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Journal of Bacteriology, August 2005, p. 5249-5258, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5249-5258.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth{dagger}

Christine L. Tavano, Angela M. Podevels, and Timothy J. Donohue*

University of Wisconsin—Madison Department of Bacteriology, 420 Henry Mall, Madison, Wisconsin 53711

Received 2 February 2005/ Accepted 3 May 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the {alpha}-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that ~50 uncharacterized genes were regulated by changes in light intensity and O2 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 H2 as an electron donor and CO2 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 CO2) 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 CO2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO2, 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 CO2, or the synthesis of other compounds. We are studying the requirements for photosynthesis in the {alpha}-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 O2 allows the aerobic respiratory chain to produce energy. When O2 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 different pathways can be used for this purpose.

Under photoautotrophic conditions, the Calvin-Benson-Bassham (Calvin) cycle both assimilates CO2 and recycles reducing power, since this pathway uses considerable reductant (34). Accordingly, in photosynthetic bacteria like R. sphaeroides, expression of genes encoding Calvin cycle functions is induced even during photosynthetic growth using a fixed carbon source (8). In addition, an R. sphaeroides Calvin cycle mutant cannot grow photoheterotrophically in the absence of the anaerobic electron acceptor DMSO (18), presumably because cells lack the ability to recycle reducing power (8). However, the ability of wild-type R. sphaeroides cells to grow photoheterotrophically in the absence of exogenous electron acceptors like DMSO or CO2 predicts that cells use other processes to recycle reductant when using fixed carbon sources.

We have identified a set of genes, RSP4157 to -4159, that are required for photoheterotrophic growth of R. sphaeroides. Clustering of global gene expression data grouped RSP4157 to RSP4159 together with genes that are known to be required for photosynthetic growth of this bacterium, since their expression is regulated by O2 tension and light intensity. A strain containing a polar insertion in the RSP4157 gene (CT01) was able to grow photoautotrophically but did not grow photoheterotrophically in the absence of compounds known to serve as outlets for excess reducing power (i.e., DMSO, CO2). CT01 also had decreased expression of so-called glc genes, which encode proteins predicted to convert glycolate to CO2. In addition, we found that the addition of glycolate or malate (a GlcB-dependent source of CO2) rescued photoheterotrophic growth of CT01. Based on these findings, we propose that glycolate degradation and one or more proteins encoded by RSP4157 to -4159 play a previously unknown role in recycling reducing power during photoheterotrophic growth of R. sphaeroides.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains, media, and growth conditions Escherichia coli DH5{alpha} was used as a plasmid host, and S17-1 (31) was used to conjugate DNA into R. sphaeroides (Table 1). E. coli cultures were grown in Luria-Bertani medium with ampicillin (100 µg/ml), kanamycin (50 µg/ml), spectinomycin (25 µg/ml), or tetracycline (20 µg/ml) as needed. R. sphaeroides was grown at 30°C in Sistrom's succinate minimal medium (32) with kanamycin (25 µg/ml), spectinomycin (25 µg/ml), or tetracycline (1 µg/ml) as needed. Where indicated, this medium was supplemented with the indicated concentrations of glycolate, glyoxylate, or malate.


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  		TABLE 1. Strains and plasmids used in this work

 
For most experiments, R. sphaeroides cultures were grown as follows: aerobic respiration, 10 ml medium in 125-ml flasks shaken at 250 rpm in the dark; anaerobic photosynthesis, in filled 16-ml tubes at a light intensity of 10 W/m2 for medium-light growth or 100 W/m2 for high-light growth; anaerobic respiration, in filled 16-ml tubes in the dark using medium supplemented with 0.625% DMSO; low O2, 500 ml medium bubbled with 3% O2 in the dark (30). Photoautotrophic cultures were grown in Sistrom's succinate medium supplemented with 40 mM NaHCO3 and 100% H2. Cultures were inoculated with anaerobically grown cells (shake flask), except for photosynthetic (10 W/m2 or 100 W/m2) conditions, for which cultures were grown in the dark with 0.625% DMSO to a density of ~1.0 x 108 cells/ml and then transferred to the appropriate light intensity for ~12 h of growth before recording cell densities.

RNA isolation. For RNA preparation, triplicate cultures were grown by aerobic respiration at 30% or 3% O2 in the dark or via photosynthesis (anaerobic, 10 W/m2) (30). High- or low-O2 cultures were inoculated from 10-ml aerobic cultures that were grown to late exponential phase, and photosynthetic cultures were inoculated from cultures in sealed 16-ml tubes grown at 10 W/m2 light. Cultures were grown in 500 ml Sistrom's medium bubbled with gas (30% O2, 30% O2-69% N2-1% CO2; 3% O2, 3% O2-96% N2-1% CO2; photosynthesis, 95% N2-5% CO2) for at least five cell doublings until they reached ~2 x 108 to 3 x 108 CFU/ml.

