Staphylococcus pasteuri-specific oligonucleotide probes derived from a random amplified DNA fragment

Abstract A rapid polymerase chain reaction method was developed to differentiate Staphylococcus pasteuri from other staphylococcal species, especially the phenotypically similar S. warneri . The oligonucleotide probes used as primers were designed from the sequence of a S. pasteuri random amplified polymorphic DNA fragment.     

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b₆f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b₆f complex.Photosynthetic organisms are subjected to constant changes in light quality and quantity and need to adapt to these changes in order to optimize, on the one hand, their photosynthetic yield, and to minimize photo-oxidative damage on the other. The photosynthetic electron transfer chain consists of photosystem II (PSII), the plastoquinone (PQ) pool, the cytochrome b₆f complex (Cyt b₆f), plastocyanin, and photosystem I (PSI). All of these complexes and components are integrated or closely associated with the thylakoid membrane. The two antenna systems of PSII and PSI capture and direct the light excitation energy to the corresponding reaction centers in which a chlorophyll dimer is oxidized and charge separation occurs across the thylakoid membrane. These processes lead to the onset of electron flow from water on the donor side of PSII to ferredoxin on the acceptor side of PSI coupled with proton translocation across the thylakoid membrane. In order to sustain optimal electron flow along this electron transfer chain, the redox poise needs to be maintained under changing environmental conditions. Several mechanisms have evolved for the maintenance of this redox balance. In the case of over-reduction of the acceptor side of PSI, excess electrons can reduce molecular oxygen through the Mehler reaction to superoxide, which is then converted to hydrogen peroxide by a plastid superoxide dismutase and ultimately to water by a peroxidase (Asada, 2000). Over-reduction of the PQ pool can be alleviated by PTOX, the plastid terminal oxidase responsible for oxidizing PQH₂ to form hydrogen peroxide, which is subsequently converted to water (Carol et al., 1999; Cournac et al., 2000; Wu et al., 1999).In addition to these electron sinks that prevent the over-reduction of the electron transfer chain, the photosynthetic apparatus is able to maintain the redox poise of the PQ pool by readjusting the relative cross sections of the light harvesting systems of PSII and PSI upon unequal excitation of the two photosystems. This readjustment can occur both in the short term through state transitions and in the long term by changing the stoichiometry between PSII and PSI (Bonaventura and Myers, 1969; Murata, 1969; Pfannschmidt, 2003). State transitions occur because of perturbations of the redox state of the PQ pool due to unequal excitation of PSII and PSI, limitations in electron acceptors downstream of PSI, and/or in CO₂ availability. Excess excitation of PSII relative to PSI leads to reduction of the PQ pool and thus favors the docking of PQH₂ to the Qo site of the Cyt b₆f complex. This process activates the Stt7/STN7 protein kinase (Vener et al., 1997; Zito et al., 1999), which is closely associated with this complex and leads to the phosphorylation of some LHCII proteins and to their detachment from PSII and binding to PSI (Depège et al., 2003; Lemeille et al., 2009). Although both Lhcb1 and Lhcb2 are phosphorylated, only the phosphorylated form of Lhcb2 is associated with PSI whereas phosphorylated Lhcb1 is excluded from this complex (Longoni et al., 2015). This state corresponds to state 2. In this way the change in the relative antenna sizes of the two photosystems restores the redox poise of the PQ pool. The process is reversible as over-excitation of PSI relative to PSII leads to the oxidation of the PQ pool and to the inactivation of the kinase. Under these conditions, phosphorylated LHCII associated with PSI is dephosphorylated by the PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and returns to PSII (state 1). It should be noted, however, that a strict causal link between LHCII phosphorylation and its migration from PSII to PSI has been questioned recently by the finding that some phosphorylated LHCII remains associated with PSII supercomplexes and that LHCII serves as antenna for both photosystems under most natural light conditions (Drop et al., 2014; Wientjes et al., 2013).State transitions are important at low light but do not occur under high light because the LHCII kinase is inactivated under these conditions (Schuster et al., 1986). It was proposed that inactivation of the kinase is mediated by the ferredoxin-thioredoxin system and that a disulfide bond in the kinase rather than in the substrate may be the target site of thioredoxin (Rintamäki et al., 1997, 2000). Analysis of the Stt7/STN7 protein sequences indeed reveals the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are conserved in all species examined and both are essential for kinase activity although they are located outside of the kinase catalytic domain (Fig. 1) (Depège et al., 2003; Lemeille et al., 2009). Based on protease protection studies, this model of the Stt7/STN7 kinase proposes that the N-terminal end of the kinase is on the lumen side of the thylakoid membrane separated from the catalytic domain on the stromal side by an unusual transmembrane domain containing several Pro residues (Lemeille et al., 2009). This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b₆f assembly (Lennartz et al., 2001; Page et al., 2004) or through the LTO1 protein (Du et al., 2015; Karamoko et al., 2011).Figure 1.Conserved Cys in the Stt7/STN7 kinase. Alignment of the sequences of the Stt7/STN protein kinase from Selaginella moelendorffii (Sm), Physcomitrella patens (Pp), Oryza sativa (Os), Populus trichocarpa (Pt), Arabidopsis thaliana (At), Chlamydomonas reinhardtii ...Here we have examined the redox state of the Stt7/STN7 kinase during state transitions and after illumination with high light to test the proposed model. We find that the Stt7/STN7 kinase contains a disulfide bridge that appears to be intramolecular and maintained not only during state transitions but also in high light when the kinase is inactive. Although these results suggest at first sight that the disulfide bridge of Stt7/STN7 is maintained during its activation and inactivation, we propose that a transient opening of this bridge occurs during the activation process followed by the formation of an intermolecular disulfide bridge and the appearance of a short-lived, covalently linked kinase dimer.     

