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.     

What is hidden in the pannexin treasure trove: the sneak peek and the guesswork

Connexins had been considered to be the only class of the vertebrate proteins capable of gap junction formation; however, new candidates for this function with no homology to connexins, termed pannexins were discovered. So far three pannexins were described in rodent and human genomes: Panx1, Panx2 and Panx3. Expressions of pannexins can be detected in numerous brain structures, and now found both in neuronal and glial cells. Hypothetical roles of pannexins in the nervous system include participating in sensory processing, hippocampal plasticity, synchronization between hippocampus and cortex, and propagation of the calcium waves supported by glial cells, which help maintain and modulate neuronal metabolism. Pannexin also may participate in pathological reactions of the neural cells, including their damage after ischemia and subsequent cell death. Recent study revealed non-gap junction function of Panx1 hemichannels in erythrocytes, where they serve as the conduits for the ATP release in response to the osmotic stress. High-throughput studies produced some evidences of the pannexin involvement in the process of tumorigenesis. According to brain cancer gene expression database REMBRANDT, PANX2 expression levels can predict post diagnosis survival for patients with glial tumors. Further investigations are needed to verify or reject hypotheses listed.     

Diversity of Polyketide Synthase (PKS) genes in metagenomic community of freshwater sponge Lubomirskia baicalensis

The screening of metagenomic DNA of the microbial community associated with the Baikalian sponge Lubomirskia baicalensis was performed in order to investigate the presence of polyketide synthase (PKS) genes. PKS enzyme systems take part in the synthesis of a great number of biologically active substances. The cloning and sequencing of amplified products of the ketosynthase domain section of the PKS gene cluster revealed 15 fragments of PKS genes with amino acid sequences differing from each other by 35?C65%. A BLASTX analysis showed that all of these sequences belong to KS domains identified in various groups of microorganisms, i.e., Alpha-, Beta-, and Deltaproteobacteria; Verrucomicrobia; Cyanobacteria; and Chlorophyta. Some sequences were related to genes that participate in the biosynthesis of curacin A (CurI, CurJ), stigmatellin (StiC, StiG), nostophycin (NpnB), and cryptophycin (CrpB). The homology of the found sequences with those of the EMBL database lies in the range of 50?C82%, which indicates that the freshwater sponge community contains genes that encode new, not yet studied polyketide substances of potential biotechnological significance.     

Distinctive features of the microbial diversity and the polyketide synthase genes spectrum in the community of the endemic Baikal sponge <Emphasis Type="Italic">Swartschewskia papyracea</Emphasis>

The diversity of the symbiotic community of the endemic Baikal sponge Swartschewskia papyracea was studied, and an analysis of the polyketide synthases genes spectrum in sponge-associated microorganisms was carried out. Six bacterial phyla were detected in the S. papyracea microbiome: Verrucomicrobia, Cyanobacteria, Actinobacteria, Bacteroidetes, Proteobacteria, and Planctomycetes. Unlike the microbial associations of other freshwater sponges, the community under study was dominated by the phylaVerrucomicrobia (42.1%) and Cyanobacteria (17.5%), while the proportion of the Proteobacteria was unusually low (9.7%). In the S. papyracea community metagenome, there were identified 18 polyketide synthases genes fragments, the closest homologues of which included the polyketide synthases of the microorganisms belonging to the bacterial phyla Cyanobacteria, Proteobacteria (classes Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria), and Acidobacteria as well as the eukaryotic algae of the phylum Heterokonta (class Eustigmatophyceae). Polyketide synthase sequences from S. papyracea formed three groups on the phylogenetic tree: a group of hybrid NRPS/PKS complexes, a group of cyanobacterial polyketide synthases, and a group of homologues of the eukaryotic alga Nannochloropsis gaditana. Notably, the identified polyketide synthase genes fragments showed only a 57–88% similarity to the sequences from the databases, which implies the presence of genes controlling the synthesis of the novel, still unstudied, polyketide compounds in the S. papyracea community. It was proposed that the habitat conditions of S. papyracea affect the taxonomic composition of the microorganisms associated with the sponge, including the diversity of the producers of secondary metabolites.     

The role of environmental factors for the composition of microbial communities of saline lakes in the Novosibirsk region (Russia)

Background

Nothing is currently known about microbial composition of saline lakes of the Novosibirsk region and its dependence on physical-chemical parameters of waters. We studied the structure of microbial communities of saline lakes of the Novosibirsk region and the effect of physical-chemical parameters of waters on microbial communities of these lakes.

Results

According to the ion content, the lakes were classified either as chloride or chloride-sulfate types. Water salinity ranges from 4.3 to 290 g L⁻¹. Many diverse microbial communities were found. Filamentous and colonial Cyanobacteria of the genera Scytonema, Aphanocapsa, and/or filamentous Algae dominated in littoral communities. Spatial and temporal organization of planktonic microbial communities and the quantities of Archaea and Bacteria were investigated using fluorescent in situ hybridization. We have found that the dominant planktonic component is represented by Archaea, or, less frequently, by Bacteria. Various phylogenetic groups (Bacteria, Archaea, Algae, and Cyanobacteria) are nonuniformly distributed. The principal component analysis was used to detect environmental factors that affect microorganism abundance. We found the principal components responsible for 71.1 % of the observed variation. It was demonstrated that two-block partial least squares was a better method than principal component analysis for analysis of the data. We observed general relationships between microbial abundance and water salinity.

Conclusions

We have performed the first-ever study of the structure of the microbial communities of eleven saline lakes in the Novosibirsk region along with their physical-chemical parameters of waters. Our study demonstrates that saline lakes in the Novosibirsk region contain a unique microbial communities that may become a prolific source of microorganisms for fundamental and applied studies in various fields of ecology, microbiology, geochemistry, and biotechnology, and deserve further metagenomic investigation.

     

Asymmetric block of the plant vacuolar Ca2+-permeable channel by organic cations

In this work we have analysed the voltage-dependent block of the slow activating channel from red beet vacuoles by Tris, quaternary ammonium ions and the natural polyamines putrescine, spermidine and spermine. All these organic cations when applied from the cytosolic side blocked the channel by binding apparently deep (zδ values in the range of 0.65–1.35) within the pore. Tetraethylammonium ion did not pass the selectivity filter, whereas the cations with a smaller cross-section and Tris could pass across the entire pore, as evidenced by a relief of block at high positive voltages. Voltage dependence of the establishment of block from cytosolic side and of its relief was anomalously strong in the sense that the total charge moved across the pore for all blockers tested, with a notable exception of spermine, was in excess of their actual valence. This behaviour is consistent with the existence of multiple binding sites within a long pore, their simultaneous occupancy and interaction between different ions. In contrast, binding of blockers from the vacuolar (lumenal) side appears to follow a single-ion handling rule, with a common binding site for all amines located at approximately 30% of the electrical distance from the lumenal side. Received: 22 February 1999 / Revised version: 6 July 1999 / Accepted: 8 July 1999     

Design and development of a series of borocycles as selective,covalent kallikrein 5 inhibitors

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.