Kinetic characterization of His-tagged CP47 Photosystem II in Synechocystis sp. PCC6803

Recently, construction of strains of Synechocystis sp. PCC6803 having a His6 extension (His-tag) of the carboxyl terminus of the CP47 protein has been reported (T.M. Bricker et al, Biochim. Biophys. Acta 1409 (1998) 50; M.J. Reifler et al., in: Garab, Pusztai (Eds.) Proc. XIth International Congress on Photosynthesis, 1998). While these initial reports suggest a minimal impact of the His-tag upon Photosystem (PS) II function, a more thorough analysis of the kinetic properties of the modified complex is essential. This communication reports on a more detailed kinetic analysis to assess possible perturbations of PS II due to the genetic addition of the His-tag on the CP47 protein. It was found that: (1) Patterns of flash O2 yield exhibited normal period four oscillations and the associated fits of the Kok-Joliot S-state cycling parameters were virtually identical to the wild type; (2) O2 release kinetics during the S3-S0 transition were experimentally indistinguishable from the wild type; (3) S-state decay measurements indicate slightly faster decays of the S2 and S3 states compared to the wild type; (4) fluorescence measurements indicate that the kinetics of the forward reaction of electron transfer from Q(A)/- to Q(B) and back-reactions of Q(A)/- with PS II electron donors are similar in the His-tag and wild-type strains. It is therefore concluded that the addition of the His-tag results in a minimal perturbation of PS II function. (C) 2000 Elsevier Science B.V

Redox changes accompanying inorganic carbon limitation in Synechocystis sp. PCC 6803

AbstractInorganic carbon (Ci) is the major sink for photosynthetic reductant in organisms capable of oxygenic photosynthesis. In the absence of abundant Ci, the cyanobacterium Synechocystis sp. strain PCC6803 expresses a high affinity Ci acquisition system, the CO2-concentrating mechanisms (CCM), controlled by the transcriptional regulator CcmR and the metabolites NADP+ and α-ketoglutarate, which act as co-repressors of CcmR by modulating its DNA binding. The CCM thus responds to internal cellular redox changes during the transition from Ci-replete to Ci-limited conditions. However, the actual changes in the metabolic state of the NADPH/NADP+ system that occur during the transition to Ci-limited conditions remain ill-defined. Analysis of changes in the redox state of cells experiencing Ci limitation reveals systematic changes associated with physiological adjustments and a trend towards the quinone and NADP pools becoming highly reduced. A rapid and persistent increase in F0 was observed in cells reaching the Ci-limited state, as was the induction of photoprotective fluorescence quenching. Systematic changes in the fluorescence induction transients were also observed. As with Chl fluorescence, a transient reduction of the NADPH pool (‘M’ peak), is assigned to State 2→State 1 transition associated with increased electron flow to NADP+. This was followed by a characteristic decline, which was abolished by Ci limitation or inhibition of the Calvin–Benson–Bassham (CBB) cycle and is thus assigned to the activation of the CBB cycle. The results are consistent with the proposed regulation of the CCM and provide new information on the nature of the Chl and NADPH fluorescence induction curves

Evaluating the effect of promoter strength on the complementation of deletion mutants in Synechocystis sp. PCC 6803

The CO2 uptake mechanism within Cyanobacteria plays a vital role in our environment due to its photosynthetic ability to uptake CO2 and produce oxygen. Cyanobacteria possesses an inorganic carbon concentrating mechanism including two CO2 specific uptake systems. One is constitutive and one is low-CO2 inducible. By limiting the CO2 availability, the bacteria will express the inducible CO2 concentrating mechanisms to survive. These systems include NDH-13, the inducible complex and NDH-14, the constitutive complex. The goal of our lab is to focus our studies on the inducible NDH-13 system, however, there are concerns that the promoter strength of our complementation strains will prove problematic to future studies. To overcome this concern, we tested three different promoters with varied expression patterns and then determined which would yield the best construct for future experiments. Included were the constitutive Rubisco and PsbA2 promoters, along with the native NdhF3 promoter. Using Gibson Assembly, plasmids containing these variations were constructed. A deletion mutant was also constructed within our lab lacking both systems, C2. Spot assays on BG-11 media were completed under conditions of air and CO2 gassing. A positive, wild-type, control and negative, M55, control was tested along with C2 and the three complementations. A phenotypic analysis was completed to observe if the promoter strength influences the cells' ability to grow within the different conditions.Lew Wentz FoundationMicrobiology and Molecular Genetic

Participation of Glutamate-354 of the CP43 Polypeptide in the Ligation of Manganese and the Binding of Substrate Water in Photosystem II

