a b s t r a c tThe Photosystem II (PS II) assembly factors Psb27 and Ycf48 are transiently associated with PS II during its biogenesis and repair pathways. We investigated the function of these proteins by constructing knockout mutants in Synechocystis sp. PCC 6803. In DYcf48 cells, PS II electron transfer and stable oxygen evolution were perturbed. Additionally, Psb27 was required for photoautotrophic growth of cells lacking Ycf48 and assembly beyond the RC47 assembly complex in DYcf48:DPsb27 cells was impeded. Our results suggest the RC47 complex formed in DYcf48 cells is defective and that this deficiency is exacerbated if CP43 binds in the absence of Psb27.

Blends of engineered polymerases enable efficient DNA-templated synthesis and reverse transcription of diverse-sequence oligonucleotide libraries composed of locked nucleic acid (LNA), 2′-O-methyl-RNA (2′OMe-RNA), or mixtures of the two.

The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.

Absorption spectroscopy is widely used to determine absorption and transmission spectra of chromophores in solution, in addition to suspensions of particles, including micro-organisms. Light scattering, caused by photons deflected from part or all of the cells or other particles in suspension, results in distortions to the absorption spectra, lost information and poor resolution. A spectrophotometer with an integrating sphere may be used to alleviate this problem. However, these instruments are not universally available in biology laboratories, for reasons such as cost. Here, we describe a novel, rapid, and inexpensive technique that minimises the effect of light scattering when performing whole-cell spectroscopy. This method involves using a custom made dual compartment cuvette containing titanium dioxide in one chamber as a scattering agent. Measurements were conducted of a range of different photosynthetic micro-organisms of varying cell size and morphology, including cyanobacteria, eukaryotic microalgae and a purple non-sulphur bacterium. A concentration of 1 mg ml−1 titanium dioxide, using a spectrophotometer with a slit width of 5 nm, produced spectra for cyanobacteria and microalgae similar (1–4% difference) to those obtained using an integrating sphere. The spectrum > 520 nm was similar to that with an integrating sphere with the purple non-sulphur bacterium. This system produced superior results to those obtained using a recently reported method, the application of the diffusing agent, Scotch™ Magic tape, to the side of the cuvette. The protocol can be completed in an equivalent period of time to standard whole-cell absorbance spectroscopy techniques, and is, in principle, suitable for any dual-beam spectrophotometer.

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