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Robert L Burnap

Publications and source records attributed to Robert L Burnap.

10 recordsLinked to original sources
PubMed2005

Multiflash experiments reveal a new kinetic phase of photosystem II manganese cluster assembly in Synechocystis sp. PCC6803 in vivo.

The assembly of Mn(2+) ions into the H(2)O oxidation complex (WOC) of the photosystem II (PSII) reaction center is a light-driven process, termed photoactivation. According to the "two-quantum" model, photoactivation involves two light-driven charge separations coupled to the photooxidation of Mn(2+) in order to form the first stable intermediate in a process that culminates in the oxidative assembly of four Mn(2+) ions and one Ca(2+) ion to form the active, higher valence (Mn(4)-Ca) center of the WOC. To better define the kinetics of the dark rearrangement and to gain some understanding of the basis for the very low quantum yield of the overall process, photoactivation experiments, involving different flash patterns, were conducted with Synechocystis sp. PCC6803. It was found that even the so-called first stable intermediate is readily lost during protracted (1-10 s) dark periods during photoactivation of Synechocystis cells. Low concentrations of the electron acceptor, DCBQ, improved the stability of the dark intermediates. The unstable photoactivation intermediates formed early in the photoactivation process were not, however, stabilized by the addition of Ca(2+), although the overall yield of photoactivation is enhanced by the additional Ca(2+). Measurements of the kinetics of fluorescence yield verify that Q(A)(-) to Q(B) electron transfer rates change during the course of photoactivation as the high potential form of Q(A)(-) is converted to the low potential form and show that DCBQ acts as an efficient electron acceptor from Q(A)(-) even while in its high potential form. In addition the approximately 150 ms phase corresponding to the originally described dark rearrangement of photoactivation, repetitive, double flash experiments, with a 10 s intervening dark period, reveals a faster, 15 ms phase that is accentuated by DCBQ.

Benzoquinones↗
PubMed2005

Expression, mutagenesis, and characterization of recombinant low-potential cytochrome c550 of photosystem II.

Cytochrome c(550) of the photosystem II complex of cyanobacteria is an unusual member of the large protein family of monoheme c-type cytochromes. Despite the fact that it shares considerable amino acid sequence similarity and has a protein fold similar to the other members of the family, Cyt.c(550) has a midpoint potential (E(m7) = -250 mV) that is much lower than the positive midpoint potentials characteristic (E(m7) = 100-300mV) of this cytochrome family. An E. coli heterologous expression system involving secretion of the recombinant protein from Synechocystis PCC6803 to the periplasm was utilized to allow production of wild-type and mutant forms of the cytochrome. For most of the variants studied, the yield of protein was significantly enhanced by growth at 28 degrees C and inclusion of sucrose and betaine, in addition to isopropyl-beta-d-thiogalactoside (IPTG), to the growth medium of the E. coli expression host. Analysis of the protein products revealed that the wild-type protein maintained the redox and visible spectroscopic characteristics of the authentic protein. Mutations in the residues engaging in hydrogen bond interactions with the heme propionate (Asn49) and the axial 6th ligand His92 (Pro93) resulted in small (12-20 mV), but reproducible, upshifts in midpoint redox potential. Substitution of the axial ligand His92 with Met produced no discernible changes in the optical spectrum relative to the wild-type despite the fact that in this mutant, unlike the others studied here, the thioether linkage either was not formed or was highly labile as evidenced by loss of the heme during SDS-PAGE. On the other hand, the midpoint potential of the C550-H92M mutant was upshifted by approximately 70 mV. This value is significantly less of a perturbation than that observed in a similar mutant that is natively expressed in Thermosynechoccocus but appears to have an intact thioether linkage between the heme and the polypeptide moiety.

Bacterial Proteins↗
PubMed2004

In situ effects of mutations of the extrinsic cytochrome c550 of photosystem II in Synechocystis sp. PCC6803.

The H(2)O oxidizing domain of the cyanobacterial photosystem II (PSII) complex contains a low potential, c-type cytochrome termed c(550) that is essential for the in vivo stability of the PSII complex. A mutant lacking cytochrome c(550) (DeltapsbV) in Synechocystis sp. PCC6803 has been further analyzed together with a construct in which the distal axial heme iron ligand, histidine 92, has been substituted with a methionine (C550-H92M). Heme staining of SDS-PAGE showed that the C550-H92M mutation did not disturb the accumulation and heme-binding properties of the cytochrome. In DeltapsbV cells, the number of charge separating PSII centers was estimated to be 56% of the wild type, but of the existing centers, 33% lacked photooxidizable Mn ions. C550-H92M did not discernibly affect the intrinsic PSII electron-transfer kinetics compared to the wild type nor did it exhibit a significant fraction of centers lacking photooxidizable Mn; however, the number of charge separating PSII centers in mutant cells was 69% of the wild type. C550-H92M lost photoautotrophic growth ability in the absence of Ca(2+), but its growth was not affected by depletion of Cl(-), which differs from DeltapsbV. Taken together, the results suggest that in the absence of cytochrome c(550) electron transfer on the donor side is retarded perhaps at the level of Y(z) to P680(+) transfer, the heme ligand. His92 is not absolutely required for assembly of functional PSII centers; however, replacement by methionine prevents normal accumulation of PSII centers in the thylakoid membranes and alters the Ca(2+) requirement of PSII. The results are discussed in terms of current understanding of the Ca(2+) site of PSII.

