Functional genomics reveals the toxin-antitoxin repertoire and AbiE activity in Serratia

This is the final version. Available on open access from the Microbiology Society via the DOI in this recordData statement: All supporting data, code and protocols have been provided within the article or through supplementary data files. Eight supplementary tables and seven supplementary figures are available with the online version of this article.Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin-antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to 'shut down', limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.University of OtagoEuropean Union Horizon 2020Ministry for Business Innovation and EmploymentTertiary Education CommissionMarsden Fun

High viral abundance and low diversity are associated with increased CRISPR-Cas prevalence across microbial ecosystems

This is the final version. Available on open access from Elsevier via the DOI in this recordData and code availability: DNA sequence data are publicly available from the SRA database. Accession numbers are listed in Data S1F. No new sequence data was generated for this study. Original code is deposited in the github repositories listed in the Key resources table and statistical analysis scripts are available at https://github.com/s-meaden/Meaden_CB_2021. Code is publicly available at the time of publication. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.CRISPR-Cas are adaptive immune systems that protect their hosts against viruses and other parasitic mobile genetic elements.1 Although widely distributed among prokaryotic taxa, CRISPR-Cas systems are not ubiquitous.2-4 Like most defense-system genes, CRISPR-Cas are frequently lost and gained, suggesting advantages are specific to particular environmental conditions.5 Selection from viruses is assumed to drive the acquisition and maintenance of these immune systems in nature, and both theory6-8 and experiments have identified phage density and diversity as key fitness determinants.9,10 However, these approaches lack the biological complexity inherent in nature. Here, we exploit metagenomic data from 324 samples across diverse ecosystems to analyze CRISPR abundance in natural environments. For each metagenome, we quantified viral abundance and diversity to test whether these contribute to CRISPR-Cas abundance across ecosystems. We find a strong positive association between CRISPR-Cas abundance and viral abundance. In addition, when controlling for differences in viral abundance, CRISPR-Cas systems are more abundant when viral diversity is low, suggesting that such adaptive immune systems may offer limited protection when required to target a diverse viral community. CRISPR-Cas abundance also differed among environments, with environmental classification explaining roughly a quarter of the variation in CRISPR-Cas relative abundance. The relationships between CRISPR-Cas abundance, viral abundance, and viral diversity are broadly consistent across environments, providing robust evidence from natural ecosystems that supports predictions of when CRISPR is beneficial. These results indicate that viral abundance and diversity are major ecological factors that drive the selection and maintenance of CRISPR-Cas in microbial ecosystems.European Union Horizon 2020Natural Environment Research Council (NERC)Netherlands Organization for Scientific Research (NWO)European Research Council (ERC

13,915 reasons for equity in sexual offences legislation: A national school-based survey in South Africa

<p>Abstract</p> <p>Objective</p> <p>Prior to 2007, forced sex with male children in South Africa did not count as rape but as "indecent assault", a much less serious offence. This study sought to document prevalence of male sexual violence among school-going youth.</p> <p>Design</p> <p>A facilitated self-administered questionnaire in nine of the 11 official languages in a stratified (province/metro/urban/rural) last stage random national sample.</p> <p>Setting</p> <p>Teams visited 5162 classes in 1191 schools, in October and November 2002.</p> <p>Participants</p> <p>A total of 269,705 learners aged 10–19 years in grades 6–11. Of these, 126,696 were male.</p> <p>Main outcome measures</p> <p>Schoolchildren answered questions about exposure in the last year to insults, beating, unwanted touching and forced sex. They indicated the sex of the perpetrator, and whether this was a family member, a fellow schoolchild, a teacher or another adult. Respondents also gave the age when they first suffered forced sex and when they first had consensual sex.</p> <p>Results</p> <p>Some 9% (weighted value based on 13915/127097) of male respondents aged 11–19 years reported forced sex in the last year. Of those aged 18 years at the time of the survey, 44% (weighted value of 5385/11450) said they had been forced to have sex in their lives and 50% reported consensual sex. Perpetrators were most frequently an adult not from their own family, followed closely in frequency by other schoolchildren. Some 32% said the perpetrator was male, 41% said she was female and 27% said they had been forced to have sex by both male and female perpetrators. Male abuse of schoolboys was more common in rural areas while female perpetration was more an urban phenomenon.</p> <p>Conclusion</p> <p>This study uncovers endemic sexual abuse of male children that was suspected but hitherto only poorly documented. Legal recognition of the criminality of rape of male children is a first step. The next steps include serious investment in supporting male victims of abuse, and in prevention of all childhood sexual abuse.</p

Regulation of Enteric vapBC Transcription: Induction by VapC Toxin Dimer-Breaking

Toxin-antitoxin (TA) loci encode inhibitors of translation, replication or cell wall synthesis and are common elements of prokaryotic plasmids and chromosomes. Ten TA loci of Escherichia coli K-12 encode mRNases that cumulatively contribute to persistence (multidrug tolerance) of the bacterial cells. The mechanisms underlying induction and reversion of the persistent state are not yet understood. The vapBC operon of Salmonalla enterica serovar Typhimurium LT2 encodes VapC, a tRNase that reversibly inhibits translation by site-specific cleavage of tRNAfMet. VapB is an antitoxin that interacts with and neutralizes VapC via its C-terminal tail and regulate TA operon transcription via its N-terminal DNA binding domain that recognize operators in the vapBC promoter region. We show here that transcription of the vapBC operon of S. enterica is controlled by a recently discovered regulatory theme referred to as ‘conditional cooperativity’: at low T/A ratios, the TA complex binds cooperatively to the promoter region and represses TA operon transcription whereas at high T/A ratios, the excess toxin leads to destabilization of the TA-operator complex and therefore, induction of transcription. We present evidence that an excess of VapC toxin leads to operator complex destabilization by breaking of toxin dimers

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by “an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. Results: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Conclusions: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems

Bacteria clustering by polymers induces the expression of quorum sense controlled phenotypes

Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one mean by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are thus a potential means to control bacterial population responses. Here we report how polymeric "bacteria sequestrants", designed to bind to bacteria through electrostatic interactions and thus inhibit bacterial adhesion to surfaces, induce the expression of quorum sensing controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterise the feedback between bacteria clustering and quorum sensing signaling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population leve

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC.  Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed.  Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells.  Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies

Individual and Situational Factors Related to Young Women’s Likelihood of Confronting Sexism in Their Everyday Lives

Factors related to young women’s reported likelihood of confronting sexism were investigated. Participants were 338 U.S. female undergraduates (M = 19 years) attending a California university. They were asked to complete questionnaire measures and to write a personal narrative about an experience with sexism. Approximately half (46%) the women reported confronting the perpetrator. Individual factors (prior experience with sexism, feminist identification, collective action) and situational factors (familiarity and status of perpetrator, type of sexism) were tested as predictors in a logistic regression. Women were less likely to report confronting sexism if (1) they did not identify as feminists, (2) the perpetrator was unfamiliar or high-status/familiar (vs. familiar/equal-status), or (3) the type of sexism involved unwanted sexual attention (vs. sexist comments)

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity