Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth

Abstract

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the ␣-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that ϳ50 uncharacterized genes were regulated by changes in light intensity and O 2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H 2 as an electron donor and CO 2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO 2 ) were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO 2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO 2 , or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions. Life on earth is dependent upon photosynthesis, either directly using energy from sunlight or indirectly through the use of organic compounds produced by photosynthetic organisms. Due to the importance of photosynthesis, much research has been devoted to understanding this process in plants, algae, and photosynthetic bacteria. The defining feature of all photosynthetic organisms is their ability to use light energy to generate reducing power, which is used to support the synthesis of ATP, the assimilation of CO 2 , or the synthesis of other compounds. We are studying the requirements for photosynthesis in the ␣-proteobacterium Rhodobacter sphaeroides, a facultative phototroph that also can produce energy by aerobic or anaerobic respiratory pathways.Since the continual flow of electrons through electron carriers is critical to energy production, cells have evolved strategies to maintain necessary electron flow by recycling or disposing of excess reducing power. For example, fermentative pathways use organic compounds as electron acceptors, and electron transport to O 2 allows the aerobic respiratory chain to produce energy. When O 2 is absent, anaerobic respiratory pathways reduce alternate electron acceptors like dimethyl sulfoxide (DMSO) to dispose of excess reducing power (18). Photosynthetic growth also requires a strategy for recycling reducing power, but di...

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

“…RNA isolation and subsequent cDNA synthesis were performed as previously described [49]. qRT-PCR experiments were conducted in triplicate for each biological replicate using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich).…”

Organization and Evolution of the Biological Response to Singlet Oxygen Stress

“…The dataset contains RNA abundance measurements from 67 GeneChip Custom Express microarrays (Affymetrix, Santa Clara, CA51) obtained from wild-type or mutant strains grown in a succinate-based minimal medium under different conditions (GSM1670, GSM1671, GSM1672, GSM1673, GSM2416, GSM2417, GSM2418, GSM2419, GSM2420, GSM2421, GSM2422, GSM2423, GSM2424, GSM2425, GSM2426, GSM2427, GSM2429, GSM2430, GSM2450, GSM25295, GSM25296, GSM25297, GSM25298, GSM25299, GSM25300, GSM25301, GSM25302, GSM25303, GSM26242, GSM26243, GSM26244, GSM26245, GSM26260, GSM26262, GSM26263, GSM26265, GSM26266, GSM27348, GSM27349, GSM27350, GSM27351, GSM27352, GSM27353, GSM3030, GSM3031, GSM3032, GSM3258, GSM3260, GSM3262, GSM3272, GSM3273, GSM3274, GSM38774, GSM38775, GSM38776, GSM38777, GSM38778, GSM38779, GSM38780, GSM38781, GSM38782, GSM38783, GSM38810, GSM40560, GSM8107, GSM8108, and GSM8109) 6,26,51–53. RNA levels in all datasets were normalized using the Robust Multichip Average method 54 to log 2 scale.…”

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

“…Total RNA was prepared from harvested cells as previously described (60) and was then treated with RNasefree DNase I (Invitrogen, Carlsbad, CA) and purified by using an RNeasy minikit (Qiagen, Valencia, CA) as described by the manufacturer. The RNA concentration was quantitated with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE).…”