Effects of Oxygen and Light Intensity on Transcriptome Expression in Rhodobacter sphaeroides 2.4.1
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m311608200
ISSN1083-351X
AutoresJung Hyeob Roh, William E. Smith, Samuel Kaplan,
Tópico(s)Algal biology and biofuel production
ResumoThe roles of oxygen and light on the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 have been well studied over the past 50 years. More recently, the effects of oxygen and light on gene regulation have been shown to involve the interacting redox chains present in R. sphaeroides under diverse growth conditions, and many of the redox carriers comprising these chains have been well studied. However, the expression patterns of those genes encoding these redox carriers, under aerobic and anaerobic photosynthetic growth, have been less well studied. Here, we provide a transcriptional analysis of many of the genes comprising the photosynthesis lifestyle, including genes corresponding to many of the known regulatory elements controlling the response of this organism to oxygen and light. The observed patterns of gene expression are evaluated and discussed in light of our knowledge of the physiology of R. sphaeroides under aerobic and photosynthetic growth conditions. Finally, this analysis has enabled to us go beyond the traditional patterns of gene expression associated with the photosynthesis lifestyle and to consider, for the first time, the full complement of genes responding to oxygen, and variations in light intensity when growing photosynthetically. The data provided here should be considered as a first step in enabling one to model electron flow in R. sphaeroides 2.4.1. The roles of oxygen and light on the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 have been well studied over the past 50 years. More recently, the effects of oxygen and light on gene regulation have been shown to involve the interacting redox chains present in R. sphaeroides under diverse growth conditions, and many of the redox carriers comprising these chains have been well studied. However, the expression patterns of those genes encoding these redox carriers, under aerobic and anaerobic photosynthetic growth, have been less well studied. Here, we provide a transcriptional analysis of many of the genes comprising the photosynthesis lifestyle, including genes corresponding to many of the known regulatory elements controlling the response of this organism to oxygen and light. The observed patterns of gene expression are evaluated and discussed in light of our knowledge of the physiology of R. sphaeroides under aerobic and photosynthetic growth conditions. Finally, this analysis has enabled to us go beyond the traditional patterns of gene expression associated with the photosynthesis lifestyle and to consider, for the first time, the full complement of genes responding to oxygen, and variations in light intensity when growing photosynthetically. The data provided here should be considered as a first step in enabling one to model electron flow in R. sphaeroides 2.4.1. Rhodobacter sphaeroides 2.4.1 is a purple nonsulfur photosynthetic bacterium that can grow aerobically, anaerobically in the light, or anaerobically in the dark in the presence of external electron acceptors such as dimethyl sulfoxide. When grown anaerobically in the light or dark, R. sphaeroides synthesizes an intracytoplasmic membrane (ICM) 1The abbreviations used are: ICM, intracytoplasmic membrane; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; ORF, open reading frame; W, watt(s); PS, photosynthesis; EF-G, elongation factor G; CSP, cold shock protein. system, which constitutes the photosynthetic apparatus and possesses the structural components necessary for light energy capture, subsequent electron transport, and energy transduction (1Kiley P.J. Kaplan S. Microbiol. Rev. 1988; 52: 50-69Crossref PubMed Google Scholar). The composition and assembly of the ICM are known to be tightly regulated, with light intensity and oxygen tension being among the most prominent environmental stimuli that serve to control the synthesis and function of the ICM (2Zeilstra-Ryalls J. Gomelsky M. Eraso J.M. Yeliseev A. O'Gara J. Kaplan S. J. Bacteriol. 