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn4Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn4Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H218O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S3 state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S1 state Mn-EXAFS spectrum, a slightly altered S2 state multiline EPR signal, a substantially altered S 2-minus-S1 FTIR difference spectrum, and an unusually long lifetime for the S2 state (>10 h) in a substantial fraction of reaction centers. In contrast, the S2 state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S2-minus-S 1 FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with 15N and specific labeling with l-[1-13C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO- group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm -1 in both the S1 and S2 states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S 2 state and that most of these can achieve the S3 state, but no evidence for advancement beyond the S3 state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn4Ca cluster in the S 2 state, the FTIR and H218O exchange data show that the mutation strongly influences other properties of the Mn4Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S1 to S2 transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S3 state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn4Ca cluster in the S1 state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S1 to S 2 transition. The H218O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S 3 state

The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

BACKGROUND: DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. RESULTS: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1(st ) round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. CONCLUSIONS: The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling

Systems and Photosystems: Cellular Limits of Autotrophic Productivity in Cyanobacteria

Recent advances in the modeling of microbial growth and metabolism have shown that growth rate critically depends upon the optimal allocation of finite proteomic resources among different cellular functions and that modeling growth rates becomes more realistic with the explicit accounting for the costs of macromolecular, most importantly, protein expression. The ‘proteomic constraint’ is considered together with its application to understanding photosynthetic microbial growth. The central hypothesis is that physical limits of cellular space (and corresponding solvation capacity) and cell surface to volume ratios represent the underlying constraints on the maximal rate of autotrophic microbial growth. The limitation of cellular space thus constrains the size the total complement of macromolecules, dissolved ions, and metabolites. To a first approximation, the upper limit in the cellular amount of the total proteome is bounded the space limit. This predicts that adaptation to osmotic stress will result in lower maximal growth rates due to decreased cellular concentrations of core metabolic proteins necessary for cell growth owing the accumulation of compatible osmolytes, as surmised previously. The finite capacity of membrane and cytoplasmic space also leads to the hypothesis that the species-specific differences in maximal growth rates likely reflect differences in the allocation of space to niche-specific proteins with the corresponding diminution of space devoted to other functions including proteins of core autotrophic metabolism, which drive cell reproduction. An optimization model for autotrophic microbial growth, the autotrophic replicator model (ARM), was developed based upon previous work investigating heterotrophic growth. The present model describes autotrophic growth in terms of the allocation protein resources among core functional groups including the photosynthetic electron transport chain, light harvesting antennae, and the ribosome groups

Cyclic Electron Flow-Coupled Proton Pumping in Synechocystis sp. PCC6803 Is Dependent upon NADPH Oxidation by the Soluble Isoform of Ferredoxin:NADP-Oxidoreductase

International audienceFerredoxin:NADP-oxidoreductase (FNR) catalyzes the reversible exchange of electrons between ferredoxin (Fd) and NADP(H). Reduction of NADP+ by Fd via FNR is essential in the terminal steps of photosynthetic electron transfer, as light-activated electron flow produces NADPH for CO2 assimilation. FNR also catalyzes the reverse reaction in photosynthetic organisms, transferring electrons from NADPH to Fd, which is important in cyanobacteria for respiration and cyclic electron flow (CEF). The cyanobacterium Synechocystis sp. PCC6803 possesses two isoforms of FNR, a large form attached to the phycobilisome (FNRL) and a small form that is soluble (FNRS). While both isoforms are capable of NADPH oxidation or NADP+ reduction, FNRL is most abundant during typical growth conditions, whereas FNRS accumulates under stressful conditions that require enhanced CEF. Because CEF-driven proton pumping in the light–dark transition is due to NDH-1 complex activity and they are powered by reduced Fd, CEF-driven proton pumping and the redox state of the PQ and NADP(H) pools were investigated in mutants possessing either FNRL or FNRS. We found that the FNRS isoform facilitates proton pumping in the dark–light transition, contributing more to CEF than FNRL. FNRL is capable of providing reducing power for CEF-driven proton pumping, but only after an adaptation period to illumination. The results support that FNRS is indeed associated with increased cyclic electron flow and proton pumping, which is consistent with the idea that stress conditions create a higher demand for ATP relative to NADPH

Regulation of CO2 Concentrating Mechanism in Cyanobacteria

In this chapter, we mainly focus on the acclimation of cyanobacteria to the changing ambient CO2 and discuss mechanisms of inorganic carbon (Ci) uptake, photorespiration, and the regulation among the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth. The structural components for several of the transport and uptake mechanisms are described and the progress towards elucidating their regulation is discussed in the context of studies, which have documented metabolomic changes in response to changes in Ci availability. Genes for several of the transport and uptake mechanisms are regulated by transcriptional regulators that are in the LysR-transcriptional regulator family and are known to act in concert with small molecule effectors, which appear to be well-known metabolites. Signals that trigger changes in gene expression and enzyme activity correspond to specific “regulatory metabolites” whose concentrations depend on the ambient Ci availability. Finally, emerging evidence for an additional layer of regulatory complexity involving small non-coding RNAs is discussed