloramphenicol-resistant bacteria have greater fitness costs at novel temperatures than their antibiotic-sensitive ancestors. In several cases, we observed no resistance cost in growth rate at the historic temperature but saw diminished growth at warmer and colder temperatures. These results were consistent across various genetic mechanisms of resistance. Thus, we propose that decreased thermal niche breadth is an under-documented fitness cost of antibiotic resistance. Furthermore, these results demonstrate that the cost of antibiotic resistance shifts rapidly as the environment changes; these context-dependent resistance costs should select for the rapid gain and loss of resistance as an evolutionary strategy.Subject terms: Bacterial evolution, Microbial ecology, Antibiotics     

细菌中RNase HI介导RNA降解的研究进展

在细菌细胞中,为了维持基因组稳定和正常的生命活动,RNase HI通常以降解RNA/DNA杂合链中RNA的方式来防止复制中引物的积累以及转录中R环的形成。RNase HI对底物的识别主要依赖于DNA与RNA结合槽,对底物的催化主要依赖于DEDD基序和位于活性位点附近柔性环中的一个组氨酸。以Mg²⁺为代表的金属离子在催化过程中发挥了至关重要的作用。杂交双链中ssDNA突出部分的类型决定了RNase HI的作用模式：在没有突出或在ssDNA的5′端存在突出部分的情况下,RNase HI作为一种非序列特异性核酸内切酶随机地降解RNA;当ssDNA的3′端存在突出部分时,RNase HI依靠5′核酸外切酶活性对RNA进行连续切割。RNase HI、Rep、DinG和UvrD通过与单链DNA结合蛋白(single-stranded DNA-binding protein, SSB)的C端尾部的6个残基相互作用被招募到复制叉附近,并可能以协作的方式解决复制-转录冲突。RNaseHI的缺失或活性降低将引起DNA结构不稳定、基因突变、转录装置回溯和复制不协调等一系列有害后果。RN...     

A role for the Saccharomyces cerevisiae Rtt109 histone acetyltransferase in R-loop homeostasis and associated genome instability

The stability of the genome is occasionally challenged by the formation of DNA–RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA–RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On the one hand, Rtt109 prevents DNA–RNA hybridization by the acetylation of histone H3 lysines 14 and 23 and, on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA–RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability.     

Out of Balance: R-loops in Human Disease

R-loops are cellular structures composed of an RNA/DNA hybrid, which is formed when the RNA hybridises to a complementary DNA strand and a displaced single-stranded DNA. R-loops have been detected in various organisms from bacteria to mammals and play crucial roles in regulating gene expression, DNA and histone modifications, immunoglobulin class switch recombination, DNA replication, and genome stability. Recent evidence suggests that R-loops are also involved in molecular mechanisms of neurological diseases and cancer. In addition, mutations in factors implicated in R-loop biology, such as RNase H and SETX (senataxin), lead to devastating human neurodegenerative disorders, highlighting the importance of correctly regulating the level of R-loops in human cells. In this review we summarise current advances in this field, with a particular focus on diseases associated with dysregulation of R-loop structures. We also discuss potential therapeutic approaches for such diseases and highlight future research directions.     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

Rapid Decline of Ceftazidime Resistance in Antibiotic-Free and Sublethal Environments Is Contingent on Genetic Background

Trade-offs of antibiotic resistance evolution, such as fitness cost and collateral sensitivity (CS), could be exploited to drive evolution toward antibiotic susceptibility. Decline of resistance may occur when resistance to other drug leads to CS to the first one and when compensatory mutations, or genetic reversion of the original ones, reduce fitness cost. Here we describe the impact of antibiotic-free and sublethal environments on declining ceftazidime resistance in different Pseudomonas aeruginosa resistant mutants. We determined that decline of ceftazidime resistance occurs within 450 generations, which is caused by newly acquired mutations and not by reversion of the original ones, and that the original CS of these mutants is preserved. In addition, we observed that the frequency and degree of this decline is contingent on genetic background. Our results are relevant to implement evolution-based therapeutic approaches, as well as to redefine global policies of antibiotic use, such as drug cycling.