1998; 180: 2801-2809Crossref PubMed Google Scholar, 3Oh J.I. Kaplan S. Mol. Microbiol. 2001; 39: 1116-1123Crossref PubMed Google Scholar). The role of oxygen in regulating the presence or absence of the ICM in R. sphaeroides has been well documented in numerous publications over the past 50 years (2Zeilstra-Ryalls J. Gomelsky M. Eraso J.M. Yeliseev A. O'Gara J. Kaplan S. J. Bacteriol. 1998; 180: 2801-2809Crossref PubMed Google Scholar, 3Oh J.I. Kaplan S. Mol. Microbiol. 2001; 39: 1116-1123Crossref PubMed Google Scholar, 4Bowyer J.R. Hunter C.N. Ohnishi T. Niederman R.A. J. Biol. Chem. 1985; 260: 3295-3304Abstract Full Text PDF PubMed Google Scholar, 5Hunter C.N. Pennoyer J.D. Niederman R.A. Prog. Clin. Biol. Res. 1982; 102: 257-265PubMed Google Scholar). Recently, studies have revealed that the role of oxygen in ICM formation is the result of the actions of key regulatory pathways, including the cbb3 terminal oxidase-PrrBA pathway (6Oh J.I. Ko I.J. Kaplan S. J. Bacteriol. 2001; 183: 6807-6814Crossref PubMed Scopus (25) Google Scholar, 7Oh J.I. Kaplan S. Biochemistry. 1999; 38: 2688-2696Crossref PubMed Scopus (89) Google Scholar, 8Eraso J.M. Kaplan S. J. Bacteriol. 1995; 177: 2695-2706Crossref PubMed Google Scholar, 9Eraso J.M. Kaplan S. J. Bacteriol. 1996; 178: 7037-7046Crossref PubMed Google Scholar, 10Lee J.K. Kaplan S. J. Bacteriol. 1992; 174: 1158-1171Crossref PubMed Google Scholar), the PpsR-AppA repressor-anti-repressor pathway (11Gomelsky M. Horne I.M. Lee H.J. Pemberton J.M. McEwan A.G. Kaplan S. J. Bacteriol. 2000; 182: 2253-2261Crossref PubMed Scopus (44) Google Scholar, 12Gomelsky M. Kaplan S. J. Biol. Chem. 1998; 273: 35319-35325Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 13Braatsch S. Gomelsky M. Kuphal S. Klug G. Mol. Microbiol. 2002; 45: 827-836Crossref PubMed Scopus (148) Google Scholar, 14Penfold R.J. Pemberton J.M. J. Bacteriol. 1994; 176: 2869-2876Crossref PubMed Google Scholar), and the FnrL pathway (15Zeilstra-Ryalls J.H. Kaplan S. J. Bacteriol. 1998; 180: 1496-1503Crossref PubMed Google Scholar, 16Zeilstra-Ryalls J.H. Kaplan S. J. Bacteriol. 1995; 177: 6422-6431Crossref PubMed Google Scholar). Light intensity has a somewhat more subtle effect on photosynthetically growing cells, whereby the cellular levels of ICM are inversely related to light intensity and the composition of the ICM is differentially altered with changing light intensity (1Kiley P.J. Kaplan S. Microbiol. Rev. 1988; 52: 50-69Crossref PubMed Google Scholar, 17Yeliseev A.A. Eraso J.M. Kaplan S. J. Bacteriol. 1996; 178: 5877-5883Crossref PubMed Google Scholar). Recent studies have implicated the cbb3-Prr system (18Oh J.I. Kaplan S. EMBO J. 2000; 19: 4237-4247Crossref PubMed Scopus (116) Google Scholar) and the PpsR-AppA system (14Penfold R.J. Pemberton J.M. J. Bacteriol. 1994; 176: 2869-2876Crossref PubMed Google Scholar, 19Oh J.I. Eraso J.M. Kaplan S. J. Bacteriol. 2000; 182: 3081-3087Crossref PubMed Scopus (55) Google Scholar) as being factors in light control of PS gene expression. Very recent work has further implicated the AppA protein as being involved in blue light control of PS gene expression (13Braatsch S. Gomelsky M. Kuphal S. Klug G. Mol. Microbiol. 2002; 45: 827-836Crossref PubMed Scopus (148) Google Scholar, 20Masuda S. Bauer C.E. Cell. 2002; 110: 613-623Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). What has been obvious from all of these studies is that they have been by necessity incremental, i.e. one gene at a time. With the advent of the DNA sequence, assembly and annotation of the R. sphaeroides 2.4.1 genome (21Mackenzie C. Choudhary M. Larimer F.W. Predki P.F. Stilwagen S. Armitage J.P. Barber R.D. Donohue T.J. Hosler J.P. Newman J.E. Shapleigh J.P. Sockett R.E. Zeilstra-Ryalls J. Kaplan S. Photo. Res. 2001; 70: 19-41Crossref PubMed Scopus (104) Google Scholar), as well as the construction of an Affymetrix GeneChip, we are in the unique position of being able not only to validate the earlier described observations, but more importantly we are in the position to extend these observations to the expression of all genes comprising the photosynthesis regulon including N2 and CO2 fixation, and taxis (22Romagnoli S. Packer H.L. Armitage J.P. J. Bacteriol. 