Cells were harvested by transferring 10 ml of culture to an ice-cold 15-ml conical tube containing 1.25 ml of 5% water-saturated phenol in ethanol. Cells were collected by centrifugation at 5,000 x g for 5 min at 4°C, and the cell pellet was frozen in a dry ice-ethanol bath prior to storage at –80°C. The pellet was resuspended in 800 µl of 0.5 mg/ml lysozyme in TE (10 mM Tris-Cl [pH 8.0], 1 mM EDTA). Cells were lysed by the addition of 1/10 volume 10% sodium dodecyl sulfate and incubation at 64°C for 2 min. Next, 1/10 volume of 1 M sodium acetate, pH 5.2, was added, followed by extraction with an equal volume of water-saturated phenol. Samples were incubated at 64°C for 6 min with mixing every 30 s and then chilled on ice before centrifugation at 16,000 x g for 10 min at 4°C. The aqueous phase was removed and extracted with phenol a second time, followed by extraction with an equal volume of chloroform. To the final aqueous phase we added 1/10 volume 3 M sodium acetate (pH 5.2), 1 mM EDTA, and 2 to 2.5 volumes cold ethanol. The samples were incubated at –80°C for ≥20 min and centrifuged at 16,000 x g for 25 min at 4°C to collect the nucleic acids. The pellet was washed with ice-cold 80% ethanol, centrifuged for 5 min, and air dried, followed by resuspension in 86 µl of water. To remove DNA, the sample was treated with RNase-free DNase (Sigma, Woodlands, TX) in 50 mM Tris-Cl (pH 7.5)-50 mM MgCl2-10 mM dithiothreitol (DTT) for 20 min at room temperature. The remaining nucleic acid was purified using an RNeasy CleanUp kit (QIAGEN, Valencia, CA), followed by a second DNase treatment with Turbo DNase (Ambion, Austin, TX) and subsequent purification of RNA using an RNeasy kit. RNA quality was assayed spectrophotometrically (i.e., A260/A280 = 1.8 to 2.0) and formamide-agarose gel electrophoresis.

cDNA synthesis and labeling. Samples for microarray analysis were prepared from 10 µg RNA using Affymetrix Antisense Genome Array Protocols with the following modifications (Affymetrix). For cDNA synthesis, RNA was mixed with 3 µg random hexamer primers (Invitrogen, Carlsbad, CA). After RNA was removed from cDNA synthesis reaction mixtures, 10 µl of 3 M sodium acetate, pH 5.4, was added and cDNA was purified using a PCR Purification Kit (QIAGEN, Valencia, CA). cDNA amounts were quantitated using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE); only reaction mixtures with yields of over 30% were used for microarray hybridization. The cDNA was fragmented with 0.2 U RQ1 DNase (Promega, Madison, WI) in 1x RQ1 buffer by incubation at 37°C for 10 min, followed by heating to 99°C for 10 min to stop the reaction. Fragmented cDNA was end labeled with biotin-16-ddUTP using Terminal Transferase (Enzo, Farmingdale, NY) for 2 h at 37°C. Before hybridization, the sample was concentrated to ≤30 µl using YM-10 columns (Millipore, Bedford, MA).

Microarray procedures, data normalization, and clustering. Labeled cDNA samples were hybridized to R. sphaeroides GeneChip CustomExpress microarrays (26) (Affymetrix) following product instructions, except that DMSO was added to 7% (vol/vol) in the hybridization solution. Samples were hybridized for 16 h at 45°C on a rotisserie spindle rotating at 60 rpm. Microarrays were washed, stained, and scanned using the Pseudomonas aeruginosa midi-array hybridization with amplification protocols (Affymetrix, Santa Clara, CA).

After data extraction using Affymetrix MAS 5.0 software, data were imported into GeneSpring (Silicon Genetics, Redwood City, CA). Normalization used the following: each microarray was normalized to the 50th percentile, and then each gene was normalized to its median value. To report differences between the absolute gene expression values for Fig. 1 to 3, data were only normalized per microarray to the 50th percentile. All microarray data sets were deposited at the Gene Expression Omnibus (GEO) website under series no. GSE2150 (http://www.ncbi.nlm.nih.gov/geo/). The same normalization methods were used for the following R. sphaeroides data sets posted at the GEO website (microarray no.): 30% O2 (GSM2420 to GSM2423, and GSM3030 to GSM3032); 3% O2 (GSM2425 and GSM2426); photosynthetic medium light, i.e., 10 W/m2 (GSM2427, GSM2428, GSM3258, GSM3260, and GSM3262); photosynthetic high light, i.e., 100 W/m2 (GSM3272 to GSM3274); and anaerobic respiration, i.e., DMSO (GSM2429 and GSM2430) (26, 30). Genes induced threefold or more under photosynthetic conditions were divided into four clusters by K-means clustering (26, 30) using a standard correlation similarity measure.