Separation of Epoxy-Embedded Cell Cultures from Plastic Flasks for Electron Microscopy

Monolayers of cells grown in ordinary plastic flasks are fixed and embedded “in situ” into Epon. When polymerized at 40 C for 4 days instead of the usual 60 C., the Epon sheet containing the cells is easily detached from the bottom of the plastic container. The Epon sheet is observed by light microscopy as a histological preparation. Ultrathin sectioning of preselected areas can then be carried out in a horizontal plane.     

Cell population kinetics of zymogen and parietal cells in the stomach of mice

Summary With autoradiography after labelling with tritiated thymidine, the kinetics of zymogen and parietal cells were studied in the gastric mucosa of mice. After one intraperitoneal injection of the DNA precursor, zymogen cells in the DNA synthesis phase were clearly identified on autoradiograms, whereas no parietal cells were seen to synthesize DNA.In another group of mice, multiple injections were used in order to obtain a greater number of labelled cells. Following the latter procedure, analysis of grain count distributions over labelled zymogen cells and of labelling indices allowed detection of two subsequent zymogen cell divisions within an interval of approximately two months. This indicates that the cell turnover of zymogen cells is at least partly assured by their own mitotic activity.By contrast, parietal cells showed no evidence of cell division, but appeared to be derived through differentiation from other cells in the neck area of the gland. Analysis of spatial distribution of the labelled parietal cells in the glandular tube indicated that, in time, most newly formed parietal cells undergo a slow migration directed downwards to the bottom of the fundic glands.These results clearly show that the zymogen and the parietal cell population of the fundic glands have a different kinetic behaviour.This work was supported by a grant of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities—I

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the αγ isozyme. The most important increase in 14-3-2 antigen and γγ enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is—during embryonic development—the decrease in the specific activity of the cerebral enolase isozyme αα. This could be explained by the replacement—in neuroblasts—of αα enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (γγ) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase.Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).     

Biodegradation of Ethylene Glycol by a Salt-Requiring Bacterium

A gram-negative nonmotile rod which was capable of using 1,2-(14)C-ethylene glycol as a sole carbon source for growth was isolated from a brine pond, Great Salt Lake, Utah. The bacterium (ATCC 27042) required at least 0.85% NaCl for growth and, although the chloride ion was replaceable by sulfate ion, the sodium ion was not replaceable by potassium ion. The maximal concentration of salt tolerated for growth was approximately 12%. The bacterium was oxidase-negative when N,N-dimethyl-p-phenylenediamine was used and weakly positive when N,N,N',N'-tetramethyl-p-phenylenediamine was used. It grows on many sugars but does not ferment them, it does not have an exogenous vitamin requirement, and it possesses a guanine plus cytosine ratio of 64.3%. Incorporation of ethylene glycol carbon into cell and respired CO(2) was quantitated by use of radioactive ethylene glycol and a force-aerated fermentor. Glucose suppressed ethylene glycol metabolism. Cells grown on ethylene and propylene glycol respired ethylene glycol in a Warburg respirometer more rapidly than cells grown on glucose. Spectrophotometric evidence was obtained for oxidation of glycolate to glyoxylate by a dialyzed cell extract.     

Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography–tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 × 185 Å; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.     

The three genomes of Chlamydomonas

During the past 50 years, the green unicellular alga Chlamydomonas reinhardtii has played a key role as model system for the study of photosynthesis and chloroplast biogenesis. This is due to its well-established nuclear and chloroplast genetics, its dispensable photosynthetic function in the presence of acetate, and its highly efficient nuclear and chloroplast transformation systems. Considerable progress has been achieved in our understanding of the structure, function, inheritance, and expression of nuclear, chloroplast, and mitochondrial genes and of the molecular cross-talk between the nuclear, chloroplast, and mitochondrial genetic systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38-40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA.