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The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO

Cyclic electron flow (CEF) around Photosystem I is vital to balancing the photosynthetic energy budget of cyanobacteria and other photosynthetic organisms. The coupling of CEF to proton pumping has long been hypothesized to occur, providing proton motive force (PMF) for the synthesis of ATP with no net cost to [NADPH]. This is thought to occur largely through the activity of NDH-1 complexes, of which cyanobacteria have four with different activities. While a much work has been done to understand the steady-state PMF in both the light and dark, and fluorescent probes have been developed to observe these fluxes in vivo, little has been done to understand the kinetics of these fluxes, particularly with regard to NDH-1 complexes. To monitor the kinetics of proton pumping in Synechocystis sp. PCC 6803, the pH sensitive dye Acridine Orange was used alongside a suite of inhibitors in order to observe light-dependent proton pumping. The assay was demonstrated to measure photosynthetically driven proton pumping and used to measure the rates of proton pumping unimpeded by dark ΔpH. Here, the cyanobacterial NDH-1 complexes are shown to pump a sizable portion of proton flux when CEF-driven and LEF-driven proton pumping rates are observed and compared in mutants lacking some or all NDH-1 complexes. It is also demonstrated that PSII and LEF are responsible for the bulk of light induced proton pumping, though CEF and NDH-1 are capable of generating ∼40% of the proton pumping rate when LEF is inactivated.Highlights statementNDH-1 is essential for proton pumping during cyclic photosynthetic electron flow in cyanobacteria

The CO2-concentrating mechanism (CCM) in cyanobacteria supports high rates of photosynthesis by greatly increasing the concentration of CO2 around the major carbon fixing enzyme, Rubisco. However, the CCM remains poorly understood, especially in regards to the enigmatic CO2-hydration enzymes which couple photosynthetically generated redox energy to the hydration of CO2 to bicarbonate. This CO2-hydration reaction is catalysed by specialized forms of NDH-1 thylakoid membrane complexes that contain phylogenetically unique extrinsic proteins that appear to couple CO2 hydration to NDH-1 proton pumping. The development of the first molecular genetic system to probe structure-function relationships of this important enzyme system is described. A CO2-hydration deficient strain was constructed as a recipient for DNA constructs containing different forms of the CO2-hydration system. This was tested by introducing a construct to an ectopic location that gives constitutive expression, rather than native inducible expression, of the ndhF3-ndhD3-cupA-cupS, (cupA operon) encoding high affinity CO2-hydration complex, NDH-13. Uptake assays show the restoration of high affinity for CO2 uptake, but demonstrate that the CupA complex can drive only modest uptake fluxes, underlining the importance of its tandem operation with the CupB-containing complex NDH-14, the complementary high flux, low affinity CO2 hydration system. Experiments with the carbonic anhydrase inhibitor, ethoxyzolamide, indicate that the NDH-13 complex is strongly inhibited, yet the remaining NDH-14 activity in the wild-type is less so, suggesting structural differences between the low affinity and high affinity CO2-hydration systems. This new construct will be an important tool to study and better understand cyanobacterial CO2 uptake systems.

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). This includes specialized Type-1 NDH complexes that function to couple photosynthetic redox energy to CO2 hydration forming the bicarbonate that accumulates to high cytoplasmic concentrations during the operation of the CCM, required for effective carbon fixation. Here we used a Synechococcus PCC7942 expression system to investigate the role of conserved histidine and cysteine residues in the CupB (also designated, ChpX) protein, which has been hypothesized to participate in a vectoral CO2 hydration reaction near the interface between CupB protein and the proton-pumping subunits of the NDH-1 complex. A homology model has been constructed and most of the targeted conserved residues are in the vicinity of a Zn ion modeled to form the catalytic site of deprotonation and CO2 hydration. Growth and CO2 uptake assays show that the most severe defects in activity among the targeted residues are due to a substitution of the predicted Zn ligand, CupB-His86. Mutations at other sites produced intermediate effects. Proteomic analysis revealed that some amino acid substitution mutations of CupB caused the induction of bicarbonate uptake proteins to a greater extent than complete deletion of CupB, despite growth under CO2-enriched conditions. The results are discussed in terms of hypotheses on the catalytic function of this unusual enzyme.