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  		FIG. 1. Physical representation of ORFs in K-means cluster 2 on each strand of linearized depictions of chromosomes 1 and 2 (C1 and C2) and each of the five R. sphaeroides plasmids (P001 to P005). Plasmid P001 is shown in three contigs based on the current genome alignments (a, b, and c). Select genes are labeled, including puc2B (RSP1556), hemA (RSP2984), nuo (RSP0101 to -0103, 0105, 0107, 0108, and 0112), hemZ (RSP0699), osp (RSP0869), dksA (RSP0166), cbbX (RSP1280), the photosynthesis gene cluster (PGC), and the RSP4157 gene cluster. The gray scale on the right indicates the relative level of expression for each gene in cells grown via photosynthesis at 10 W/m2. (The data used in this analysis are in the GEO database under microarray numbers GSM2427, GSM2428, GSM3258, GSM3260, and GSM3262.)

 


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  		FIG. 3. Normalized (see Materials and Methods) expression values for select genes from R. sphaeroides 2.4.1 microarray data sets. Growth conditions are as follows: respiration at 30% or 3% O2 (dark), PS10 (medium-light photosynthesis, 10 W/m2), PS100 (high-light photosynthesis, 100 W/m2), and DMSO (anaerobic dark, 0.625% DMSO). RSP numbers for genes are as follows: (A) pucB, 0314; puhA, 0291; pufA, 0258; pufL, 0257; cycA, 0296; (B) crtD, 0266; crtI, 0271; bchC, 0263; cbbFI, 1285 cbbFII, 3266; dorA, 3048. Genes encoding the photosynthetic apparatus (A) or pigment biosynthesis, Calvin cycle, and anaerobic respiration (DMSO) functions (B) are marked. The asterisk indicates that the expression value for dorA in the DMSO condition is 107.5. Note that the expression values for panels A and B use different scales.

 
Primer extension assays. Primers (100 pmol) specific to RSP4157 (5'-GGTTCTTCGCTTCGCTTCGCATTG-3'), pucB (5'-GTTCAGATCGTCAGTCACTGTGTCGTC-3'), or coxII (5'-AGGTCGTGGAATGTCTCATG-3') were radiolabeled with [{gamma}-32P]ATP (35). {alpha}-32P-labeled primers (6.7 pmol) were annealed to 10 µg RNA in 50 mM Tris-Cl (pH 8.3)-60 mM NaCl-10 mM DTT by heating to 95°C for 2 min and slowly cooled to 40°C. Primer extension reaction mixtures contained the RNA-primer mix, 0.5 mM deoxynucleoside triphosphates, 40 U RNasin (Promega, Madison, WI), and 0.1 mg actinomycin D in 1x RT buffer (25 mM Tris-Cl [pH 8.3], 30 mM NaCl, 5 mM MgCl2, 5 mM DTT) for a 35-µl total volume. Reactions were initiated by addition of 2 U avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and incubated at 42°C for 40 min. Reactions were terminated by adding 3x stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF). To map the transcription initiation site, a DNA sequence ladder was generated using the {alpha}-32P-labeled pucB primer annealed to pUI601 (12). Samples were heated to 95°C for 5 min and analyzed alongside on a 6% polyacrylamide-8 M urea gel in order to map transcription initiation sites. The gel was dried and visualized using a PhosphorImager (Molecular Dynamics).

Cloning and analysis of the RSP4157 gene cluster. The RSP4157 to RSP4164 region was amplified using Pfu Turbo (Stratagene, La Jolla, CA) (5'-GACCTCGGCCTGAGCCACCTG-3' and 5'-CTCAAGAGCGGAGAGGTCGGTAATTC-3'). The PCR product was digested with PstI, and the resulting 4.1-kb fragment was cloned into the PstI site of pUC19 (22) to make pCT101. A 2.0-kb {Omega}Spr/Smr cassette, excised from pHP45{Omega} (29) with SmaI, was inserted into the EcoRV site of RSP4157 in pCT101 to create pCT104. A PstI digest of pCT104 generated a 6.1-kb fragment that was cloned into the PstI site of pSUP202 (31) to make pCT122.

