Many prokaryotes employ CRISPR–Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I–F (Pseudomonas, Pectobacterium, and Serratia), I–E (Pseudomonas and Serratia), and I–C (Pseudomonas). We uncover 11 type I–F and/or I–E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.

CRISPR-Cas systems are widespread in bacterial and archaeal genomes, and in their canonical role in phage defence they confer a fitness advantage. However, CRISPR-Cas may also hinder the uptake of potentially beneficial genes. This is particularly true under antibiotic selection, where preventing the uptake of antibiotic resistance genes could be detrimental. Newly discovered features within these evolutionary dynamics are anti-CRISPR genes, which inhibit specific CRISPR-Cas systems. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally acquired antibiotic resistance genes. To assess that question, we analysed correlations between the CRISPR-Cas, anti-CRISPR and antibiotic resistance gene content of 104 947 reference genomes, including 5677 different species. In most species, the presence of CRISPR-Cas systems did not correlate with the presence of antibiotic resistance genes. However, in some clinically important species, we observed either a positive or negative correlation of CRISPR-Cas with antibiotic resistance genes. Anti-CRISPR genes were common enough in four species to be analysed. In Pseudomonas aeruginosa , the presence of anti-CRISPRs was associated with antibiotic resistance genes. This analysis indicates that the role of CRISPR-Cas and anti-CRISPRs in the spread of antibiotic resistance is likely to be very different in particular pathogenic species and clinical environments. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

Background Gut microbes play a crucial role in the maintenance of human health. Components in the diet of the host affect their metabolism and diversity. Here, we investigated the influences of three commonly used non-caloric artificial sweeteners-aspartame, acesulfame K and sucralose-on the growth and metabolism of an omnipresent gut microbe Escherichia c oli K-12. Methods : Growth of E. coli in the presence of aspartame, acesulfame K and sucralose in media was assessed and the influences of these artificial sweeteners on metabolism were investigated by relative expression analysis of genes encoding the rate limiting steps of important metabolic pathways as well as their global metabolomic profiles. Results: As a whole, E. coli growth was inhibited by aspartame and induced by acesulfame potassium, while the effect of sucralose on growth was less prominent. Although the expressions of multiple key enzymes that regulate important metabolic pathways were significantly altered by all three sweeteners, acesulfame K caused the most notable changes in this regard. In multivariate analysis with the metabolite profiles, the sucralose-treated cells clustered the closest to the untreated cells, while the acesulfame potassium treated cells were the most distant. These sweeteners affect multiple metabolic pathways in E. coli , which include propanoate, phosphonate, phosphinate and fatty acid metabolism, pentose phosphate pathway, and biosynthesis of several amino acids including lysine and the aromatic amino acids. Similar to the gene expression pattern, acesulfame potassium treated E. coli showed the largest deviation in their metabolite profiles compared to the untreated cells.

The human gut is inhabited by several hundred different bacterial species. These bacteria are closely associated with our health and well-being. The composition of these diverse commensals is influenced by our dietary intakes. Non-caloric artificial sweeteners (NAS) have gained global popularity, particularly among diabetic patients, due to their perceived health benefits, such as reduction of body weight and maintenance of blood glucose level compared to caloric sugars. Recent studies have reported that these artificial sweeteners can alter the composition of gut microbiota and, thus, affect our normal physiological state. Here, we investigated the effect of aspartame and acesulfame potassium (ace-K), two popular NAS, in a commercial formulation on the growth and metabolic pathways of omnipresent gut commensal Escherichia coli by analyzing the relative expression levels of the key genes, which control over twenty important metabolic pathways. Treatment with NAS preparation (aspartame and ace-K) modulates the growth of E. coli as well as inducing the expression of important metabolic genes associated with glucose (pfkA, sucA, aceE, pfkB, lpdA), nucleotide (tmk, adk, tdk, thyA), and fatty acid (fabI) metabolisms, among others. Several of the affected genes were previously reported to be important for the colonization of the microbes in the gut. These findings may shed light on the mechanism of alteration of gut microbes and their metabolism by NAS.

Many bacteria use CRISPR-Cas systems to defend against invasive mobile genetic elements (MGEs). In response, MGEs have developed strategies to resist CRISPR-Cas, including the use of anti-CRISPR (Acr) proteins. Known acr genes may be followed in an operon by a putative regulatory Acr-associated gene (aca), suggesting the importance of regulation. Although ten families of helix-turn-helix (HTH) motif containing Aca proteins have been identified (Aca1-10), only three have been tested and shown to be transcriptional repressors of acr-aca expression. The AcrIIA1 protein (a Cas9 inhibitor) also contains a functionally similar HTH containing repressor domain. Here, we identified and analysed Aca and AcrIIA1 homologs across all bacterial genomes. Using HMM models we found aca-like genes are widely distributed in bacteria, both with and without known acr genes. The putative promoter regions of acr-aca operons were analysed and members of each family of bacterial Aca tested for regulatory function. For each Aca family, we predicted a conserved inverted repeat binding site within a core promoter. Promoters containing these sites directed reporter expression in E. coli and were repressed by the cognate Aca protein. These data demonstrate that acr repression by Aca proteins is widely conserved in nature.