Peptidoglycan fragments stimulate resuscitation of “non-culturable” mycobacteria

Resuscitation promoting factors (Rpfs), belonging to a family of secreted actinobacterial proteins with predicted peptidoglycan (PG) hydrolytic activities, participate in the reactivation of dormant cells. In the present study we demonstrate that a recombinant truncated form of Micrococcus luteus Rpf hydrolyzes isolated PG of Mycobacterium smegmatis and Mycobacterium tuberculosis liberating PG fragments of different size. These fragments possess stimulatory activity toward “non-culturable” dormant M. smegmatis and M. tuberculosis cells, similar to the activity of recombinant Rpf. Relatively large PG fragments (0.1–0.5 μm) obtained either by Rpf digestion or by PG ultrasonication revealed resuscitation activities when added in concentrations 0.1–0.2 μg/ml to the resuscitation medium. It is suggested that PG fragments could either directly activate the resuscitation pathway of dormant mycobacteria or serve as a substrate for endogenous Rpf, resulting in low molecular weight products with resuscitation activity. Whilst both suggestions are plausible, it was observed that PG-dependent resuscitation activity was suppressed by means of a specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate), which provides additional support for the second of these possibilities.     

Eutrophication and Dreissena Invasion as Drivers of Biodiversity: A Century of Change in the Mollusc Community of Oneida Lake

Changes in nutrient loading and invasive species are among the strongest human-driven disturbances in freshwater ecosystems, but our knowledge on how they affect the biodiversity of lakes is still limited. We conducted a detailed historical analysis of the mollusc community of Oneida Lake based on our comprehensive lakewide study in 2012 and previous surveys dating back to 1915. In the early 20th century, the lake had a high water clarity, with abundant macrophytes and benthic algae, and hosted the most diverse molluscan community in New York State, including 32 gastropod and 9 unionid species. By the 1960s, lake turbidity increased during a period of anthropogenic eutrophication, resulting in a 38% decline in species richness and a 95% reduction in abundance of native gastropods grazing on benthic algae. Following the invasion of Dreissena spp. in 1991 and subsequent increases in water clarity, native gastropod species richness expanded by 37% and abundance increased 20-fold by 2012. In contrast, filter-feeding unionids were unaffected by increased turbidity during the period of eutrophication but were extirpated by dreissenids. Through contrasting effects on turbidity, eutrophication and Dreissena spp. have likely driven the observed changes in native grazing gastropods by affecting the abundance of light-limited benthic algae. Given the high species richness and ecological importance of benthic grazers, monitoring and managing turbidity is important in preserving molluscan diversity.     

Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element

Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.     

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure-activity relationships in the bacterial membrane disruptor betapep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of betapep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel beta-sheet structure is the bioactive conformation of betapep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, betapep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.     

Ceruloplasmin: Macromolecular Assemblies with Iron-Containing Acute Phase Proteins

Copper-containing ferroxidase ceruloplasmin (Cp) forms binary and ternary complexes with cationic proteins lactoferrin (Lf) and myeloperoxidase (Mpo) during inflammation. We present an X-ray crystal structure of a 2Cp-Mpo complex at 4.7 Å resolution. This structure allows one to identify major protein–protein interaction areas and provides an explanation for a competitive inhibition of Mpo by Cp and for the activation of p-phenylenediamine oxidation by Mpo. Small angle X-ray scattering was employed to construct low-resolution models of the Cp-Lf complex and, for the first time, of the ternary 2Cp-2Lf-Mpo complex in solution. The SAXS-based model of Cp-Lf supports the predicted 1∶1 stoichiometry of the complex and demonstrates that both lobes of Lf contact domains 1 and 6 of Cp. The 2Cp-2Lf-Mpo SAXS model reveals the absence of interaction between Mpo and Lf in the ternary complex, so Cp can serve as a mediator of protein interactions in complex architecture. Mpo protects antioxidant properties of Cp by isolating its sensitive loop from proteases. The latter is important for incorporation of Fe³⁺ into Lf, which activates ferroxidase activity of Cp and precludes oxidation of Cp substrates. Our models provide the structural basis for possible regulatory role of these complexes in preventing iron-induced oxidative damage.     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Rahnella aquatilis 95 U003

The O-polysaccharide of Rahnella aquatilis 95 U003 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure:     

Membrane protein crystallization in lipidic mesophases with tailored bilayers

Monoacylglycerols have been used as bilayered hosts for growing crystals of membrane proteins. To date, the lipids used have had chains 16 and 18 carbon atoms long. We hypothesized that a shorter-chained lipid producing a thinner bilayer would facilitate the so-called in meso crystallization process. A 14 carbon monoacylglycerol was chosen as the lipid with which to test the proposal. To be compatible with the in meso method, a cis olefinic bond was placed in its acyl chain at a location arrived at by rational design. The target lipid was synthesized and was shown to form the requisite mesophase at room temperature. In support of the hypothesis, it produced crystals of bacteriorhodopsin and the outer membrane transporter, BtuB. The latter is the first beta barrel protein to be crystallized by the in meso method. Protein stability in the short-chain lipid and how this relates to crystallogenesis are discussed.     

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.