An R. sphaeroides strain with an insertion in RSP4157 was made by mating pCT122 into wild-type cells and screening spectinomycin-resistant cells for tetracycline sensitivity. Primers 5'-CGTGTATGAAATCTAACAATGCGCTC-3' and 5'-GTCCAGTGATCGAAGTTAGGCTGG-3' did not detect a tetracycline resistance gene; 5'-CACGGCGTACCCGATGAAGGGAATC-3' and 5'-CAGTAAACACAATCGTAGTTGCACACC-3' showed that a {Omega}Spr/Smr cassette was present in RSP4157.

Genomic DNA flanking RSP4157 to -4162 was amplified with Pfu Turbo (Stratagene, La Jolla, CA) and primers comp1 (5'-GCTCTAGAAGGATCATCGCCTTCAGCGCAGCA-3') and comp4 (5'-GCTCTAGACGTTCAGGAACCGTGCACCGTCGAA-3'). The product was digested with XbaI (sites present in the primers) and cloned into pCM66 (17) to create pCT132.

Southern blot analysis of an R. sphaeroides genomic library identified cosmid pUI8593 as containing RSP4157 (4). DNA sequencing of the cloned R. sphaeroides DNA determined that pUI8593 contained RSP4157 to -4164 plus ~3.7 kb of DNA upstream and ~8.7 kb of DNA downstream of this region (primers: 5'-AAAGTAAACTGGATGGCTTTCTTGC-3' and 5'-TGCGAAACGATCCTCATCCTGT-3').


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of candidate photosynthesis genes. In R. sphaeroides, the expression of many genes known to be required for photosynthetic growth is low at high O2 tensions, but it is increased by a decline in either O2 tension or light intensity under anaerobic conditions (15, 23). There are many uncharacterized genes in the R. sphaeroides genome, and we hypothesized that some of the uncharacterized genes that may be required for photosynthetic growth would have expression patterns paralleling those of genes known to be required under these conditions. To test this hypothesis, we monitored global gene expression patterns in wild-type cells grown by respiration at 30% O2 (which lack the photosynthetic apparatus) (13) or photosynthetically at 10 W/m2 light intensity. Microarray analysis called ~87% of the 4,966 annotated genes as being expressed (average raw value, ≥100) under each condition. These data also identified 282 genes that had ≥3-fold higher RNA levels in photosynthetic cultures, including 42 that were located within the so-called photosynthesis gene cluster (2, 24). Photosynthetically grown cells also showed increased expression of other functionally characterized genes (i.e., hemA, hemZ, puc2B, osp, and dksA), as well as genes encoding motility or chemotaxis functions, Calvin cycle enzymes, or NADH dehydrogenase (nuo) subunits (5, 10, 20, 26, 30, 39, 40, 41).

To identify candidates for additional gene products that might be involved in photosynthetic growth, a K-means algorithm was used to group the 282 photosynthesis-induced genes based on similarities in expression patterns (K = four clusters). To assemble a larger data set for this cluster analysis, we included gene expression data for wild-type cells grown by aerobic respiration at 30% or 3% O2 tension, photosynthetically at medium (10 W/m2) or high (100 W/m2) light intensity, and by anaerobic respiration (dark with 0.625% DMSO) (26, 30; see supplementary data tables under series entry GSE2150 at the GEO website and Table S1 in the supplemental material for lists of genes in clusters 1 to 4).

Each of the four clusters contained genes that shared a distinctive gene expression pattern. Genes in clusters 1 (65 genes) and 3 (26 genes) were not consistently induced during photosynthetic growth at either light intensity. Genes in cluster 4 (60 genes) were induced during photosynthesis at high or medium light but not under low (3%)-O2 or anaerobic respiratory conditions. Cluster 4 included 11 genes that encode Calvin cycle enzymes (RSP3266 to -3271 and RSP1281 to -1285), which are required for CO2 fixation. With the exception of the Calvin cycle genes, these three clusters did not contain any genes known to be required for photosynthetic growth.

The 131 genes in cluster 2 had expression patterns characteristic of genes that are known to be required for photosynthetic growth (13, 23). Of the genes in cluster 2, 37 were located in the photosynthesis gene cluster (Fig. 1); others included hemA, hemZ, puc2B, osp, dksA, several genes encoding electron transport proteins (RSP2687, cytochrome bc1, Fig. 1), one Calvin cycle gene (cbbX), and ~50 uncharacterized genes encoding proteins of unknown function (Table 2). Of the uncharacterized genes in cluster 2, RSP4157 to RSP4164 were located on the self-transmissible 101-kb plasmid P004 (33). These genes were flanked by open reading frames (ORFs) predicted to encode transposase proteins (RSP4156 and RSP4165 to RSP4166), which were transcribed from the opposite strand and exhibited low or unchanged levels of RNA under all conditions analyzed (Fig. 2). Like the other genes in cluster 2, the expression of RSP4157 to RSP4164 was increased during photosynthetic growth compared to cells grown at 30% O2 tension (Fig. 2 and 3A), and under low-O2 or anaerobic respiratory (DMSO) conditions (Fig. 3A). RSP4157 to -4164 also had decreased expression at high light intensity compared to medium-light photosynthesis, as was the case for genes encoding known components of the photosynthetic apparatus (i.e., pucB, puhA, pufA, pufL, and cycA; Fig. 3A) (3, 11, 13, 30, 39). In contrast, genes encoding photopigment biosynthesis enzymes (crtDI and bchC) showed similar RNA abundance regardless of light intensity (Fig. 3B) (21, 37), while RNA levels from Calvin cycle genes (cbbFI or cbbFII) were only marginally increased at low O2 tensions and were not altered significantly in response to changes in light intensity (Fig. 3B). As expected, dorA (DMSO reductase) expression was only induced under anaerobic conditions in the presence of DMSO (Fig. 3B) (18, 19).