2002; 184: 5590-5598Crossref PubMed Scopus (29) Google Scholar, 23Gibson J.L. Dubbs J.M. Tabita F.R. J. Bacteriol. 2002; 184: 6654-6664Crossref PubMed Scopus (22) Google Scholar, 24Joshi H.M. Tabita F.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14515-14520Crossref PubMed Scopus (166) Google Scholar, 25Eraso J.M. Kaplan S. Biochemistry. 2000; 39: 2052-2062Crossref PubMed Scopus (71) Google Scholar), as well as other physiologic activities for which genetic elements are regulated by oxygen and light intensity. Finally, we are in the position of being able to follow the expression of well studied regulatory elements controlling the photosynthesis lifestyle, as these respond to oxygen- and light-induced regulation. For the first time, we are able to observe the expression of many genes for which encoded proteins are responsible for the flow of redox in R. sphaeroides, with the goal being to model these complex interacting pathways. In the present work we have extended our studies of oxygen control of gene expression as well as the role of high (100 W/m2) versus low (3 W/m2) light intensity on gene expression in R. sphaeroides 2.4.1. We directly address the hypothesis that oxygen and light regulation of gene expression in R. sphaeroides extend well beyond those systems, giving rise to the photosynthetic lifestyle. R. sphaeroides Growth Conditions—R. sphaeroides strains were grown at 30 ± 0.5 °C on Sistrom's minimal medium A containing succinate as a carbon source. Aerobic cultures were grown sparging with a gas mixture of 30% O2, 69% N2, and 1% CO2 and harvested at an A600 nm of 0.2 ± 0.02 to ensure oxygen saturation. Photosynthetic cultures were grown at a light intensity of either 3 or 100 W/m2 measured at the surface of the growth vessel and sparging with 95% N2, 5% CO2 and harvested at A600 nm of 0.45 ± 0.05, which insures that self-shading is not problem. Light intensity was measured using an YSI-Kettering model 65A radiometer (Simpson Electric Co.). DNA Manipulation—To prepare hybridization probes for Northern blot analysis, genes encoding the translation elongation factor G (RSP2248) and two encoding cold shock proteins (RSP2346 and RSP3621) were PCR-amplified using chromosomal DNA of R. sphaeroides 2.4.1 as template. RSP2248 was amplified with primers 5′-GAT CCT GCG CTT TCT CGA CGA CCG CAA-3′ and 5′-GAA GCT CGG CCA TCA GCG CAT CGT CGA-3′. RSP2346 and 3621 were amplified with primers 5′-GTG GAA GAC GAA AAG GCC CTG CAG CTT-3′ and 5′-GGC CGG TTG CTG ATG CTG ACG CGC TGC-3′, and with primers 5′-ACA GGA GAG ATC ACG ATG GCC AAT GGC ACC GTG-3′ and 5′-GGC GAG AAC GAG GTT CGT CGC CGA CTC GCG GC-3′, respectively. PCR-amplified DNA fragments were subcloned in pGEM-T easy vector (Promega), and the DNA sequence was confirmed. Each subclone was digested with NotI, and the DNA fragments containing the PCR-amplified DNA were isolated following agarose gel electrophoresis, and labeled with [α-32P]dATP using the RadPrime DNA labeling system (Invitrogen). Hybridization and washing was performed with QuikHyb hybridization solution following the instructions from the manufacturer (Stratagene). Quantitation of specific signals from the Northern blot analyses was performed by subtracting background for each lane from the values for the specific band using the spot density tools of the AlphaEaseTC™ imaging system (Alpha Innotech). RNA Manipulation—A previously described RNA isolation procedure (26Roh J.H. Kaplan S. J. Bacteriol. 