DNA Photolyase Research Articles

Polarized Transient Absorption To Resolve Electron Transfer between Tryptophans in DNA Photolyase

Transient absorption spectroscopy is a powerful tool for studying biological electron-transfer chains, provided that their members give rise to distinct changes of their absorption spectra. There are, however, chains that contain identical molecules, so that electron transfer between them does not change net absorption. An example is the chain flavin adenine dinucleotide (FAD)-W382-W359-W306 in DNA photolyase from E. coli. Upon absorption of a photon, the excited state of FADH* (neutral FAD radical) abstracts an electron from the tryptophan residue W382 in approximately 30 ps (monitored by transient absorption). The cation radical W382*+ is presumably reduced by W359 and W359*+ by W306. The latter two reactions could not be monitored directly so far because the absorption changes of the partners compensate in each step. To overcome this difficulty, we used linearly polarized flashes for excitation of FADH*, thus inducing a preferential axis in the a priori unoriented sample (photoselection). Because W359 and W306 are very differently oriented within the protein, detection with polarized light should allow us to distinguish them. To demonstrate this, W306 was mutated to redox-inert phenylalanine. We show that the resulting anisotropy spectrum of the initial absorption changes (measured at 10 ns time resolution) is in line with W359 being oxidized. The corresponding spectrum in wildtype photolyase is clearly different and identifies W306 as the oxidized species. These findings set an upper limit of 10 ns for electron transfer from W306 to W359*+ in wildtype DNA photolyase, consistent with previous, more indirect evidence [Aubert, C.; Vos, M. H.; Mathis, P.; Eker, A. P. M.; Brettel, K. Nature 2000, 405, 586-590].

Structures and Energetics of Base Flipping of the Thymine Dimer Depend on DNA Sequence

The cis,syn-cyclobutane pyrimidine dimer (CPD) is a photoinduced DNA lesion leading to a significant distortion of the DNA structure. Its repair by DNA photolyase requires a flip of the damaged base into an extrahelical position. This base flip is expected to be sequence-dependent, but the structures and energetics as a function of the bases 3' and 5' to the CPD lesion are unknown. Eight-nanosecond MD simulations of four different hexadecamer duplexes with the CPD were performed for the flipped-in and flipped-out structures. Analysis of these results indicates clear sequence-dependent differences. Significant disruptions of the base pairs to the 3' side of the CPD are observed for the flipped-out structures with adjacent A-T pairs, whereas those with G-C pairs adjacent show no such distortions. The conformational spaces occupied by these two duplexes are significantly different. The structural differences correlate well with the free energy differences for base flipping calculated using the previously established 2D potential of mean force (PMF) method. The energy differences for base flipping in duplexes containing A, T, G, and C pairs adjacent to the CPD were found to be 6.25-6.5, 5.25-5.5, 7.25-7.5, and 6.5-6.75 kcal/mol, respectively. These energy differences of up to 2 kcal/mol should be large enough to be detected experimentally using sensitive probes.

Discrimination of Class I Cyclobutane Pyrimidine Dimer Photolyase from Blue Light Photoreceptors by Single Methionine Residue

DNA photolyase recognizes ultraviolet-damaged DNA and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. Theoretical analysis of the electron-tunneling pathways of the DNA photolyase derived from Anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. Here, we report the unexpectedly important role of the single methionine residue, Met-353, where busy trafficking of electron-tunneling currents is observed. The amino acid conservation pattern of Met-353 in the homologous sequences perfectly correlates with experimentally verified annotation as photolyases. The bioinformatics sequence analysis also suggests that the residue plays a pivotal role in biological function. Consistent findings from different disciplines of computational biology strongly suggest the pivotal role of Met-353 in the biological function of DNA photolyase.

The Extent of DNA Deformation in DNA Photolyase– Substrate Complexes: A Solution State Fluorescence Study

Cyclobutylpyrimidine dimers (CPDs) are the major UV photoproduct formed in DNA containing adjacent pyrimidines. These lesions can be repaired by DNA photolyase, a flavoprotein that utilizes blue light in a direct reversal of the cyclobutane ring. Previous studies have shown that the CPD is base flipped into the protein, with concomitant disruption of the substrate around the CPD. In this study, we use a fluorescent cytidine analog, pyrrolo-dC (PC), to probe how far base flipping propagates along the duplex. From these measurements, the degree of base destacking in the two bases flanking the adenines opposing the CPD appears to be minimal, which was consistent with the protein:substrate crystal structure. Fluorescence-detected melting temperatures for duplexes with and without a CPD were obtained, suggesting that a 5'-pyrimidine-PC-purine-3' motif is more stable than the 5'-purine-PC-pyrimidine-3' motif. This stability trend was reflected in the fluorescence intensities of ss-PC oligos but not for duplexes. The melting point depression due to the PC probe was found to be comparable to other popular fluorescent base analogs.

