Spontaneous mutations and mutational responses to penicillin treatment in the bacterial pathogen Streptococcus pneumoniae D39

Bacteria with functional DNA repair systems are expected to have low mutation rates due to strong natural selection for genomic stability.However,our study of the wild-type Streptococcus pneumoniae D39,a pathogen responsible for many common diseases,revealed a high spontaneous mutation rate of 0.02 per genome per cell division in mutation-accumulation(MA)lines.This rate is orders of magnitude higher than that of other non-mutator bacteria and is characterized by a high mutation bias in the A/T direction.The high mutation rate may have resulted from a reduction in the overall efficiency of selection,conferred by the tiny effective population size in nature.In line with this,S.pneumoniae D39 also exhibited the lowest DNA mismatch-repair(MMR)efficiency among bacteria.Treatment with the antibiotic penicillin did not elevate the mutation rate,as penicillin did not induce DNA damage and S.pneumoniae lacks a stress response pathway.Our findings suggested that the MA results are applicable to within-host scenarios and provide insights into pathogen evolution.

Evolution of sex determination in crustaceans

Sex determination(SD)involves mechanisms that determine whether an individual will develop into a male,female,or in rare cases,hermaphrodite.Crustaceans harbor extremely diverse SD systems,including hermaphroditism,environmental sex determination(ESD),genetic sex determination(GSD),and cytoplasmic sex determination(e.g.,Wolbachia controlled SD systems).Such diversity lays the groundwork for researching the evolution of SD in crustaceans,i.e.,transitions among different SD systems.However,most previous research has focused on understanding the mechanism of SD within a sin-gle lineage or species,overlooking the transition across different SD systems.To help bridge this gap,we summarize the understanding of SD in various clades of crustaceans,and discuss how different SD systems might evolve from one another.Furthermore,we review the genetic basis for transitions between different SD systems(i.e.,Dmrt genes)and propose the microcrustacean Daphnia(clade Branchiopoda)as a model to study the transition from ESD to GSD.

Contribution of the SOS response and the DNA repair systems to norfloxacin induced mutations in E.coli

Antibiotic-resistant bacteria severely threaten human health.Besides spontaneous mutations generated by endogenous factors,the resistance might also originate from mutations induced by certain antibiotics,such as the fluoroquinolones.Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems.However,the relative contributions of these molecular mechanisms remain unclear,hindering understanding of the generation of resistant pathogens.Here,using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains,as well as those of the strains deficient for the mismatch repair(MMR)and the oxidative damage repair pathways,we find that the SOS response is the major mutagenesis contributor in mutation elevation,responsible for~30–50%of the total base-pair substitution(BPS)mutation-rate elevation upon treatment with sublethal levels of norfloxacin(0~50 ng/mL).We further estimate the significance of the effects on other mutational features of these mechanisms(i.e.,transversions,structural variations,and mutation spectrum)in E.coli using linear models.The SOS response plays a positive role in all three mutational features(mutation rates of BPSs,transversions,structural variations)and affects the mutational spectrum.The repair systems significantly reduce the BPS mutation rate and the transversion rate,regardless of whether antibiotics are present,while significantly increasing the structural variation rate in E.coli.Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.

An integrative protocol for one‑step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.