2002; 184: 5330-5338Crossref PubMed Scopus (15) Google Scholar) was modified to optimize the isolation of intact mRNA for DNA microarray analysis. We modified the earlier procedure by eliminating cell collection by centrifugation. A volume of cells grown as described was directly pipetted into an equal volume of 2 × lysis buffer (100 °C). After thorough mixing, lysed cells were immediately transferred to an equal volume of hot phenol solution (65 °C). Total time required to transfer from the culture vessel to hot phenol was kept to less than 1 min to minimize mRNA degradation and to maximize the yield of intact mRNA. The remainder of the RNA purification procedure was identical to that described previously (26Roh J.H. Kaplan S. J. Bacteriol. 2002; 184: 5330-5338Crossref PubMed Scopus (15) Google Scholar). Each isolated RNA sample was treated with 50 μl of RQ1 RNase-free DNase (1 unit/μl, Promega) and 50 μl of 10× buffer in a total volume of 500 μl. Samples were incubated for 1 h at 37 °C; extracted with acidic phenol, acidic phenol/chloroform, and chloroform; and then precipitated by adding 1 ml of ethanol. The pellet was washed with 75% ethanol and suspended in diethylpyrocarbonate-treated water. Total RNA was pelleted again by adding the same volume of 4 m LiCl, washed with 75% ethanol, and resuspended in diethylpyrocarbonate-treated water. Chromosomal DNA contamination was tested by PCR amplification using the rdxB-specific primers (a and b) as described previously (26Roh J.H. Kaplan S. J. Bacteriol. 2002; 184: 5330-5338Crossref PubMed Scopus (15) Google Scholar). Microarray Experiment and Data Analysis—The R. sphaeroides 2.4.1 GeneChip was custom-designed and manufactured by Affymetrix Inc. 2R. sphaeroides 2.4.1, which was used for genomic sequencing and construction of the GeneChip, is available through the ATCC as strain number BAA-808. The manuscript describing construction of the Affymetrix GeneChip will be published elsewhere. The GeneChip contains information for 4292 ORFs, 47 rRNA and tRNAs, 65 selected ORFs from R. sphaeroides strains other than R. sphaeroides 2.4.1, and 394 intergenic regions. Intergenic regions are designated for DNA regions larger than 0.5 kb situated between ORFs. Almost 75% of these are shown to be actual ORFs. Total RNA was prepared from three independent cultures involving R. sphaeroides 2.4.1 grown under aerobic or photosynthetic conditions. cDNA synthesis, fragmentation, labeling, and hybridization were adapted, with few modifications, from the methods optimized for the GeneChip designed for the Pseudomonas aeruginosa Genome Array by Affymetrix Inc (www.affymetrix.com/support/technical/manuals.affx). Briefly, 10 μg of total RNA was annealed with 750 ng of random primers (New England Biolabs) and incubated at 70 °C for 10 min, and then at 25 °C for 1 h. First strand cDNA was synthesized with 200 units/μl SuperScript II with 5× first strand buffer (Invitrogen) in the presence of 10 mm dithiothreitol, 0.5 mm dNTPs, and 0.5 units/μl SUPERase·In (Ambion) RNase inhibitor (25 °C for 20 min, 37 °C for 1 h, 42 °C for 1 h, 70 °C for 10 min). After removal of RNA by alkaline treatment and neutralization, the cDNA synthesis product was purified using the QIAquick PCR purification kit (Qiagen). For fragmentation, 7–9 μg of cDNA and 1 unit of RQ1 DNase I (Promega) were incubated at 37 °C. One third of the cDNA from the cDNA-DNase mixture was heat inactivated for every 1 min after adding the RQI DNase. The desired cDNA size range of 50–200 bases was selected after 3% agarose gel electrophoresis using 200 ng of fragmented cDNA. The fragmented cDNA was 3′ end-labeled using the Enzo BioArray Terminal Labeling Kit (Affymetrix) with biotin-ddUTP. Target hybridization, washing, staining, and scanning were performed according to the protocol supplied by the manufacturer using a GeneChip Hybridization Oven 640, a Fluidics Station 400, and the Agilent GeneArray Scanner under the control of the Affymetrix Microarray Suite 5.0. Computer Programs—The scanned image was analyzed using the Affymetrix Microarray Suite 5.0 to obtain signal value, detection calls (present/absent/marginal), and p values. The relative -fold change and hierarchical clustering of the hybridization intensity of the experimental probe set to that of the control were calculated using dChip 1.2 software (biosun1.harvard.edu/complab/dchip1) (27Li C. Hung Wong W. Genome Biol. 2001; 2: 1-11Google Scholar, 28Li C. Wong W.