Genetic engineering of photosynthetic organisms typically redirects native metabolism towards desirable products, which thereby represent new metabolic sinks. There is limited information on how these modifications impact the evolved mechanisms of photosynthetic energy metabolism and cellular growth. Two engineered strains of Synechocystis sp. PCC 6803 with altered carbon sink capacity were assayed for their photosynthetic and CO2 concentrating mechanism properties in conditions of high and low inorganic carbon (Ci) availability. In the ΔglgC mutant, glycogen cannot be synthesized and a carbon sink pathway has been effectively removed. The JU547 strain has been engineered by integration of the Pseudomonas syringae ethylene forming enzyme and provides a new sink. When cultured under high carbon conditions, ΔglgC displayed diminished photochemical efficiency, a more reduced NADPH pool, delayed initiation of the Calvin-Benson-Bassham cycle, and impairment of linear and cyclic electron flows. It also exhibited a large decrease in photochemical quenching indicative of the accumulation of QA −, normally associated with a reduced PQ pool, but appears instead to be the result of an undefined dissipative mechanism to spill excess energy. In the case of carbon sink integration, JU547 displayed slightly more oxidized PQ and NADPH pools and increased rates of cyclic electron flow and an enhanced demand for inorganic carbon as suggested by increase in the expression of the bicarbonate transporter, SbtA. Overall, the results highlight the importance of the native regulatory network of autotrophic metabolism in governing photosynthetic performance and provide cogent examples of both predicable and difficult to predict phenotypic consequences upon installation of new pathways in autotrophs.

What are the cellular limits that constrain the maximal rate of microbial cell duplication? What are the genetic determinants conferring maximal microbial growth rates? In the case of Cyanobacteria—an ecologically ubiquitous and diverse phylum of prokaryotes capable of oxygenic photosynthesis—these have increasingly become questions of both applied and fundamental significance. In PNAS, in a technical tour de force of reciprocal genome editing by Ungerer et al. (1), two genetically similar cyanobacterial strains with very different growth phenotypes are shown to have their large phenotypic differences controlled by remarkably few alleles. What is perhaps even more remarkable is the nature of the relationship between these alleles and the surprising case of allele synergy that may help illustrate how broad phenotypes such as growth rate and overall metabolic capacity can be controlled. Cyanobacteria have long proved to be excellent experimental models for the study of photosynthesis and other aspects of autotrophic growth. More recently, cyanobacteria have emerged as important targets for sustainable biotechnology due to their amenability to genetic manipulation and their ability to grow on little more than sunlight, carbon dioxide, and minerals (2). Different species of cyanobacteria exhibit a very wide range of maximal rates of photosynthesis and, correspondingly, a wide range of maximal growth rates (reviewed in ref. 3). This is important since “fast growth” is often an important characteristic in choosing a cyanobacterial strain for engineering. An especially interesting example of how different strains of cyanobacteria can have very different growth rates are recent comparisons of two closely related strains of the rod-shaped unicellular cyanobacteria, each described as Synechococcus elongatus (4⇓–6). Understanding these strains’ background is important to the whole story. One of the two strains, Synechococcus elongatus PCC 7942 (Pasteur Culture Collection designation) ( Synechococcus 7942), was isolated in the early 1970s in California and … [↵][1]1To whom correspondence should be addressed. Email: rob.burnap{at}okstate.edu. [1]: #xref-corresp-1-1