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

Abstract The elucidation of structural and electronic aspects of paramagnetic organic cofactors in their protein surroundings and related model compounds is a topic of widespread interest. In this field, electron paramagnetic resonance (EPR) spectroscopy, especially in conjunction with high-resolution electron-nuclear double resonance (ENDOR) is an important tool, allowing us to analyze details of the distribution of the unpaired electron spin, which is influenced by the interaction between the cofactor and its immediate environment, as well as to characterize paramagnetic intermediate states in the course of the protein action. In this account, investigations are reported in which EPR/ENDOR methods have contributed significantly to answering questions on mechanistic and structural aspects of protein function. Emphasis is given to in-depth characterizations of electronic and spatial structures of radical cofactors and their positioning relative to substrate molecules. After a brief introduction, covering some of the trends of modern EPR/ENDOR method development, we will focus, as examples, on characterizations of quinone-type and flavin cofactors in two proteins, (i) quinoprotein alcohol dehydrogenase, and (ii) DNA photolyase.

Base Flipping of the Thymine Dimer in Duplex DNA

Exposure of two adjacent thymines in DNA to UV light of 260-320 nm can result in the formation of the cis,syn-cyclobutane pyrimidine dimer (CPD). The structure of DNA containing an intrahelical CPD lesion has been previously studied experimentally and computationally. However, the structure of the extrahelical, flipped-out, CPD lesion, which has been shown to be the structure that binds to the CPD repair enzyme, DNA photolyase, has yet to be reported. In this work the structure of both the flipped-in and the flipped-out CPD lesions in duplex DNA is reported. These structures were calculated using 8 ns molecular dynamics (MD) simulations. These structures are then used to define the starting and ending points for the base-flipping process for the CPD lesion. Using a complex, two-dimensional pseudodihedral coordinate, the potential of mean force (PMF) for the base-flipping process was calculcated using novel methodology. The free energy of the flipped-out CPD is roughly 6.5 kcal/mol higher than that of the flipped-in state, indicating that the barrier to flipping out is much lower for CPD than for undamaged DNA. This may indicate that the flipped-out CPD lesion may be recognized by its repair enzyme, DNA photolyase, whereas previous studies of other damaged, as well as nondamaged, bases indicate that they are recognized by enzymes in the intrahelical, flipped-in state.

Do Photolyases Need To Provide Considerable Activation Energy for the Splitting of Cyclobutane Pyrimidine Dimer Radical Anions?

cis-syn Cyclobutane pyrimidine dimers, major UV-induced DNA lesions, are efficiently repaired by DNA photolyases. The key step of the repair reaction is a light-driven electron transfer from the FADH(-) cofactor to the dimer; the resulting radical anion splits spontaneously. Whether the splitting reaction requires considerable activation energy is still under dispute. Recent reports show that the splitting reaction of a dimer radical anion has a significant activation barrier (0.45 eV), and so photolyases have to provide considerable energy. However, these results contradict observations that cis-syn dimer radical anions split into monomers at -196 degrees C, and that the full process of DNA photoreactivation was fast (1.5-2 ns). To investigate the activation energies of dimer radical anions, three model compounds 1-3 were prepared. These include a covalently linked cyclobutane thymine dimer and a tryptophan residue (1) or a flavin unit (3), and the covalently linked uracil dimer and tryptophan (2). Their properties of photosensitised splitting of the dimer units by tryptophan or flavin unit were investigated over a large temperature range, -196 to 70 degrees C. The activation energies were obtained from the temperature dependency of splitting reactions for 1 and 2, 1.9 kJ mol(-1) and 0.9 kJ mol(-1) for the thymine and uracil dimer radical anions, respectively. These values are much lower than that obtained for E. coli photolyase (0.45 eV), and are surmountable at -196 degrees C. The activation energies provide support for previous observations that repair efficiencies for uracil dimers are higher than thymine dimers, both in enzymatic and model systems. The mechanisms of highly efficient enzymatic DNA repair are discussed.