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 31-36Crossref PubMed Scopus (2713) Google Scholar). For group comparisons, filtering criterion between the group means was a 1.5-fold change using the 90% confidence boundary for 0-fold change, which is calculated using the standard error of the group means (27Li C. Hung Wong W. Genome Biol. 2001; 2: 1-11Google Scholar, 28Li C. Wong W.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 31-36Crossref PubMed Scopus (2713) Google Scholar). The Pearson correlation coefficient value (r value) was calculated using the Microsoft Excel program. Overview of Microarray Analysis—The transcriptome profiles from three independent cultures grown and processed independently under high and low light intensities, as well as under aerobic conditions, were analyzed. The present/absent/marginal percentage and the reliability between triplicate samples are summarized in Table I. "Present" calls by MAS5 for both 100 W/m2 and aerobic growth conditions were approximately the same percentage values; these calls were considerably greater than those observed for cells grown at 3 W/m2. Conversely, the percentage "absent" was greater at 3 W/m2 than observed for aerobic or high light grown cells. "Marginal" calls were approximately the same regardless of growth condition. The experimental reproducibility between samples of the same data set is indicated by the r value (Table I). There are two different forms of control genes on the R. sphaeroides 2.4.1 GeneChip in addition to genes from other organisms, namely 24 probe sets from non-related organisms such as E. coli, Bacillus subtilis, and bacteriophage P1, as well as nine ORF from R. sphaeroides 2.4.1 which are represented by five copies each. The 24 control probe sets from the different organisms were all detected as "absent," and the standard deviation for the five copies each of nine ORF from R. sphaeroides 2.4.1 did not exceed 25% of the average value for the set. The mean intensity levels for each probe set were cross-compared using dChip 1.2 software, and the -fold change between the group means was calculated using the standard deviation of the group means (27Li C. Hung Wong W. Genome Biol. 2001; 2: 1-11Google Scholar, 28Li C. Wong W.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 31-36Crossref PubMed Scopus (2713) Google Scholar). Those genes/ORFs that showed a greater than 1.5-fold change with 90% confidence boundaries were used for further analysis.Table ISummary of DNA microarray analyses Present/absent/marginal calls were obtained using the MAS5 (Affymetrix Inc.), and indicated by lowest and highest percentage for triplicate samples. Gap genes (generally incomplete genes at the ends of contigs) represent ≈5% of total genome prior to final assembly and represent ≈1.3% after assembly. All gap genes are located at their correct position on the assembled genome. r value is the Pearson correlation coefficient and obtained by comparison of the 5306 probe sets to one another within the triplicate set.CulturePresentAbsentMarginalr value%%%3 W/m2ORF58–6432–393–40.978, 0.984GAP31–3858–654–50.986100 W/m2ORF68–7225–293–40.988, 0.994GAP39–4451–564–50.994AerobicORF65–7125–313–40.980, 0.988GAP34–4154–615–60.991 Open table in a new tab In Fig. 1, we have depicted the numbers of genes/ORFs that either increased or decreased relative to one of the other culture conditions, with all combinations being provided. Several points are noteworthy. There is the greatest degree of variability in gene expression when the 3 W/m2 condition is viewed relative to aerobic growth as opposed to when high light (100 W/m2) is similarly compared with aerobic growth. In the former comparison, there were 543 up-regulated and 911 down-regulated genes. This observation is consistent with earlier, although much more limited observations that high light grown photosynthetic cells are more similar to aerobic grown cells, as least with regard to the percentage of genes up- or down-regulated, but not necessarily involving the same genes when the comparison is made to low light grown photosynthetic cells (29Tai S.P. Kaplan S. J. Bacteriol. 