To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO 2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the DNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700 þ rereduction in darkness in the DNdhD1/D2 mutant grown at low CO 2 were similar to those in the wild type, whereas in the M55 mutant (DNdhB), lacking both NDH-1L and NDH-1M, the rate of P700 þ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO 2 in the wild type as well as in DNdhD1/D2 and M55. In contrast with the wild type and DNdhD1/D2, which show normal CO 2 uptake, M55 is unable to take up CO 2 even when the NDH-1S complex is present. Conversely, the DNdhD3/D4 mutant, also unable to take up CO 2 , lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO 2 . These results demonstrate that both NDH-1S and NDH-1M are essential for CO 2 uptake and that NDH-1M is a functional complex. We also show that the Na þ /HCO 3 ÿ transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO 2 .

No abstract

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.

Background: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies. Methods: The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with α1-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells. The Fab fragment that detects only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-resolved fluorescence is used to measure both europium and terbium signals in one well. Results: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 μg/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2–11% and 4–10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0.99) were not significantly different from zero, and the slopes were 0.95–1.01. In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay. Conclusions: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.