Patent Application
20070004043
The invention relates generally to modulating physiological responses to singlet oxygen in a bacterial cells, algae or plant phototrophs. Several sources of singlet oxygen in biological systems, including enzymes such as peroxidases and oxidases, as well as processes such as photosynthesis. Kochevar I, “Singlet oxygen signaling: from intimate to global,” STKE 204:pe7 (2004). In the photosynthetic process, input light energy converts water (H2O) and carbon dioxide (CO2) to oxygen (O2) and sugar. Cellular respiration subsequently converts some of the sugar into chemical energy in the form of ATP. The conversion is associated with chlorophyll, a green pigment common to all photosynthetic cells. Although O2 is a relatively non-reactive chemical, when exposed to high-energy or electron-transferring chemical reactions, it can be converted to highly reactive chemical forms collectively designated as “reactive oxygen species” (ROS). ROS are generally considered toxic to organisms because they oxidize carbohydrates, DNA, lipids and proteins, breaking down normal cellular, membrane and reproductive functions. Ultimately, at toxic ROS levels, a chain reaction of cellular oxidation can result in disease or lethality.
Singlet oxygen (1O2) is an ROS produced as a photosynthetic byproduct. In phototrophs, including plants, light energy excites chlorophyll pigments in the light harvesting complexes to a triplet state. At some frequency, an energy transfer from the excited triple state chlorophyll pigments to ground-state O2 generates 1O2 which, as a strong oxidant, can destroy membrane integrity, abolish biomolecular function, and reduce photochemical activity by inactivating photosynthetic enzymes.
Because excited triplet-state chlorophyll pigments and ground-state oxygen are found in close proximity to one another, many phototrophs exhibit some natural defenses against 1O2. For example, carotenoids, fat-soluble, anti-oxidant pigments found within the photosynthetic apparatus, quench 1O2. Telfer A, “What is β-carotene doing in the photosystem II reaction centre,” Phil. Tans. R. Soc. Lond. 357:1431-1440 (2002). Carotenoids include, but are not limited to, β-carotene, zeaxanthin and tocopherols. If not completely quenched by carotenoids, 1O2 can specifically trigger upregulation of genes that encode proteins involved in the molecular defense against photo-oxidative stress. For example, a network of upregulated plant genes maintains a balance between ROS-scavenging proteins and ROS-producing proteins. Mittler R, “Reactive oxygen gene network of plants,” TRENDS in Plant Sci. 9:490-498 (2004). In bacteria, a set of sigma factors, interchangeable RNA polymerase subunits responsible for recognizing transcriptional promoters, maintain essential housekeeping functions and facilitate host response to specific environmental stresses, including ROS. A constitutively-expressed, principal sigma factor is responsible for transcribing essential housekeeping genes. Other sigma factors, transcriptionally- or post-translationally-activated in response to stresses, recognize promoters upstream of genes involved in the response to stresses. Sigma factors are themselves regulated by anti-sigma factors that bind to a specific sigma factor and inhibit that sigma factor's ability to recognize a promoter.
Activation of sigma factors has been studied, inter alia, in Rhodobacter sphaeroides, a member of the α-subdivision of Proteobacteria and a facultative phototroph. R. sphaeroides is among the most metabolically diverse organisms known, being capable of growth under a wide variety of growth conditions. In addition to being photosynthetic, R. sphaeroides possesses additional energy-acquiring mechanisms including lithotrophy, aerobic respiration and anaerobic respiration. SigmaE (σE), a 19.2 kDa alternative sigma factor encoded by rpoE and related to members of the extra-cytoplasmic function (ECF) subfamily of eubacterial RNA polymerase sigma factors, is increased following environmental stress in R. sphaeroides. σE directs transcription from rpoE P1, a promoter for the rpoEchrR operon, and from cycA P3, a promoter for cytochrome c2. Newman J. et al, “The Rhodobacter sphaeroides ECF sigma factor, σE, and the target promoters cycA P3 and rpoE P1,” J. Mol. Biol. 294:307-320 (1999), incorporated herein by reference as if set forth in its entirety. Basal σE activity, however, is quite low because it is complexed with a zinc-dependent anti-sigma factor, ChrR. ChrR loses its ability to inhibit σE if zinc is removed, or if a zinc-binding domain of the N-terminal domain is removed. Newman J, et al., “The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor,” J. Mol. Biol. 313:485-499 (2001), incorporated herein by reference as if set forth in its entirety.
GenBank Accession No. AAB17905 (SEQ ID NO:1), discloses the fuill-length R. sphaeroides ChrR sequence. ChrR with a C38R mutation prevented binding to σE. See Newman et al. (1999), supra. Likewise, ChrR with a C35S or a C38S mutation prevented binding to σE. See Newman et al. (2001), supra. Furthermore, a ChrR with a C187/189S mutation was shown to prevent binding to σE. Id. In addition, ChrR with a H6A mutation, a H31A mutation, a C35A mutation or a C38A mutation cannot bind zinc and ultimately cannot bind σE.
GenBank Accession No. AAB17906 (SEQ ID NO:2) discloses the full-length R. sphaeroides σE sequence. Mutations in region 2.1 (amino acids 22 to 46 of SEQ ID NO:2) of σE alter the interaction between ChrR and σE. Anthony J, et al., “Interactions between the Rhodobacter sphaeroides ECF sigma factor, σE, and its anti-sigma factor, ChrR,” J. Mol. Biol. 341:345-360 (2004), incorporated herein by reference as if set forth in its entirety. In particular, σE with a K38E mutation, a K38R mutation or a M42A mutation were less sensitive to ChrR both in vivo and in vitro.
Because 1O2 affects many organisms (including, but not limited to, bacteria, plants, animals and humans), the components of the biological response to 1O2 find application in medicine, agriculture, biotechnology and bioenergy production systems. Animals and plants use 1O2 to defend against microbial pathogens. Davies M, “Reactive species formed on proteins exposed to singlet oxygen,” Photochem. Photobiol. Sci. 3:17-25 (2004), incorporated herein by reference as if set forth in its entirety. For the foregoing reasons, there is a desire to manipulate physiological responses to 1O2 in animals, bacteria and plants. There are many advantages of studying responses to 1O2 in R. sphaeroides. First, one can control the formation of significant amounts of 1O2. Also, biochemical and genetic systems are available to study the response to 1O2 in vivo and in vitro, including an Affymetrix gene chip (Affymetrix; Santa Clara, Calif.), LC/MS proteomics and computation approaches.
The present invention relates to observations by the inventors relating to genes required for viability of R. sphaeroides in the presence of 1O2 which can be generated during photosynthesis. Specifically, changes in the interaction between alternative sigma factor σE and its anti-sigma factor ChrR affects expression of genes required for viability of R. sphaeroides in the presence of 1O2. Although homologs of σE and ChrR have been identified computationally in other bacteria, their involvement in a cellular response to 1O2 has not heretofore been noted.
As the inventors detail below, 1O2 typically has detrimental effects upon cells, but cells can avoid or overcome the effects by increasing σE, which is ordinarily complexed with ChrR. In the presence of 1O2, ChrR and σE dissociate and synthesis of σE increases, allowing free σE to bind to a core RNA polymerase, facilitating transcription of a regulon involved in attenuating physiological effects of 1O2. This observation suggests that σE or ChrR can be manipulated to exploit the response of cells and organisms to 1O2. Even though the application refers to observations made in R. sphaeroides, the invention is not intended to be limited to this single prokaryote, as responses to 1O2 are present in many other species, including both photosynthetic and non-photosynthetic prokaryotes and eukaryotes.
The observation can be exploited to inhibit or prevent microbial survival, by preventing dissociation between ChrR and σE or by reducing the extent of dissociation in the microbes. In the presence of 1O2, the microbes would succumb to damage caused by increased oxidative stress.
The observation can alternatively be exploited to increase efficiency of commercial processes for generating commodity chemicals such as, but not limited to, acetic acid and other organic acids, acetone, acrylamide, butanol, ethanol, glycerol, isoprenoids, quinines, and pigments. Nagasawa T & Yamada H, “Microbial production of commodity chemicals,” 67 Pure & Appl. Chem. 1241-1256 (1995), incorporated herein by reference as if set forth in its entirety. In particular, photosynthetic organisms for use in such processes can be engineered to inhibit or eliminate binding between ChrR and σE, such that when the microbe finds itself in the presence of 1O2, it readily overcomes any toxic effects by mobilizing its increased available supply of σE to initiate transcription of the protective regulon, ensuring robust production from the process, notwithstanding the presence of 1O2. Increasing production of these or other commodity chemicals involves inhibiting the interaction between ChrR and σE in the presence of 1O2 so that the microbe continues to produce a desired commodity chemical notwithstanding oxidative stress. In one approach, the microbe can be engineered either to contain a mutated ChrR that cannot bind σE, or to lack ChrR entirely. A similar effect can be obtained by engineering a microbe for use in the process where the microbe contains a mutated σE relative to wild-type σE such that the mutated sigma factor cannot bind ChrR, or binds ChrR only weakly. In some embodiments, the photosynthetic organism is a bacterium, an alga or a plant. In some embodiments, the photosynthetic organism is R. sphaeroides. In some embodiments, σE is modified relative to wild type by engineering a K38E mutation, a K38R mutation or a M42A mutation in σE. In some embodiments, ChrR is modified relative to wild type by engineering a C35S mutation, a C38S mutation, a C38R mutation or a C187/189S in ChrR.