1985; 164: 181-186Crossref PubMed Google Scholar, 30Chory J. Kaplan S. J. Bacteriol. 1983; 153: 465-474Crossref PubMed Google Scholar, 31Kaplan S. Arntzen C.J. Photosynthetic Energy Conversion by Plants and Bacteria. Academic Press, Orlando1982: 67-151Google Scholar). Low light grown cells show an approximate 60–70% slower growth rate compared with high light grown cells (1Kiley P.J. Kaplan S. Microbiol. Rev. 1988; 52: 50-69Crossref PubMed Google Scholar). However, such cells contain 3–4-fold higher levels of photosynthetic membrane, as well as a greater differential increase in the B800–850 spectral complex relative to the B875 spectral complex, and a volume nearly 70% greater than high light grown cells (1Kiley P.J. Kaplan S. Microbiol. Rev. 1988; 52: 50-69Crossref PubMed Google Scholar, 29Tai S.P. Kaplan S. J. Bacteriol. 1985; 164: 181-186Crossref PubMed Google Scholar). Second, the data reveal that the median range in increased -fold change in gene/ORF expression is in the 3–5 range for aerobic versus 3 W/m2 or 100 W/m2, declining to the 2–3-fold range for 3 W/m2 versus 100 W/m2. Similarly, when one looks at those genes/ORFs showing a decline in expression, the median values decrease from -2 to -3 range to -3 to -5 range as one goes from aerobic versus 3 W/m2 to 3 W/m2 versus 100 W/m2. A comparison between the two photosynthetically grown data sets shows 636 genes/ORFs up-regulated and 427 down-regulated. Because there are between 1200–1500 genes/ORFs showing changes under these conditions, it implies that most of the genes/ORFs affected by oxygen and light intensity have never been observed. If we are overly generous in compiling those genes known to be part of the photosynthetic lifestyle, we come up with ∼150–175. A detailed list of genes/ORFs that comprise the data described in Fig. 1 can be found at our web site (www.rhodobacter.org) and has been included here as supplementary data, available in the on-line version of this article. As described above there are many published studies that have followed, individually, the expression of a few, but not most of the genes encoding the photosystem in R. sphaeroides (32Lee J.K. DeHoff B.S. Donohue T.J. Gumport R.I. Kaplan S. J. Biol. Chem. 1989; 264: 19354-19365Abstract Full Text PDF PubMed Google Scholar, 33Lee J.K. Kiley P.J. Kaplan S. J. Bacteriol. 1989; 171: 3391-3405Crossref PubMed Google Scholar, 34Addlesee H.A. Fiedor L. 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Microbiol. 2002; 46: 1081-1094Crossref PubMed Scopus (64) Google Scholar), or assorted transcriptional regulators (16Zeilstra-Ryalls J.H. Kaplan S. J. Bacteriol. 1995; 177: 6422-6431Crossref PubMed Google Scholar, 49Eraso J.M. Kaplan S. J. Bacteriol. 1994; 176: 32-43Crossref PubMed Google Scholar, 50Gomelsky M. Kaplan S. J. Bacteriol. 1995; 177: 4609-4618Crossref PubMed Google Scholar), etc., using more traditional approaches such as Northern hybridization, lacZ fusions, and even phoA fusions. However, these studies have been by necessity selective, and for the most part the overwhelming majority of these genetic elements comprising the photosynthetic lifestyle have never been investigated under the diverse environmental conditions described here. Photosynthesis Gene Cluster—As previously described for those genes encoding the apoproteins of the spectral complexes (19Oh J.I. Eraso J.M. Kaplan S. J. Bacteriol. 