An improved TM110 resonator for continuous-wave ENDOR studies at X-band

An improved TM110 resonator for continuous-wave and time-resolved electron-nuclear double resonance (ENDOR) studies at X-band frequencies is described that has been designed with small samples and/or light excitation in mind. The filling factor is increased by reducing the resonator length to only 16 mm. The radio-frequency field is generated by either a conventional solenoid for stable samples or by a pair of coils for samples which require photoexcitation. This arrangement leaves the central volume of the resonator free for optimal sample illumination, which is achieved through a slit in the wall of the resonator. Microwave coupling is achieved by the incorporation of an iris in the top of the resonator, and magnetic field modulation is applied by external coils. The resonator’s high sensitivity is illustrated by a study of the temperature dependence of the continuous-wave ENDOR signal of the stable neutral flavin radical in DNA photolyase and an investigation of the photoexcited triplet state of free-base tetraphenylbacteriochlorin by time-resolved ENDOR.

6MAP, a Fluorescent Adenine Analogue, Is a Probe of Base Flipping by DNA Photolyase

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) in the presence of visible light. How PL recognizes and binds its substrate is still not well understood. Fluorescent nucleic acid base analogues are powerful probes of DNA structure. We have used the fluorescent adenine analogue 6MAP, a pteridone, to probe the local double helical structure of the CPD substrate when bound by photolyase. Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability. Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted. 6MAP shows a different quenching pattern compared to 2-aminopurine, another important adenine analogue, although both probes show that the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes. We also show that 6MAP/CPD duplexes are substrates for PL. Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory. Using these results, the Förster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern. These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe. However, no experimental evidence for this process was observed in the steady-state emission spectra.

Observation of an Intermediate Tryptophanyl Radical in W306F Mutant DNA Photolyase from Escherichia coli Supports Electron Hopping along the Triple Tryptophan Chain

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees . The catalytically potent state FADH- can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees , in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH degrees * <-- W382 <-- W359 <-- W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH degrees and leads to the formation of FADH- and a deprotonated tryptophanyl radical, most likely W359 degrees. These photoproducts are formed in less than 10 ns and recombine to the dark state in approximately 1 micros. These results support the electron hopping mechanism.

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.

Femtochemistry in enzyme catalysis: DNA photolyase

Photolyase uses light energy to split UV-induced cyclobutane pyrimidine dimers in damaged DNA. This photoenzyme encompasses a series of elementary dynamical processes during repair function from early photoinitiation by a photoantenna molecule to enhance repair efficiency, to in vitro photoreduction through aromatic residues to reconvert the cofactor to the active form, and to final photorepair to fix damaged DNA. The corresponding series of dynamics include resonance energy transfer, intraprotein electron transfer, and intermolecular electron transfer, bond breaking-making rearrangements and back electron return, respectively. We review here our recent direct studies of these dynamical processes in real time, which showed that all these elementary reactions in the enzyme occur within subnanosecond timescale. Active-site solvation was observed to play a critical role in the continuous modulation of catalytic reactions. As a model system for enzyme catalysis, we isolated the enzyme-substrate complex in the transition-state region and mapped out the entire evolution of unmasked catalytic reactions of DNA repair. These observed synergistic motions in the active site reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.

A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.

Induction and inhibition of cyclobutane pyrimidine dimer photolyase in etiolated cucumber (Cucumis sativus) cotyledons after ultraviolet irradiation depends on wavelength

Under polychromatic ultraviolet (UV) irradiation (maximum energy at 327 nm) the activity of DNA photolyase specific to cyclobutane pyrimidine dimers (CPDs), CPD photolyase, increased by an amount which depended on UV irradiance, and the level of CPD photolyase gene (CsPHR) transcripts temporarily increased before the activity reached a constant level. UV light (>320 nm) was more effective than visible light at increasing CPD photolyase activity. In contrast, monochromatic UV irradiation at wavelengths <300 nm increased the level of CsPHR transcripts similarly to irradiation at wavelengths >320 nm, but reduced CPD photolyase activity compared with the dark control. Exposure of a CPD photolyase solution to UV-C (254 nm) reduced enzyme activity and induced accumulation of H(2)O(2). Addition of H(2)O(2) to the enzyme solution also inactivated CPD photolyase activity. These results suggest the possibility that reactive oxygen species participate in the inactivation of CPD photolyase in cotyledons exposed to UV irradiation of <300 nm.

