Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve

Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA

Dual effect of organic acids as a function of external pH in Oenococcus oeni

International audienceIn this study we analyzed under various pH conditions including low pH, the effects of l-malic acid and citric acid, combined or not, on the growth, the proton motive force components and the transcription level of selected genes of the heterolactic bacterium Oenococcus oeni. It is shown here that l-malate enhanced the growth yield at pH equal or below 4.5 while the presence of citrate in media led to a complete and unexpected inhibition of the growth at pH 3.2. Nevertheless, whatever the growth conditions, both l-malate and citrate participated in the enhancement of the transmembrane pH gradient, whereas the membrane potential decreased with the pH. These results suggested that it was not citrate that was directly responsible for the inhibition observed in cultures done at low pH, but probably its end products. This was confirmed since, in media containing l-malate, the addition of acetate substantially impaired the growth rate of the bacterium and slightly the membrane potential and pH gradient. Finally, study of the expression of genes involved in the metabolism of organic acids showed that at pH 4.5 and 3.2 the presence of l-malate led to an increased amount of mRNA of mleP encoding a malate transporter

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

International audienceThe human pathogen Mycobacterium tuberculosis requires a P-1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.The human pathogen Mycobacterium tuberculosis requires a metal exporter, CtpC, for resistance to zinc poisoning. Here, the authors show that zinc resistance also depends on a chaperone-like protein that binds zinc ions, forms high-molecular-weight complexes with CtpC in the cytoplasmic membrane, and is required for CtpC function

Climatic variations in the Lake Abhe area since the end of the Pleistocene (Djibouti): the transition from the last hunter-gatherers-fishers to the first herders in the Horn of Africa and their resiliency

International audienc

Climatic variations in the Lake Abhe area since the end of the Pleistocene (Djibouti): the transition from the last hunter-gatherers-fishers to the first herders in the Horn of Africa and their resiliency

International audienc

Climatic variations in the Lake Abhe area since the end of the Pleistocene (Djibouti): the transition from the last hunter-gatherers-fishers to the first herders in the Horn of Africa and their resiliency

International audienc

Climatic variations in the Lake Abhe area since the end of the Pleistocene (Djibouti): the transition from the last hunter-gatherers-fishers to the first herders in the Horn of Africa and their resiliency

International audienc