Photoconversion kinetics of chloroplast Photosystems I and II. Effect of Mg2+

The effect of divalent cations on the primary photoconversion kinetics of chloroplast Photosystems (PS) I and II was investigated by absorbance difference spectrophotometry in the ultraviolet (ΔA₃₂₀) and red (ΔA₇₀₀) regions and by fluorescence at room temperature. Three main chlorophyll (Chl) a fluorescence emission components were identified. Addition of 5 mM MgCl₂ to unstacked chloroplasts caused a 5–7-fold increase in F_vα, the variable fluorescence yield controlled by the α-centers. The fluorescence yield F_vβ controlled by the β-centers and the nonvariable fluorescence yield F₀ were only slightly changed by the treatment. The absolute number of α- and β-centers remained unchanged and independent of divalent cations. The rate constants K_α, K_β and K_P-700 determined from the photoconversion kinetics of Q_α, Q_β and P-700 were also unchanged by divalent cations, suggesting a constancy of the respective absorption cross-sections. Evidence is presented that the Mg²⁺ effect on Chl a fluorescence is not due simply to unstacking. Conclusion: (1) In the absence of divalent cations from the chloroplast suspending medium, the variable fluorescence yield is not complementary to the rate of PS II photochemistry. (2) A spillover of excitation from PS II to PS I in the absence of Mg²⁺ cannot account for the 7-fold lowering of the variable fluorescence yield F_vα at room temperature. The results are discussed in view of a model of excitation transfer and fluorescence emission in the pigment bed of PS II_α and PS II_β.     

Recombinant P4 bacteriophages propagate as viable lytic phages or as autonomous plasmids in Klebsiella pneumoniae

Summary We demonstrate the use of bacteriophage P4 as a molecular cloning vector in Klebsiella pneumoniae. A hybrid P4 phage, constructed in vitro, that contains a K. pneumoniae hisDG DNA fragment can be propagated either as a lytic viable specialized transducing phage or as an autonomous, self-replicating plasmid. Hybrid P4 genomes existing as plasmids can be readily converted into non-defective P4-hybrid phage particles by superinfection with helper phage P2. Infection of a K. pneumoniae hisD non-P2 lysogen with P4-hisD hybrid phage results in approximately 90% of the infected cells becoming stably transduced to HisD⁺. Because P4 interferes with P2 growth, high titre stocks of P4 hybrid phages are relatively free (10^-6) of P2 contamination. The hisG gene product was detected in ultraviolet light irradiated host cells infected by the P4-hisDG hybrid phage. A mutant of P4 (P4sidl) that directs the packaging of P4 DNA into P2 sized capsids should permit the construction of hybrid phages carrying 26 kilobase inserts.     

Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.     

Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe.

Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.     

Thein vivo pattern of firefly luciferase expression in transgenic plants

Expression of the firefly luciferase gene in transgenic plants produces light emission patterns when the plants are supplied with luciferin. We explored whether inin vivo pattern of light emission truly reveals the pattern of luciferase gene expression or whether it reflects other parameters such as the availability of the substrate, luciferin, or the tissue-specific distribution of organelles in which luciferase was localized. The tissue-specific distribution of luciferase activity and thein vivo pattern of light were examined when the luciferase gene was driven by different promoters and when luciferase was redirected from the peroxisome, where it is normally targeted, to the chloroplast compartment. It was found that the distribution of luciferase activity closely correlated with the tissue-specific pattern of luciferase mRNA. However, thein vivo light pattern appeared to reflect not only tissue-specific distribution of luciferase activity, but also the pattern of luciferin uptake.     

Metabolic adaptations in a H2 producing heterocyst-forming cyanobacterium: potentials and implications for biological engineering

Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the ability to fix atmospheric nitrogen and photoproduce hydrogen through the enzyme nitrogenase. The H(2) produced is reoxidized by an uptake hydrogenase. Inactivation of the uptake hydrogenase in N. punctiforme leads to increased H(2) release but unchanged rates of N(2) fixation, indicating redirected metabolism. System-wide understanding of the mechanisms of this metabolic redirection was obtained using complementary quantitative proteomic approaches, at both the filament and the heterocyst level. Of the total 1070 identified and quantified proteins, 239 were differentially expressed in the uptake hydrogenase mutant (NHM5) as compared to wild type. Our results indicate that the inactivation of uptake hydrogenase in N. punctiforme changes the overall metabolic equilibrium, affecting both oxygen reduction mechanisms in heterocysts as well as processes providing reducing equivalents for metabolic functions such as N(2) fixation. We identify specific metabolic processes used by NHM5 to maintain a high rate of N(2) fixation, and thereby potential targets for further improvement of nitrogenase based H(2) photogeneration. These targets include, but are not limited to, components of the oxygen scavenging capacity and cell envelope of heterocysts and proteins directly or indirectly involved in reduced carbon transport from vegetative cells to heterocysts.     

Minimising iTRAQ ratio compression through understanding LC-MS elution dependence and high-resolution HILIC fractionation

Application of iTRAQ-based workflows for protein profiling has become widespread. Concomitantly, the idiosyncratic limitations of iTRAQ, such as its tendency to underestimate quantifications, have been studied and recognised. This report shows that the influence of ratio compression and limiting transmission in iTRAQ MS/MS in high-complexity mixtures (iTRAQ-labelled lysates) can be partly alleviated using high-resolution sample fractionation. Here, we also investigate in greater detail the dependency of iTRAQ quantification on the dynamics of online chromatography in low-complexity mixtures (iTRAQ-labelled standards). These findings will allow more efficient strategies to be designed for iTRAQ proteomics, alleviating iTRAQ underestimation and thus facilitating the detection of subtle abundance changes.