Organic Matter Preservation and Microbial Community Accumulations in Deep-Hypersaline Anoxic Basins

The Eastern Mediterranean Sea hosts several deep hypersaline anoxic basins (DHABs) such as the Bannock, L'Atalante, Discovery, and Urania which, due to strong salinity gradients, have a limited exchange with the overlying seawater. In the present study, a series of environmental variables associated with the origin and quality of organic matter were thoroughly investigated in an attempt to understand the function of these unique ecosystems. The redox potential of sediments collected from the brines as well as from reference sites varied from ?136 to 543 mV and salinity varied from 38 to 380 psu. Principal component analysis of chemical characteristics, including salinity, redox potential, organic carbon and nitrogen content, and C/N ratio grouped the sediments into two major clusters according to their redox state. Aliphatic hydrocarbon analysis revealed that the organic matter in the DHABs was predominantly of terrestrial origin but there was also evidence for petroleum inputs and for organic matter of phototrophic origin. Phospholipid linked fatty acids (PLFA) which were employed to assess the composition of microbial communities were found in greater abundance in stations situated inside the anoxic basins providing also strong evidence for the presence of methanotrophs and sulfate reducers. These results may represent an enhanced preservation of organic matter and an accumulation of microorganisms in these extreme environments. Heterogeneity in microbial community fatty acid profiles was documented between the anoxic sediments and the oxic and suboxic stations. However there were no significant correlations between PLFA and organic matter parameters. Redox conditions appear to influence microbial community composition, highlighting the role of the redox state as a regulator of organic matter preservation and microbial community accumulations in these ancient hypersaline anoxic lakes.     

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA.     

Exploring the DNA mimicry of the Ocr protein of phage T7

DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.     

Adaptation of Drosophila to temperature: Heat-shock proteins and survival in Drosophila melanogaster

Two stocks of Drosophila melanogaster, one sensitive (6.5% survival) and one resistant (76.24%) to heat shock (40°C/25 min) were derived through indirect selection [1]. Genetic analysis of heat-sensitive and heat-resistant lines we had selected revealed that the survival rate is chiefly determined by cytoplasmic inheritance but also depends to some extent on the nucleus [1]. The ability of the fly to survive thermal stress was found to have an excellent correlation with the kinetics of protein synthesis in ovaries or glands subjected to heat treatment. The incorporation rate of ³⁵S-methionine into proteins was found to be higher for strains exhibiting higher survival (R₁, R₁S₁) than for strains with a lesser ability (S₁, S₁ R₁) to survive heat shock. Moreover, the intensity of labeling of the proteins synthesized and especially of the hsps (heat-shock proteins) after the heat shock is higher in the R₁ and R₁S₁ stocks than in the S₁ and S₁R₁ stocks. This convergence between survival and the cellular level of hsps (both manipulated by selection) bears on the physiological significance of these proteins which seems to participate in the control of the survival as an additive component.     

A mutational analysis of DNA mimicry by ocr, the gene 0.3 antirestriction protein of bacteriophage T7

The ocr protein of bacteriophage T7 is a structural and electrostatic mimic of approximately 24 base pairs of double-stranded B-form DNA. As such, it inhibits all Type I restriction and modification (R/M) enzymes by blocking their DNA binding grooves and inactivates them. This allows the infection of the bacterial cell by T7 to proceed unhindered by the action of the R/M defence system. We have mutated aspartate and glutamate residues on the surface of ocr to investigate their contribution to the tight binding between the EcoKI Type I R/M enzyme and ocr. Contrary to expectations, all of the single and double site mutations of ocr constructed were active as anti-R/M proteins in vivo and in vitro indicating that the mimicry of DNA by ocr is very resistant to change.     

Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.     

Cardiac release of urocortin precedes the occurrence of irreversible myocardial damage in the rat heart exposed to ischemia/reperfusion injury

This study evaluates whether cardiac ischemia induces release of urocortin, before and independently from myocyte cell death. Urocortin levels rose after 5-min ischemia and peaked after 10-min ischemia, when cell death was not detected. However, myocyte apoptosis and/or necrosis occurred following 20- and 30-min ischemia, which paralleled a fall in urocortin levels, suggesting that urocortin expression and release are mainly sustained by metabolically challenged, though still viable myocytes. Hence, since cardiac release of urocortin, unlike that of conventional biomarkers, occurs before and apart from cell death, urocortin levels may be clinically useful in the diagnosis of sublethal myocardial ischemia.     

Antiapoptotic activity of the free caspase recruitment domain of procaspase-9: a novel endogenous rescue pathway in cell death

Mitochondrial injury initiates proteolytic processing of procaspase-9 into the large and small subunits, leading to apoptotic cell death. Here we show that the free caspase recruitment domain (CARD) released by procaspase-9 processing activates nuclear factor kappaB expression. A procaspase-9 construct with a point mutation that abrogates the release of the CARD abolished nuclear factor kappaB activation. Most importantly, the free CARD is shown to enhance the expression of the gene encoding the antiapoptotic Bcl-x protein and to strongly inhibit apoptosis. This is the first demonstration that different domains of the same caspase protein have proapoptotic and antiapoptotic effects and suggests that the relative effects of these domains are important in regulating the balance between death and survival.