A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

An immuno-chemo-proteomics method for drug target deconvolution

Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-alpha inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-alpha, GSK3-beta, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-alpha, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.     

Pyrosequencing-based analysis of the mucosal microbiota in healthy individuals reveals ubiquitous bacterial groups and micro-heterogeneity

This study used 16S rRNA-based pyrosequencing to examine the microbial community that is closely associated with the colonic mucosa of five healthy individuals. Spatial heterogeneity in microbiota was measured at right colon, left colon and rectum, and between biopsy duplicates spaced 1 cm apart. The data demonstrate that mucosal-associated microbiota is comprised of Firmicutes (50.9% ± 21.3%), Bacteroidetes (40.2% ± 23.8%) and Proteobacteria (8.6%± 4.7%), and that interindividual differences were apparent. Among the genera, Bacteroides, Leuconostoc and Weissella were present at high abundance (4.6% to 41.2%) in more than 90% of the studied biopsy samples. Lactococcus, Streptococcus, Acidovorax, Acinetobacter, Blautia, Faecalibacterium, Veillonella, and several unclassified bacterial groups were also ubiquitously present at an abundance <7.0% of total microbial community. With the exception of one individual, the mucosal-associated microbiota was relatively homogeneous along the colon (average 61% Bray-Curtis similarity). However, micro-heterogeneity was observed in biopsy duplicates within defined colonic sites for three of the individuals. A weak but significant Mantel correlation of 0.13 was observed between the abundance of acidomucins and mucosal-associated microbiota (P-value = 0.04), indicating that the localized biochemical differences may contribute in part to the micro-heterogeneity. This study provided a detailed insight to the baseline mucosal microbiota along the colon, and revealed the existence of micro-heterogeneity within defined colonic sites for certain individuals.     

Characterization of Aerosolized Bacteria and Fungi From Desert Dust Events in Mali, West Africa

Millions of metric tons of African desert dust blow across the Atlantic Ocean each year, blanketing the Caribbean and southeastern United States. Previous work in the Caribbean has shown that atmospheric samples collected during dust events contain living microbes, including plant and opportunistic human pathogens. To better understand the potential downwind public health and ecosystem effects of the dust microbes, it is important to characterize the source population. We describe 19 genera of bacteria and 3 genera of fungi isolated from air samples collected in Mali, a known source region for dust storms, and over which large dust storms travel.     

Origins and evolution of eukaryotic RNA interference

Small interfering RNAs (siRNAs) and genome-encoded microRNAs (miRNAs) silence genes via complementary interactions with mRNAs. With thousands of miRNA genes identified and genome sequences of diverse eukaryotes available for comparison, the opportunity emerges for insights into the origin and evolution of RNA interference (RNAi). The miRNA repertoires of plants and animals appear to have evolved independently. However, conservation of the key proteins involved in RNAi suggests that the last common ancestor of modern eukaryotes possessed siRNA-based mechanisms. Prokaryotes have an RNAi-like defense system that is functionally analogous but not homologous to eukaryotic RNAi. The protein machinery of eukaryotic RNAi seems to have been pieced together from ancestral archaeal, bacterial and phage proteins that are involved in DNA repair and RNA processing.