Amino Acid Substitution↗
PubMed2004

Consequences of a deletion in dspA on transcript accumulation in Synechocystis sp. strain PCC6803.

A sensor histidine kinase of Synechococcus sp. strain PCC7942, designated nblS, was previously identified and shown to be critical for the acclimation of cells to high-light and nutrient limitation conditions and to influence the expression of a number of light-responsive genes. The nblS orthologue in Synechocystis sp. strain PCC6803 is designated dspA (also called hik33). We have generated a dspA null mutant and analyzed global gene expression in both the mutant and wild-type strains under high- and low-light conditions. The mutant is aberrant for the expression of many genes encoding proteins critical for photosynthesis, phosphate and carbon acquisition, and the amelioration of stress conditions. Furthermore, transcripts from a number of genes normally detected only during exposure of wild-type cells to high-light conditions become partially constitutive in the low-light-grown dspA mutant. Other genes for which transcripts decline upon exposure of wild-type cells to high light are already lower in the mutant during growth in low light. These results suggest that DspA may influence gene expression in both a positive and a negative manner and that the dspA mutant behaves as if it were experiencing stress conditions (e.g., high-light exposure) even when maintained at near-optimal growth conditions for wild-type cells. This is discussed with respect to the importance of DspA for regulating the responses of the cell to environmental cues.

Bacterial Proteins↗
PubMed2003

Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator.

The cyanobacterium Synechocystis sp. PCC 6803 possesses multiple inorganic carbon (Ci) uptake systems that are regulated by Ci availability. The control mechanisms of these systems and their integration with other cell functions remain to be clarified. An analysis of the changes in global gene expression in response to Ci downshift and the inactivation of ndhR (sll1594), a LysR family regulator of Ci uptake is presented in this report. Mild Ci limitation (3% CO2 (v/v) in air to air alone) induced a dramatic up-regulation of genes encoding both inducible CO2 and HCO3- uptake systems. An induction of ndhD5/ndhD6 and other genes in a probable transcriptional unit was observed, suggesting a function in inducible Ci uptake. The expression of slr1513 and sll1735, physically clustered with sbtA and ndhF3/ndhD3/cupA, respectively, were also coordinated with upstream genes encoding the essential components for HCO3- and CO2 uptake. Ci limitation induced the regulatory genes slr1214, sll1292, slr1594, sigD, sigG, and sigH, among which slr1214, a two-component response regulator, showed the earliest induction, implying a role for the early response to Ci limitation. Opposite regulation of genes encoding the assimilation of carbon and nitrogen demonstrated a striking coordination of expression to balance C- and N-fluxes. The analyses revealed that ndhR inactivation up-regulated the expression of sbtA/sbtB, ndhF3/ndhD3/cupA/sll1735, and slr2006-13 including ndhD5 and ndhD6, indicating a vital role of this regulatory gene in both CO2 and HCO3- acquisition of the cyanobacterium. We therefore suggest that ndhR be renamed ccmR to better represent its broader regulatory characteristics.

Bacterial Proteins↗
PubMed2003

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 1st 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.

Cyanobacteria↗
PubMed2003

Introduction.

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Journal Article↗
PubMed2002

Mutations of basic arginine residue 334 in the D1 protein of Photosystem II lead to unusual S(2) state properties in Synechocystis sp. PCC 6803.

The C-terminus region of the D1 protein of Photosystem II (PS II) is situated on the lumenal side of the complex and is likely to be involved in the coordination of the active site Mn atoms of the water oxidation complex (WOC). The strictly conserved arginine at position 334 (D1-334) was targeted for site-directed mutagenesis to explore the hypothesis that it is involved in the PS II extrinsic protein binding, chloride binding, or proton transfer. Although it was found that D1-R334 probably not essential for these functions, mutations at this position were found to uniquely alter the kinetics of S-state cycling in general and the properties of the S(2) state in particular. Substitutions of a glutamate (D1-R334E) and a valine (D1-R334V) for D1-R334 lead to an unusually stable (t (1/2) >30 min at room temp) S(2) state, but not S(3), as measured by double flash measurements on the bare platinum electrode. However, measurements of fluorescence decay in the presence of DCMU suggest the S(2) state is only modestly affected by the mutations. Possible reasons for these apparently contradictory results are discussed.

Journal Article↗
PubMed2002

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803.

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.

ATP-Binding Cassette Transporters↗