Plant LHC-like proteins show robust folding and static non-photochemical quenching

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)—abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.

Bacterial carotenoids suppress Caenorhabditis elegans surveillance and defense of translational dysfunction

Microbial toxins and virulence factors often target the eukaryotic translation machinery. Caenorhabditis elegans surveils for such microbial attacks by monitoring translational competence, and if a deficit is detected, particular drug detoxification and bacterial defense genes are induced. The bacteria Kocuria rhizophila has evolved countermeasures to animal translational surveillance and defense pathways. Here, we used comprehensive genetic analysis of Kocuria rhizophila to identify the bacterial genetic pathways that inhibit C. elegans translational toxin surveillance and defense. Kocuria rhizophila mutations that disrupt its ability to disable animal immunity and defense map to multiple steps in the biosynthesis of a 50-carbon bacterial carotenoid from 5 carbon precursors. Extracts of the C50 carotenoid from wild type K. rhizophila could restore this bacterial anti-immunity activity to K. rhizophila carotenoid biosynthetic mutant. Corynebacterium glutamicum, also inhibits the C. elegans translation detoxification response by producing the C50 carotenoid decaprenoxanthin, and C. glutamicum carotenoid mutants are defective in this suppression of C. elegans detoxification. Consistent with the salience of these bacterial countermeasures to animal drug responses, bacterial carotenoids sensitize C. elegans to drugs that target translation and inhibit food aversion behaviors normally induced by protein translation toxins or mutations. The surveillance and response to toxins is mediated by signaling pathways conserved across animal phylogeny, suggesting that these bacterial carotenoids may also suppress such human immune and toxin responses.

The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides

Coloured carotenoids play some undefined role in the assembly of a functional light-harvesting 2 (LH2) complex in photosynthetic bacteria. We have used a series of transposon Tn5 insertion mutants disrupted at various stages of the carotenoid-biosynthetic pathway, together with an LH2 deletion/insertion mutant, to investigate this effect in Rhodobacter sphaeroides. Mutants were initially characterized by low-temperature absorbance spectroscopy and ultrastructural analysis: Northern-blot analysis demonstrated normal pucBA transcripts for LH2 polypeptides in all the carotenoid mutants. Analysis of translation of the puc transcript and investigation of the fate of any resulting LH2 polypeptides by SDS/PAGE, Western-blot and pulse-chase experiments clearly demonstrated that, in the absence of coloured carotenoids, the LH2 alpha- and beta-polypeptides are synthesized but are rapidly turned over and do not become stably integrated into the membrane. Complementation of mutants with lesions in the crtB and crtI genes, encoding phytoene synthase and phytoene desaturase respectively, with the cloned R. sphaeroides crtI gene, resulted in restoration of carotenoid biosynthesis and stable assembly of the LH2 complex in the crtI mutant but not in the crtB mutant, despite the presence of the CrtI protein.

Photoregulation of Carotenoid Biosynthesis in Mutants of Neurospora crassa: Activities of Enzymes Involved in the Synthesis and Conversion of Phytoene

Synthesis of carotenoids is photoregulated in many fungi including Neurospora crassa. In order to investigate the regulatory mechanism at the enzyme level, several carotenoid mutants of Neurospora were used to determine the activities of enzymes involved in the carotenoid bio synthetic pathway after growth under illumination or in darkness. Light stimulation of carotenoid formation was due to enhanced activities of three subsequent enzymes, geranylgeranyl pyrophosphate synthase, phytoene synthase, and phytoene desaturase indicating a coordinated regulation at the enzyme level. Farnesyl pyrophosphate synthase and lycopene cyclase were not involved in light regulation. Immunological studies showed that in the case of phytoene desaturase higher activity in the light originated from an increased amount of this enzyme in light-grown cultures.