Levels and Characteristics of mRNAs in Spores of Firmicute Species.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

The AcrAB-TolC Efflux Pump Impacts Persistence and Resistance Development in Stationary-Phase Escherichia coli following Delafloxacin Treatment.

Bacteria have a repertoire of strategies to overcome antibiotics in clinical use, complicating our ability to treat and cure infectious diseases. In addition to evolving resistance, bacteria within genetically clonal cultures can undergo transient phenotypic changes and tolerate high doses of antibiotics. These cells, termed persisters, exhibit heterogeneous phenotypes; the strategies that a bacterial population deploys to overcome one class of antibiotics can be distinct from those needed to survive treatment with drugs with another mode of action. It was previously reported that fluoroquinolones, which target DNA topoisomerases, retain the capacity to kill nongrowing bacteria that tolerate other classes of antibiotics. Here, we show that in Escherichia coli stationary-phase cultures and colony biofilms, persisters that survive treatment with the anionic fluoroquinolone delafloxacin depend on the AcrAB-TolC efflux pump. In contrast, we did not detect this dependence on AcrAB-TolC in E. coli persisters that survive treatment with three other fluoroquinolone compounds. We found that the loss of AcrAB-TolC activity via genetic mutations or chemical inhibition not only reduces delafloxacin persistence in nongrowing E. coli MG1655 or EDL933 (an E. coli O157:H7 strain), but it limits resistance development in progenies derived from delafloxacin persisters that were given the opportunity to recover in nutritive medium following antibiotic treatment. Our findings highlight the heterogeneity in defense mechanisms that persisters use to overcome different compounds within the same class of antibiotics. They further indicate that efflux pump inhibitors can potentiate the activity of delafloxacin against stationary-phase E. coli and block resistance development in delafloxacin persister progenies.

The social network: Impact of host and microbial interactions on bacterial antibiotic tolerance and persistence.

Antibiotics have vastly improved our quality of life since their discovery and introduction into modern medicine. Yet, widespread use and misuse have compromised the efficacy of these compounds and put our ability to cure infectious diseases in jeopardy. To defend themselves against antibiotics, bacteria have evolved an arsenal of survival strategies. In addition to acquiring mutations and genetic determinants that confer antibiotic resistance, bacteria can respond to environmental cues and adopt reversible phenotypic changes that transiently enhance their ability to survive adverse conditions, including those brought on by antibiotics. These antibiotic tolerant and persistent bacteria, which are prevalent in biofilms and can survive antimicrobial therapy without inheriting resistance, are thought to underlie treatment failure and infection relapse. At infection sites, bacteria encounter a range of signals originating from host immunity and the local microbiota that can induce transcriptomic and metabolic reprogramming. In this review, we will focus on the impact of host factors and microbial interactions on antibiotic tolerance and persistence. We will also outline current efforts in leveraging the knowledge of host-microbe and microbe-microbe interactions in designing therapies that potentiate antibiotic activity and reduce the burden caused by recurrent infections.

Leishmania DNA Replication Timing: A Stochastic Event?

For eukaryotic genomes, DNA synthesis initiates at multiple discrete regions known as replication origins in a dynamic yet regulated manner to ensure genomic stability. Two recent studies using different approaches reveal few Leishmania origins and that origin firing may proceed in a mainly stochastic manner.

Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification.

Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. "Flame seedless" stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.