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Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.

ABSTRACT: The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.

SUBMITTER: McNeely K

PROVIDER: S-EPMC3067432 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.

McNeely Kelsey K Xu Yu Y Ananyev Gennady G Bennette Nicholas N Bryant Donald A DA Dismukes G Charles GC

Applied and environmental microbiology 20110211 7

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attri ...[more]

PMID: 21317262

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Project description:RNA degradation is an important process that influences the ultimate concentration of individual proteins inside cells. While the main enzymes that facilitate this process have been identified, global maps of RNA turnover are available for only a few species. Even in these cases, there are few sequence elements that are known to enhance or destabilize a native transcript; even fewer confer the same effect when added to a heterologous transcript. To address this knowledge gap, we assayed genome-wide RNA degradation in the cyanobacterium Synechococcus sp. strain PCC 7002 by collecting total RNA samples after stopping nascent transcription with rifampin. We quantified the abundance of each position in the transcriptome as a function of time using RNA-sequencing data and later analyzed the global mRNA decay map using machine learning principles. Half-lives, calculated on a per-ORF (open reading frame) basis, were extremely short, with a median half-life of only 0.97 min. Despite extremely rapid turnover of most mRNA, transcripts encoding proteins involved in photosynthesis were both highly expressed and highly stable. Upon inspection of these stable transcripts, we identified an enriched motif in the 3' untranslated region (UTR) that had similarity to Rho-independent terminators. We built statistical models for half-life prediction and used them to systematically identify sequence motifs in both 5' and 3' UTRs that correlate with stabilized transcripts. We found that transcripts linked to a terminator containing a poly(U) tract had a longer half-life than both those without a poly(U) tract and those without a terminator.IMPORTANCE RNA degradation is an important process that affects the final concentration of individual mRNAs, affecting protein expression and cellular physiology. Studies of how RNA is degraded increase our knowledge of this fundamental process as well as enable the creation of genetic tools to manipulate RNA stability. By studying global transcript turnover, we searched for sequence elements that correlated with transcript (in)stability and used these sequences to guide tool design. This study probes global RNA turnover in a cyanobacterium, Synechococcus sp. strain PCC 7002, that both has a unique array of RNases that facilitate RNA degradation and is an industrially relevant strain that could be used to convert CO2 and sunlight into useful products.

Dataset Information

Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.

Publications

Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.

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