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Understanding the persistence of SARS-CoV-2 biomarkers in wastewater should guide wastewater-based epidemiology users in selecting best RNA biomarkers for reliable detection of the virus during current and future waves of the pandemic. In the present study, the persistence of endogenous SARS-CoV-2 were assessed during one month for six different RNA biomarkers and for the pepper mild mottle virus (PMMoV) at three different temperatures (4, 12 and 20 °C) in one wastewater sample. All SARS-CoV-2 RNA biomarkers were consistently detected during 6 days at 4° and differences in signal persistence among RNA biomarkers were mostly observed at 20 °C with N biomarkers being globally more persistent than RdRP, E and ORF1ab ones. SARS-CoV-2 signal persistence further decreased in a temperature dependent manner. At 12 and 20 °C, RNA biomarker losses of 1-log

Toxin-antitoxin (TA) systems are broadly distributed modules whose biological roles remain mostly unknown. The mqsRA system is a noncanonical TA system in which the toxin and antitoxins genes are organized in operon but with the particularity that the toxin gene precedes that of the antitoxin. This system was shown to regulate global processes such as resistance to bile salts, motility, and biofilm formation. In addition, the MqsA antitoxin was shown to be a master regulator that represses the transcription of the csgD, cspD, and rpoS global regulator genes, thereby displaying a pleiotropic regulatory role. Here, we identified two promoters located in the toxin sequence driving the constitutive expression of mqsA, allowing thereby excess production of the MqsA antitoxin compared to the MqsR toxin. Our results show that both antitoxin-specific and operon promoters are not regulated by stresses such as amino acid starvation, oxidative shock, or bile salts. Moreover, we show that the MqsA antitoxin is not a global regulator as suggested, since the expression of csgD, cspD and rpoS is similar in wild-type and ΔmqsRA mutant strains. Moreover, these two strains behave similarly in terms of biofilm formation and sensitivity to oxidative stress or bile salts.IMPORTANCE There is growing controversy regarding the role of chromosomal toxin-antitoxin systems in bacterial physiology. mqsRA is a peculiar toxin-antitoxin system, as the gene encoding the toxin precedes that of the antitoxin. This system was previously shown to play a role in stress response and biofilm formation. In this work, we identified two promoters specifically driving the constitutive expression of the antitoxin, thereby decoupling the expression of antitoxin from the toxin. We also showed that mqsRA contributes neither to the regulation of biofilm formation nor to the sensitivity to oxidative stress and bile salts. Finally, we were unable to confirm that the MqsA antitoxin is a global regulator. Altogether, our data are ruling out the involvement of the mqsRA system in Escherichia coli regulatory networks., info:eu-repo/semantics/published

Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

We discovered a chromosomal locus containing 2 toxin-antitoxin modules (TAs) with an antisense transcriptional organization in the E. faecalis clinical isolate V583. These TAs are homologous to the type I txpA-ratA system and the type II mazEF, respectively. We have shown that the putative MazF is toxic for E. coli and triggers RNA degradation, and its cognate antitoxin MazE counteracts toxicity. The second module, adjacent to mazEF, expresses a toxin predicted to belong to the TxpA type I family found in Firmicutes, and the antisense RNA antidote, RatA. Genomic analysis indicates that the cis-association of mazEF and txpA-ratA modules has been favored during evolution, suggesting a selective advantage for this TA organization in the E. faecalis species. We showed regulatory interplays between the 2 modules, involving transcription control and RNA stability. Remarkably, our data reveal that MazE and MazEF have a dual transcriptional activity: they act as autorepressors and activate ratA transcription, most likely in a direct manner. RatA controls txpA RNA levels through stability. Our data suggest a pivotal role of MazEF in the coordinated expression of mazEF and txpA-ratA modules in V583. To our knowledge, this is the first report describing a crosstalk between type I and II TAs.

Bacterial type II toxin-antitoxin systems are widespread in bacteria. Among them, the RelE toxin family is one of the most abundant. The RelE K-12 toxin of Escherichia coli K-12 represents the paradigm for this family and has been extensively studied, both in vivo and in vitro . RelE K-12 is an endoribonuclease that cleaves mRNAs that are translated by the ribosome machinery as these transcripts enter the A site. Earlier in vivo reports showed that RelE K-12 cleaves preferentially in the 5′-end coding region of the transcripts in a codon-independent manner. To investigate whether the molecular activity as well as the cleavage pattern are conserved within the members of this toxin family, RelE-like sequences were selected in Proteobacteria , Cyanobacteria , Actinobacteria , and Spirochaetes and tested in E. coli . Our results show that these RelE-like sequences are part of toxin-antitoxin gene pairs, and that they inhibit translation in E. coli by cleaving transcripts that are being translated. Primer extension analyses show that these toxins exhibit specific cleavage patterns in vivo , both in terms of frequency and location of cleavage sites. We did not observe codon-dependent cleavage but rather a trend to cleave upstream purines and between the second and third positions of codons, except for the actinobacterial toxin. Our results suggest that RelE-like toxins have evolved to rapidly and efficiently shut down translation in a large spectrum of bacterial species, which correlates with the observation that toxin-antitoxin systems are spreading by horizontal gene transfer.

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems are categorised into different types. Type III TA systems, the focus of this review, are composed of a toxic endoribonuclease neutralised by a non-coding RNA antitoxin in a pseudoknotted configuration. Bioinformatic analysis shows that the Type III systems can be classified into subtypes. These TA systems were originally discovered through a phage resistance phenotype arising due to a process akin to an altruistic suicide; the phenomenon of abortive infection. Some Type III TA systems are bifunctional and can stabilise plasmids during vegetative growth and sporulation. Features particular to Type III systems are explored here, emphasising some of the characteristics of the RNA antitoxin and how these may affect the co-evolutionary relationship between toxins and cognate antitoxins in their quaternary structures. Finally, an updated analysis of the distribution and diversity of these systems are presented and discussed.

The ccd F and parDE RK2 systems were originally identified on the E. coli F plasmid and on the RK2 broad-host-range plasmid, respectively. Early reports indicated that these two loci promote plasmid maintenance in growing population by killing daughter bacteria that did not inherit a plasmid copy at cell division by a mechanism termed ‘post-segregational killing’. The toxins encoded by these two loci target DNA-gyrase, an essential topoisomerase II, although the molecular details appear to be different. Interestingly, CcdBF and ParERK2 do not share a common ancestor, and therefore appear to have evolved independently. CcdB and ParE protein families share structural similarity with toxins belonging to the MazF and RelE toxin families, respectively, members of which inhibit translation by different mechanisms. In addition to focus on the molecular mechanism of poisoning of DNA-gyrase by CcdBF and ParERK2 as well as structural data, we describe here the different systems encoding CcdB-like and ParE-like toxins and highlight the specific features of those systems in terms of antitoxin structure and properties, autoregulation, biological functions, and evolution.