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  		TABLE 2. Putative ORFs in cluster 2a

 


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  		FIG. 2. Expression values (normalized per chip; see Materials and Methods) are shown for RSP4156 to -4166. Gray bars, 30% O2; black bars, photosynthetic (10 W/m2) condition. The representation of RSP4156 and -4166 depicts ORFs with predicted functions in gray, hypothetical ORFs (encoding unknown functions) in white, and ORFs encoding putative transposases in black. (These data have been deposited in the GEO database under series number GSE2145.)

 
As an independent test of the global gene expression results, primer extension assays showed that RSP4157-specific mRNA was 7- to 12-fold more abundant in the photosynthetic (10 W/m2) cultures compared to cells grown in 30% O2 (Fig. 4). The start of transcription (+1 site) for RSP4157 was mapped to 44 nucleotides upstream of the predicted ATG start of RSP4157 (putative promoter: TTGACA-17 nucleotides-TCAAAT). As controls for this primer extension analysis, we monitored mRNA abundance from pucB, which encodes a light-harvesting complex subunit in the photosynthetic apparatus, and coxII, which encodes a cytochrome aa3 terminal oxidase subunit (Fig. 4) (6, 12). In both cases, we observed primer extension products of the expected size and abundance, based on previous experiments (6, 12). For example, pucB mRNA was undetectable by primer extension assays in cells grown at 30% O2, but it was in high abundance in cells grown by photosynthesis (Fig. 4). The opposite was true of coxII mRNA, which only produced a detectable product using RNA from cells grown at 30% O2 (Fig. 4). Having confirmed that a promoter upstream of RSP4157 to -4164 was induced during photosynthetic growth, we investigated the potential role of these genes in photosynthesis.



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  		FIG. 4. (A) Primer extension products (marked with arrows) specific to coxII, RSP4157, and pucB. Predicted sizes of primer extension products (in nucleotides) are noted (data not shown). P, products obtained using RNA from photosynthetically grown cells (10 W/m2); A, products obtained from cells grown by aerobic respiration at 30% O2; nt, nucleotides. (B) Graph showing relative amounts of primer extension products (signal intensity). Extracted data are from representative samples of three independent replicates. Aero, aerobic (30% O2) condition; Photo, photosynthetic (10 W/m2) growth condition; n.d., none detected.

 
Characterization of an RSP4157 mutant. To test whether one or more of these genes were needed for photosynthetic growth, we constructed a strain, CT01, containing an insertion in RSP4157 that should be polar onto any cotranscribed genes. In liquid cultures, CT01 grew as well as wild-type cells by aerobic respiration in shake flasks (generation time [tgen], ~4 h) (Fig. 5) or by anaerobic respiration (data not shown). When CT01 was grown by respiration at 3% O2, its tgen (~4.0 h) was also similar to that of wild-type cells (tgen, ~3.3 h) (Table 3). The wild-type colony morphology of CT01 when grown by anaerobic respiration suggested that it synthesized a normal complement of pigment-protein complexes. In fact, whole-cell absorbance spectra indicated that CT01 produced wild-type levels of light-harvesting complexes when grown by aerobic respiration at 3% O2 (data not shown).



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  		FIG. 5. Growth of wild-type and CT01 cells by photosynthesis or by respiration at 30% O2. Late exponential phase aerobic cultures were inoculated as follows: for aerobic cultures, cells were diluted to ~2.5 x 106 cells/ml, and for photosynthetic growth, cells were diluted to ~2 x 107 cells/ml. PS, photosynthetic (10 W/m2) growth condition; aerobic, 30% O2 condition.

