Phenotypic Variation in the Group A Streptococcus Due to Natural Mutation of the Accessory Protein-Encoding Gene rocA

Populations of a bacterial pathogen, whether recovered from a single patient or from a worldwide study, are often a heterogeneous mix of genetically and phenotypically divergent strains. Such heterogeneity is of value in changing environments and arises via mechanisms such as gene gain or gene mutation. Here, we identify an isolate of serotype M12 group A Streptococcus (GAS) (Streptococcus pyogenes) that has a natural mutation in rocA, which encodes an accessory protein to the virulence-regulating two-component system CovR/CovS (CovR/S). Disruption of RocA activity results in the differential expression of multiple GAS virulence factors, including the anti-phagocytic hyaluronic acid capsule and the chemokine protease SpyCEP. While some of our data regarding RocA-regulated genes overlaps with previous studies, which were performed with isolates of alternate GAS serotypes, some variability was also observed. Perhaps as a consequence of this alternate regulatory activity, we discovered that the contribution of RocA to the ability of the M12 isolate to survive and proliferate in human blood ex vivo is opposite that previously observed in M1, M3, and M18 GAS strains. Specifically, rocA mutation reduced, rather than enhanced, survival of the isolate. Finally, we also present data from an analysis of rocA transcription and show that rocA is transcribed in both mono- and polycistronic mRNAs. In aggregate, our data provide insight into the important regulatory role of RocA and into the mechanisms and consequences of GAS phenotypic heterogeneity. IMPORTANCE This study investigates the regulatory and phenotypic consequences of a naturally occurring mutation in a strain of the bacterial pathogen the group A Streptococcus (Streptococcus pyogenes). We show that this mutation, which occurs in a regulator-encoding gene, rocA, leads to altered virulence factor expression and reduces the ability of this isolate to survive in human blood. Critically, the blood survival phenotype and the assortment of genes regulated by RocA differ compared to previous studies into RocA activity. The data are consistent with there being strain-or serotype-specific variability in RocA function. Given that phenotypic variants can lead to treatment failures and escape from preventative regimes, our data provide information with regard to a mechanism of phenotypic variation in a prevalent Gram-positive pathogen

A Formalism for the Systematic Treatment of Rapidity Logarithms in Quantum Field Theory

Many observables in QCD rely upon the resummation of perturbation theory to retain predictive power. Resummation follows after one factorizes the cross section into the rele- vant modes. The class of observables which are sensitive to soft recoil effects are particularly challenging to factorize and resum since they involve rapidity logarithms. In this paper we will present a formalism which allows one to factorize and resum the perturbative series for such observables in a systematic fashion through the notion of a "rapidity renormalization group". That is, a Collin-Soper like equation is realized as a renormalization group equation, but has a more universal applicability to observables beyond the traditional transverse momentum dependent parton distribution functions (TMDPDFs) and the Sudakov form factor. This formalism has the feature that it allows one to track the (non-standard) scheme dependence which is inherent in any scenario where one performs a resummation of rapidity divergences. We present a pedagogical introduction to the formalism by applying it to the well-known massive Sudakov form factor. The formalism is then used to study observables of current interest. A factorization theorem for the transverse momentum distribution of Higgs production is presented along with the result for the resummed cross section at NLL. Our formalism allows one to define gauge invariant TMDPDFs which are independent of both the hard scattering amplitude and the soft function, i.e. they are uni- versal. We present details of the factorization and resummation of the jet broadening cross section including a renormalization in pT space. We furthermore show how to regulate and renormalize exclusive processes which are plagued by endpoint singularities in such a way as to allow for a consistent resummation.Comment: Typos in Appendix C corrected, as well as a typo in eq. 5.6

Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair

Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5 ' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs

Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition

Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair

Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA–independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair

Extensive DNA End Processing by Exo1 and Sgs1 Inhibits Break-Induced Replication

Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination