David Lea-Smith - Academia.edu (original) (raw)

Papers by David Lea-Smith

Plant physiology, 2016

Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and... more Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1...

Journal of Visualized Experiments, 2016

Cyanobacteria are ecologically important organisms and potential platforms for production of biof... more Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2015

Proceedings of the National Academy of Sciences, 2015

Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can b... more Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2-540 pg alkanes per mL per day, which translates into a global ocean yield of ∼308-771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities.

ACS Chemical Biology, 2014

An enantioselective synthesis of (+)-corynomycolic acid, and its elaboration to esters of trehalo... more An enantioselective synthesis of (+)-corynomycolic acid, and its elaboration to esters of trehalose, glucose and glycerol, is described.

PLANT PHYSIOLOGY, 2013

CB2 1PZ United Kingdom (S.A.S.)

Physical Chemistry Chemical Physics, 2013

Biological photo-voltaic systems are a type of microbial fuel cell employing photosynthetic micro... more Biological photo-voltaic systems are a type of microbial fuel cell employing photosynthetic microbes at the anode, enabling the direct transduction of light energy to electrical power. Unlike the anaerobic bacteria found in conventional microbial fuel cells that use metals in the environment as terminal electron acceptors, oxygenic photosynthetic organisms are poorly adapted for electron transfer out of the cell. Mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 were created in which all combinations of the three respiratory terminal oxidase complexes had been inactivated. These strains were screened for the ability to reduce the membrane-impermeable soluble electron acceptor ferricyanide, and the results were compared to the performance of the mutants in a biological photovoltaic system. Deletion of the two thylakoid-localised terminal oxidases, the bd-quinol oxidase and cytochrome c oxidase resulted in a 16-fold increase in ferricyanide reduction rate in the dark compared to the wild-type. A further improvement to a 24-fold increase was seen upon deletion of the remaining ''alternative respiratory terminal oxidase''. These increases were reflected in the peak power generated in the biological photo-voltaic systems. Inactivation of all three terminal oxidase complexes resulted in a substantial redirection of reducing power; in the dark the equivalent of 10% of the respiratory electron flux was channelled to ferricyanide, compared to less than 0.2% in the wild-type. Only minor improvements in ferricyanide reduction rates over the wild-type were seen in illuminated conditions, where carbon dioxide is preferentially used as an electron sink. This study demonstrates the potential for optimising photosynthetic microbes for direct electrical current production.

Journal of Biological Chemistry, 2007

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Co... more Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced ␤-keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.

Journal of Biological Chemistry, 2008

The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacteriu... more The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum ⌬NCgl2106 was restored by complementation with M. tuberculosis Rv2188c.

Journal of Bacteriology, 2005

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three pos... more The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc 1 complex and terminating in the aa 3 -type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc 1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O 2 -limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc 1 -deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc 1 -aa 3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR twocomponent regulator.

Energy & Environmental Science, 2013

Microbial electrolysis cells (MECs) represent an emerging technology that uses heterotrophic micr... more Microbial electrolysis cells (MECs) represent an emerging technology that uses heterotrophic microbes to convert organic substrates into fuel products, such as hydrogen gas (H 2 ). The recent development of biophotovoltaic cells (BPVs), which use autotrophic microbes to produce electricity with only light as a substrate, raises the possibility of exploiting similar systems to harness photosynthesis to drive the production of H 2 . In the current study we explore the capacity of the cyanobacterium Synechocystis sp.

Current Opinion in Biotechnology, 2010

Plant Physiology, 2016

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, ... more Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here we demonstrate that hydrocarbon deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modelling of membranes suggests these compounds aggregate in the centre of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild-type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.