 

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  		TABLE 3. Growth of strains under different conditionsa

 
We also found that CT01 was able to grow photoautotrophically in liquid cultures using H2 as an electron donor and CO2 as its sole carbon source (Table 3). Under this condition, CT01 had a doubling time comparable to wild-type cells even though it had a longer growth lag (by ~12 h) after transfer from anaerobic respiration conditions (with DMSO). This lag in growth may reflect altered expression or function of H2 oxidation or CO2 assimilation pathways in cells lacking RSP4157 to -4159. The ability of CT01 to grow under photoautotrophic conditions indicates that the insertion in RSP4157 does not prevent formation of a functional photosynthetic apparatus, a hydrogen oxidation pathway, or the Calvin cycle.

Despite apparently normal photosynthetic apparatus production, we found that CT01 was unable to grow photoheterotrophically in a succinate-based minimal medium (Table 3). No growth was observed on petri plates after 2 to 3 days under photoheterotrophic conditions, a time frame in which wild-type R. sphaeroides produced colonies. Dilution plating of an aerobically grown culture of CT01 under aerobic or photosynthetic conditions on a succinate minimal medium indicated that this strain reverted to photoheterotrophic competence at a frequency of 10–4 to 10–5. Incubation of CT01 under photoheterotrophic conditions was apparently bacteriostatic, since a plate showing no colonies after being incubated anaerobically in the light for 2 to 3 days produced the expected number of colonies after being incubated under aerobic growth conditions for another 2 to 3 days.

The growth defect and reversion of CT01 could be monitored in liquid photoheterotrophic cultures. Under photoheterotrophic conditions, CT01 had an apparent tgen of 5.8 h, compared to 3.0 h for wild-type cells (Fig. 5). Dilution plating under aerobic and photosynthetic conditions showed that the apparent growth of CT01 under photoheterotrophic conditions was correlated with the time-dependent accumulation of photoheterotrophically competent cells. For example, the inoculum contained ~1 to 10 in 105 photoheterotrophic competent cells, but the number of these cells had increased 100-fold (to ~1 to 10 in 103 cells) by the end of exponential growth. Cells in the CT01 cultures also aggregated during photoheterotrophic growth, presumably due to the growth defect of this strain. We also tested whether photoheterotrophic growth of CT01 could be rescued by high light intensity, as is the case for strains lacking light-harvesting complexes that revert at a similar frequency (14). We found that high light intensity did not allow photoheterotrophic growth of CT01 (Table 3).

To ensure that these properties of CT01 were due to the insertion in RSP4157, we tested the ability of a cosmid (pUI8593) containing this region from wild-type cells to rescue the growth defect. Photoheterotrophic growth of CT01 was restored by pUI8593, since the tgen of this strain (~4.6 h) was comparable to that of wild-type cells (~4.2 h). In addition, a plasmid (pCT132) containing only RSP4157 to RSP4162 under control of their native promoter(s) complemented CT01, but one containing only RSP4157 did not (data not shown). From these results, we conclude that one or more genes in this region are required for photoheterotrophic growth in the presence of succinate.

Cells containing an insertion in RSP4157 have a defect in recycling reducing power under photosynthetic conditions. One possible explanation for the inability of CT01 to grow photosynthetically in the presence of a fixed carbon source like succinate is a problem with recycling excess reducing power (7, 8, 36). To test if CT01 had a defect in recycling reducing power during photosynthetic growth, we asked whether addition of exogenous electron acceptors restored growth. DMSO is known to serve as an alternate electron acceptor under anaerobic conditions in R. sphaeroides (7, 18). We found that the addition of DMSO to succinate minimal medium allowed CT01 to grow photoheterotrophically with wild-type doubling times (Table 3). When CO2 is present during photoheterotrophic growth, the Calvin cycle also can serve as a sink for excess reducing power (7, 8). We also found that the addition of 5% CO2 restored photoheterotrophic growth of CT01 (Table 3). In comparison, a previously described Calvin cycle mutant, CfxA–B–, could not grow photoheterotrophically unless an external electron acceptor like DMSO was added (Table 3) (8). Since CfxA–B– is unable to use the Calvin cycle, the presence of 5% CO2 does not restore growth under photoheterotrophic conditions (8). From these properties of CT01, we conclude that the RSP4157 gene cluster plays a role in recycling reducing power under photosynthetic conditions.

How might loss of the RSP4157 cluster gene products alter recycling of reducing power? To gain additional insight into the role of the RSP4157 gene cluster, we examined global gene expression changes caused by the mutation in CT01 using cells grown by respiration at 3% O2. This growth condition results in production of the photosynthetic apparatus but does not cause growth differences between wild-type cells and CT01. In this analysis, four genes had ≥3-fold increased expression and 25 genes had ≥3-fold decreased expression in CT01, relative to wild-type cells. The genes with increased RNA abundance in CT01 were predicted to encode prophage proteins or hypothetical proteins with no known function (Table 4) and so did not provide insight into the properties of this strain. Genes with ≥3-fold less RNA in CT01 included RSP4157 to -4159, presumably resulting from the polar mutation in RSP4157. This result suggests that RSP4157 to -4159 are cotranscribed and that loss of one or more of these gene products is responsible for the photoheterotrophic growth defect of CT01. Several genes located on chromosome 1 (including RSP1018 to -1020 and RSP1980) also had ≥3-fold less RNA in CT01. These genes encode homologs of E. coli glycolate oxidase (GlcEF) and malate synthase G (GlcB) (25, 27).