Electron-Transfer Induced Repair of 6-4 Photoproducts in DNA: A Computational Study

The mechanism employed by DNA photolyase to repair 6-4 photoproducts in UV-damaged DNA is explored by means of quantum chemical calculations. Considering the repair of both oxetane and azetidine lesions, it is demonstrated that reduction as well as oxidation enables a reversion reaction by creating anionic or cationic radicals that readily fragment into monomeric pyrimidines. However, on the basis of calculated reaction energies indicating that electron transfer from the enzyme to the lesion is a much more favorable process than electron transfer in the opposite direction, it is suggested that the photoenzymic repair can only occur by way of an anionic mechanism. Furthermore, it is shown that reduction of the oxetane facilitates a mechanism involving cleavage of the C-O bond followed by cleavage of the C-C bond, whereas reductive fragmentation of the azetidine may proceed with either of the intermonomeric C-N and C-C bonds cleaved as the first step. From calculations on neutral azetidine radicals, a significant increase in the free-energy barrier for the initial fragmentation step upon protonation of the carbonylic oxygens is predicted. This effect can be attributed to protonation serving to stabilize reactant complexes more than transition structures.

Cryptochrome Blue Light Photoreceptors Are Activated through Interconversion of Flavin Redox States

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.

Photoselected electron transfer pathways in DNA photolyase.

Cyclobutane dimer photolyases are proteins that bind to UV-damaged DNA containing cyclobutane pyrimidine dimer lesions. They repair these lesions by photo-induced electron transfer. The electron donor cofactor of a photolyase is a two-electron-reduced flavin adenine dinucleotide (FADH(-)). When FADH(-) is photo-excited, it transfers an electron from an excited pi --> pi* singlet state to the pyrimidine dimer lesion of DNA. We compute the lowest excited singlet states of FADH(-) using ab initio (time-dependent density functional theory and time-dependent Hartree-Fock), and semiempirical (INDO/S configuration interaction) methods. The calculations show that the two lowest pi --> pi* singlet states of FADH(-) are localized on the side of the flavin ring that is proximal to the dimer lesion of DNA. For the lowest-energy donor excited state of FADH(-), we compute the conformationally averaged electronic coupling to acceptor states of the thymine dimer. The coupling calculations are performed at the INDO/S level, on donor-acceptor cofactor conformations obtained from molecular dynamics simulations of the solvated protein with a thymine dimer docked in its active site. These calculations demonstrate that the localization of the (1)FADH(-)* donor state on the flavin ring enhances the electronic coupling between the flavin and the dimer by permitting shorter electron-transfer pathways to the dimer that have single through-space jumps. Therefore, in photolyase, the photo-excitation itself enhances the electron transfer rate by moving the electron towards the dimer.

Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars

Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.

Photoactivated excited states of DNA repair photolyase: Dynamical and semiempircal identification

AbstractDNA damage caused by UV light radiation is often naturally repaired in a process initiated by excited state electron transfer from the photoactivated photolyase enzyme to the DNA cyclobutane pyrimidine dimer lesion. The active cofactor in the excited state electron transfer in the photolyase is the two‐electron fully reduced form of the flavin adenine dinucleotide (FADH−). To calculate electron tunneling matrix element and model the DNA binding with photolyase, the LUMO of the FADH− calculated using extended Huckel method was previously chosen from the SCF wavefunctions. Recently, the DNA–photolyase complex was crystallized in its bound form, in good agreement with our previous model in even minute details at the active site. Here we carry out molecular dynamics simulation of the entire complex using the new experimental structure of Anacystis nidulans and identify the low‐lying photoactivated states of the enzyme for the dynamical confirmations. Our results from ZINDO/S CIS calculations are compared with experimental UV spectra, and their implications for excited state electron transfer and energy transfer are discussed. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

Growth-phase dependent differential gene expression in Synechocystis sp. strain PCC 6803 and regulation by a group 2 sigma factor

Cyanobacteria must continually alter their physiological growth state in response to changes in light intensity and their nutritional and physical environment. Under typical laboratory batch growth conditions, cyanobacteria grow exponentially, then transition to a light-limited stage of linear growth before finally reaching a non-growth stationary phase. In this study, we utilized DNA microarrays to profile the expression of genes in the cyanobacterium Synechocystis sp. PCC 6803 to compare exponential and linear growth. We also studied the importance of SigB, a group 2 sigma factor in this cyanobacterium, during the different growth phases. The transcription of approximately 10% of the genes in the wild type were different in the linear, compared to the exponential phase, and our results showed that: (1) many photosynthesis and regulatory genes had lowered transcript levels; (2) individual genes, such as sigH, phrA, and isiA, which encode a group 4 sigma factor, a DNA photolyase, and a Chl-binding protein, respectively, were strongly induced; and, (3) the loss of SigB significantly impacted the differential expression of genes and modulated the changes seen in the wild type in regard to photosynthesis, regulatory and the unknown genes.