2000; 182: 3081-3087Crossref PubMed Scopus (55) Google Scholar, 32Lee J.K. DeHoff B.S. Donohue T.J. Gumport R.I. Kaplan S. J. Biol. Chem. 1989; 264: 19354-19365Abstract Full Text PDF PubMed Google Scholar, 33Lee J.K. Kiley P.J. Kaplan S. J. Bacteriol. 1989; 171: 3391-3405Crossref PubMed Google Scholar), there is a wealth of expression data, but for the most part the majority of the genes of the PS gene cluster of R. sphaeroides 2.4.1 have never been studied, except to note that their expression is higher under photosynthetic growth conditions relative to aerobic growth. In Fig. 2, in the left panel we show the expression profiles for those genes encoding the apoproteins of the reaction center (RC, puhA, pufL, M), the B875 complex (pufBA), and the B800–850 complex (pucBA) in the presence of oxygen and high and low light intensity under anaerobic growth. Although these data are a repeat of many earlier studies, they serve a very useful and unique purpose, namely helping to validate the use of the GeneChip. When comparisons are made between gene expression profiles for cells grown at 3 W/m2versus 100 W/m2, we also observe, for those genes encoding the apoproteins of the spectral complexes, that these are expressed to much greater levels at low relative to high light. Note that cells grown aerobically constitute our base line for this analysis. Although there are a few genes encoding enzymes in the pigment biosynthetic pathways, which are expressed at moderately high levels and therefore appear on the left panel of Fig. 2, most of the genes in this category are represented in the right panel where expression levels are greatly decreased. The data reveal that for almost all of the pigment encoding genes, even those appearing in the left panel, their expression levels decline at 3 W/m2 relative to 100 W/m2. The consistency of this observation, when coupled to the fact that there are higher cellular pigment levels at low light by a factor of ∼3 (1Kiley P.J. Kaplan S. Microbiol. Rev. 1988; 52: 50-69Crossref PubMed Google Scholar), is unexpected and appears paradoxical. However, we suggest that these data may be explained by concluding that post-transcriptional controls, at the level of enzyme activity, may play a more prominent role in pigment production than previously suggested (51Zhu Y.S. Cook D.N. Leach F. Armstrong G.A. Alberti M. Hearst J.E. J. Bacteriol. 1986; 168: 1180-1188Crossref PubMed Google Scholar, 52Zhu Y.S. Hearst J.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7613-7617Crossref PubMed Scopus (40) Google Scholar). Such a conclusion is also supported by some earlier studies in R. sphaeroides on PucC (25Eraso J.M. Kaplan S. Biochemistry. 2000; 39: 2052-2062Crossref PubMed Scopus (71) Google Scholar, 53Zeng X. Choudhary M. Kaplan S. J. Bacteriol. 2003; 185: 6171-6184Crossref PubMed Scopus (44) Google Scholar), CrtA (54O'Gara J.P. Kaplan S. J. Bacteriol. 1997; 179: 1951-1961Crossref PubMed Google Scholar), and TspO (55Yeliseev A.A. Kaplan S. J. Biol. Chem. 2000; 275: 5657-5667Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Throughout these investigations, we caution that we are cognizant that posttranscriptional analyses, such as an analysis of the proteome, corresponding to most of these genes is lacking. However, many of the pigment-encoding genes that appear to reside in the same transcription unit show similar levels of expression and behavior. Nonetheless, there are a few genes that defy this general trend, e.g. bchM, bchP, crtE, crtA, etc. Evidence exists that for certain of these genes there are additional regulatory factors (19Oh J.I. Eraso J.M. Kaplan S. J. Bacteriol. 2000; 182: 3081-3087Crossref PubMed Scopus (55) Google Scholar, 56Yeliseev A.A. Kaplan S. J. Biol. Chem. 1995; 270: 21167-21175Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The CrtA protein, which has been observed to be highly expressed, is under redox control (56Yeliseev A.A. Kaplan S. J. Biol. 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