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  		TABLE 4. ORFs with changed expression in CT01 versus wild type in 3% oxygen

 
One explanation for the decreased expression from glc genes in CT01 is an unknown link between glycolate metabolism and one or more of the RSP4157 to -4159 gene products. The E. coli Glc proteins oxidize glycolate to glyoxylate, with subsequent release of CO2 as glyoxylate is converted either to malate (by GlcB) or to 3-phosphoglycerate (25, 27). Further oxidation of malate also produces CO2 (see Discussion). Given the ability of glycolate degradation to generate CO2, we reasoned that decreased expression of the R. sphaeroides glc genes in CT01 may limit CO2 production under photoheterotrophic conditions. If this were the case, the addition of glycolate to CT01 might allow production of sufficient CO2 to restore photoheterotrophic growth. As predicted, photoheterotrophic growth of CT01 was restored by adding 30 mM glycolate to succinate-based minimal medium (Table 3): the growth rate of CT01 was comparable to that of wild-type cells, and the proportion of photoheterotrophically competent cells in the population remained at 1 to 10 in 105. By testing a range of glycolate concentrations, we found that >15 mM glycolate was required to rescue photoheterotrophic growth of CT01 (data not shown); this is reminiscent of the large amounts of glycolate required to rescue growth of E. coli glc mutants (25, 27). Neither wild-type nor CT01 mutant cells were able to use glycolate as a sole source of carbon, so it is unlikely that glycolate is serving as a major carbon source during photoheterotrophic growth (data not shown). Malate also can generate CO2 via Glc-independent enzymes (36), and we found that addition of 34 mM malate rescued photoheterotrophic growth of CT01 in succinate-containing medium (Table 3). For an unknown reason, the addition of 30 mM glyoxylate did not rescue photoheterotrophic growth of CT01, even though it is a predicted intermediate of the glycolate degradation pathway. As expected, the addition of glycolate or malate did not rescue photoheterotrophic growth of CfxA–B–, which cannot use the Calvin cycle (Table 3). When taken together, our results suggest that Glc proteins can produce CO2, which can be used to recycle reducing power under photoheterotrophic conditions. Our data also predict that one or more products of RSP4157 to -4159 are needed for efficient operation of the Glc pathway in R. sphaeroides (see Discussion).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
All cells need to recycle reducing power to balance energy production with biosynthesis and other critical functions. The Calvin cycle and anaerobic respiration with DMSO are two known pathways that serve to recycle reducing power in photosynthetic bacteria like R. sphaeroides (7, 8, 36). In this work, global gene expression analysis was used to search for uncharacterized genes that could be involved in photosynthetic growth of this bacterium. Our data predict that RSP4157 to -4159 and glycolate degradation serve important roles in recycling reducing power generated during the photosynthetic lifestyle.

Cells lacking RSP4157 to -4159 have defects in recycling reducing power during photoheterotrophic growth. We found that cells containing a polar mutation in RSP4157 (CT01) had a defect in recycling reducing power during photoheterotrophic growth. CT01 had normal levels of light-harvesting complexes during growth at low O2 tension and was able to grow photoautotrophically. In some respects, CT01 behaved like a Calvin cycle mutant (CfxA–B–) that was unable to use CO2 as an electron acceptor during photosynthetic growth (8), since both strains required an external electron acceptor like DMSO to grow under photoheterotrophic conditions. However, the ability of CT01 to grow photoautotrophically indicates that it has a functional Calvin cycle. Furthermore, addition of exogenous CO2 restored photoheterotrophic growth of CT01, indicating that this mutant contains a functional Calvin cycle to serve as an electron sink for reducing power generated during photosynthetic growth (36).

CT01 also had decreased levels of RNA specific for genes that encode homologs of enzymes for glycolate degradation in E. coli (27), prompting us to test the role of glycolate in recycling reducing power during photosynthesis. Our data lead us to propose that glycolate metabolism by GlcEF and malate oxidation by GlcB produce CO2, which can be used by the Calvin cycle to recycle excess reducing power generated under photosynthetic conditions by R. sphaeroides. Activity of either R. sphaeroides GlcEF (glycolate dehydrogenase) or GlcB (malate synthase) (see the EcoCyc glycol metabolism and degradation superpathway at http://www.ecocyc.org) could generate CO2 for subsequent use by the Calvin cycle during photosynthetic growth (8, 34). A pathway similar to that found in enteric bacteria appears to function in R. sphaeroides since either glycolate or malate rescued photoheterotrophic growth of CT01.