The CO

Based on the colloid chemistry of a suspension of ODS packing material, a new packing technique using particle sedimentation has been developed. With this approach, a slurry consisting of the packing and a deflocculating solvent is poured into the column, the bed was formed by sedimentation. The bed is then solidified by a flocculating solvent. Using acetone as the slurry solvent and methanol—water (1:1) as the solidification solvent, high-quality columns (150 mm × 21 mm I.D., 16-μm C18-silica packing) were successfully packed with reduced plate heights of 2.5–3.6 and typical A terms in the Knox plot below 1.0. The normalized sedimentation volume is introduced as a basis for the selection of sedimentation and solidification solvents. Various experimental factors, including slurry concentration, solidification flow-rate programming and temperature, are studied and discussed.

The applicability of high-performance hydrophobic interaction chromatography using newly developed silica-based ether-bonded phases is demonstrated in the purification of the rat liver enzymes carbamoylphosphate synthetase I and ornithine transcarbamoylase from crude mitochondrial extracts. As a result of the mild adsorption/elution conditions in this high-performance chromatographic mode, the enzymes are recovered in 20 min with 3- to 15-fold increases in specific activity. Since the enzymes are labile and may aggregate in solution, in one case up to Mr 330,000, this rapid purification demonstrates the potential of hydrophobic interaction chromatography in complex biological systems.

This paper is a continuation of the evaluation of hydrophilic ether columns for the separation of proteins by hydrophobic-interaction chromatography. In this approach, linear salt gradients of decreasing concentration of ammonium sulfate yield sharp chromatographic peaks with high mass recovery and maintenance of biological activity. Mild adsorption conditions are indicated by the minimal changes in chromatographic peak area of native proteins as a function of contact time by the biopolymer with the stationary phase. Further evidence of minimal kinetic processes under the chromatographic conditions are seen in the constantly of isocratic retention with mobile phase flow-rate and sample load up to 2 mg. Based on the well-behaved chromatographic characteristics, we have explored gradient optimization in terms of the Snyder model for gradient elution. It is shown that changes in retention, peak capacity and peak height follow predicted gradient time dependencies. Moreover, the influence of particle diameter and column length are found to be in agreement with expected behavior, based on the model. As a consequence of the agreement, prediction of conditions for optimum separation for a particular problem are possible. Other studies examine the influence of specific ion effects, e.g. , Mg 2+ binding to protein, which can override retention based on predicted surface tension behavior. Some characteristics of the ether column for hydrophobic-interaction chromatography are shown, e.g. , column stability at pH 8 and sample capacity.

A TLC-densitometric method for determination of the triazine herbicides atrazine and simazine in natural and tap water is described. Separation was carried out on silica gel G, followed by detection with silver nitrate and UV exposure. Alumina column cleanup was required for tap water. Recovery of samples fortified at 10 ppb was > 80%, and the relative standard deviation was better than 7.5%.

Practical aspects of the analysis of amino acids are addressed in this paper. Conditions for the resolution of enantionmers of all teh common amino acids as their Dns derivatives using the chiral mobile phase additive: l-prolyl-n-octylamide-Ni(II) are reported. Variable d,l selectivity is obsserved, while maintaining good column efficiency, l-prolyl-n-dodecylamide-Ni(II) is found to distribute on the surface of a n-alkyl-bonded stationary phase and may act as a chiral immobilized phase. The separation of all the common Dns-amino acids within ≈ 35 min using reversed-phase conditions and a linear gradient is presented. Approaches to enantiomeric composition analysis of amino acids in a mixture are discussed, and a coupled column strategy linking the two separations reported above is proposed. In the combined system amino acid analysis is performed in the first part, with switching oof individual amino acids of interest to the second part of the system for d,l-enantiomeric composition determination.

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure  Si-(CH 2 ) 3 -O-(CH 2 -CH 2 -O) n -R, where n  1, 2, 3 and R  methyl, ethyl or n -butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g. , from 3 M ammonium sulfate (pH 6.0, 25°C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g. , 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[ n ]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.