Our model predicts that glycolate should be formed under photoheterotrophic conditions. Although the metabolic source(s) of glycolate in photosynthetic bacteria is unknown, significant amounts of glycolate can be formed by the oxygenase activity of 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a reaction that converts ribulose-1,5-bisphosphate into 3-phosphoglycerate and phosphoglycolate. Glycolate production by RuBisCO is an energetically wasteful and potentially deleterious process, since this compound is a potent inhibitor of triose phosphate isomerase (1, 16). In many phototrophs, consequent oxidation of glycolate to glyoxylate and ultimately 3-phosphoglycerate is an essential part of photorespiration (16). While photosynthetic bacteria like R. sphaeroides do not evolve O2 as a by-product of photochemical activity, there is evidence that O2 is present to support carotenoid biosynthesis in anoxic laboratory cultures (38). In addition, R. sphaeroides and other photosynthetic bacteria are found in low-O2 environments (28), so formation of glycolate by RuBisCO and its oxidation to CO2 by the Glc pathway may be a means to recycle reducing power that is relevant in nature. Homologs of the glc genes also are predicted to exist in photosynthetic bacteria like Rhodopseudomonas palustris and Jannaschia sp. strain CCS1 (http://img.jgi.doe.gov/v1.0/main.cgi). Thus, it is possible that glycolate oxidation can remove an unwanted metabolite and provide CO2 to recycle reducing power in both R. sphaeroides and other photosynthetic bacteria.

Function of the RSP4157 to -4164 gene products. Based on what is known about RSP4157 to -4159, we propose that these proteins synthesize a compound required for either glycolate formation (by an unknown, RuBisCO-independent pathway) or some other step in recycling reducing power under photoheterotrophic conditions. RSP4157 is predicted to be a radical S-adenosylmethionine (SAM) enzyme that contains an Fe-S cluster. However, bioinformatic comparison of RSP4157 to other known radical SAM enzymes fails to make specific predictions about the reaction catalyzed by this protein. RSP4158 is predicted to be a homolog of a generic methyltransferase, while RSP4159 is similar to isopropyl-malate synthase or NifV-like enzymes (9). While RSP4157 to -4159 appear to be homologs of characterized enzymes, additional experiments are needed to identify their function in photoheterotrophic growth.

In summary, K-means clustering of global gene expression data sets was able to identify ~50 uncharacterized genes with expression patterns that parallel those of genes required for photosynthetic growth of R. sphaeroides. This approach can identify other genes that are involved in the photosynthetic lifestyle since one group of genes, RSP4157 to -4159, was necessary for photoheterotrophic growth. We propose that one or more genes in the RSP4157 to -4159 cluster provide an unknown metabolite that is critical for recycling reducing power under photosynthetic conditions. This role of RSP4157 to -4159 may help explain why plasmid P004 is maintained in R. sphaeroides strain 2.4.1. In nature, R. sphaeroides and other purple photosynthetic bacteria may use a number of pathways, including glycolate degradation, the Calvin cycle, or anaerobic respiration, to balance reducing power during photosynthetic growth, depending upon how much excess reductant is generated and the availability of various electron acceptors. In the future, it will be interesting to learn the function of RSP4157 to -4159 and test the role of the other uncharacterized genes in photosynthesis by R. sphaeroides.


   ACKNOWLEDGMENTS
 
This research was funded by grant ER 63232-1018220-0007 203 from the U.S. Department of Energy Genomes to Life program to T.J.D. C.L.T. also was supported by NIH Biotechnology Training Program grant T32 GM08349 and the University of Wisconsin—Madison Ira L. Baldwin fellowship. A.M.P. was supported by a University of Wisconsin—Madison Hilldale scholarship.

We thank the Kaplan lab at the University of Texas—Houston and the Gomelsky lab at the University of Wyoming for depositing data publicly at the National Center for Biotechnology Information GEO website. Microarrays were processed at the Genome Expression Center at the University of Wisconsin—Madison, and microarray data were analyzed using GeneSpring Software from Silicon Genetics.


   FOOTNOTES
 
* Corresponding author. Mailing address: University of Wisconsin—Madison Department of Bacteriology, 420 Henry Mall, Madison, WI 53711. Phone: (608) 263-4663. Fax: (608) 262-9865. E-mail: tdonohue{at}bact.wisc.edu. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, August 2005, p. 5249-5258, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5249-5258.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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