In another aspect, the observation can be exploited by protecting the phototroph from toxic effects of 1O2 by looking beyond the direct interaction of σE and ChrR, to the genes transcribed directly by σE or genes whose expression is increased by a σE-dependent transcription factor. For example, where the available supply of σE is not, or cannot, be increased as noted above, the phototroph, especially a plant, can be engineered to increase expression of genes that encode protective proteins. For example, CfaS, shown herein to be upregulated by σE, encodes cyclopropane-fatty-acyl-phospholipid synthase which catalyzes the generation of cyclopropane fatty acids by adding a methylene bridge across a double bond of a fatty acid. In lipid bilayers, 1O2 can hydroxylate unsaturated fatty acids and membrane-destabilizing lipid peroxides can form. On the other hand, 1O2 cannot hydroxylate cyclopropane fatty acids in the bilayers, so lipid peroxides cannot form and the phototrophs are protected from oxidative stress. Conversely, unwanted or invasive plant species can be made more susceptible to oxidative stress by engineering the phototroph to downregulate genes that encode protective proteins, such as CfaS.
In another aspect, the present invention is summarized in that a consensus promoter responsive to σE is disclosed as SEQ ID NO:3. In some embodiments, the nucleic acid residues at positions 2, 6, 12, 16, 17 and 20 are G; at position 11 is A; and at positions 19, 22 and 26 are C. The isolated nucleic acid sequence of SEQ ID NO:3 can be operably linked to a heterologous reporter gene or gene of interest to produce a genetic construct suitable for transfer into cells of a phototroph. Expression of the operably linked gene can thereby be placed under the control of σE. In so doing, not only can protective proteins be produced in the presence of 1O2, but any other protein, polypeptide, peptide or oligonucleotide of interest can be induced under such conditions. Similarly, a reporter gene can be provided so that the presence of 1O2 can be detected, observed and monitored.
It is an advantage of the present invention that it provides the skilled person with the tools for efficiently producing products in phototrophic organisms while avoiding longstanding issues arising from the presence of 1O2.
A second advantage is that the methods and compositions are non-toxic to the environment.
These and other features, aspects and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.
Abbreviations used in the drawings: PS—photosynthetically grown cells, Aero—cells grown by aerobic respiration, WT—wild type, ΔChrR—cells lacking the anti-sigma factor ChrR, ΔσE—cells lacking both σE and ChrR.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
As used herein, a “phototroph” or “photosynthetic organism” refers to any organism that is capable of photosynthesis.
Methods
Bacterial strains and plasmids: R. sphaeroides 2.4.1 (wild-type, WT), R. sphaeroides with a mutant ChrR (ΔChrR) (chrR-1::drf; see Newman et al., supra.) or R. sphaeroides with both a mutant σE (ΔσE) and ΔChrR (TF18; rpoEchrR-1::drf, see Schilke B & Donohue T, “ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene,” J. Bacteriol. 177:1929-1937 (1995), incorporated herein by reference as if set forth in its entirety) were grown at 30° C. in Sistrom's succinate-based minimal medium A. Media used for growth of strains containing low-copy lacZ reporter plasmids was supplemented with 25 μg/ml kanamycin.
Growth conditions: For aerobic respiratory growth, 500 ml of media was bubbled with a mixture of 69% N2, 30% O2 and 1% CO2 in the dark. Conversely, for photosynthetic growth, 500 ml of media was bubbled with a mixture of 95% N2 and 5% CO2 in front of an incandescent light source (10 W/m2 as measured with a Yellow-Springs-Kettering model 6.5A radiometer through a Corning 7-69, 620 to 110 nm filter).
To test the effects of 1O2, photosynthetic cultures were exposed to aerobic growth conditions (69% N2, 30% O2 and 1% CO2) in the presence or in the absence of light (10 W/m2). Where indicated, light was passed through a 1283 filter (Kopp Glass; Pittsburgh, Pa.) that impedes >99% of light at wavelengths <770 nm, but transmits >45% of light at 830 nm and >80% of light at 900 nm. When using methylene blue (Sigma-Aldrich; St. Louis, Mo.) to generate 1O2, a final concentration of 1 μM was added to aerobic cultures in the presence or in the absence of incandescent light (10 W/m2). To test the effects of other ROS, 0.5 mM H2O2, 1 mM diamide or 1 mM paraquat (Sigma-Aldrich) was added to aerobic cultures.
All experiments were initiated when cultures reached ˜2×108 cfu/ml to minimize light or O2 limitation to photosynthetic and aerobic cells, respectively. To measure cell viability, samples were removed, diluted and plated in media supplemented with 25 μg/ml kanamycin to select for the rpoE P1::lacZ reporter plasmid. The whole cell abundance of carotenoids was measured as described in Cohen-Bazire G, et al., “Kinetic studies of pigment synthesis by non-sulfur purple bacteria,” J. Cell Physiol. 49:25-68 (1957).
Determining promoter activity: Promoter activity was determined by measuring β-galactosidase activity from a low copy rpoE P1::lacZ reporter plasmid or a trxA::lacZ reporter plasmid. The promoter for the thioredoxin gene (trxA, −214 to +27 relative to the known transcription initiation site) was fused to lacZ and mobilized into R. sphaeroides.
β-galactosidase activity (units/ml of culture) was calculated as follows: (A420×1000)/(Cell volume in assay (ml)×Time of assay (min)). Culture density was typically monitored by measuring A600 in a BioSpec 1601 spectrophotometer (Schimatzu; Columbia, Md.). The density of cultures treated with methylene blue was monitored at 500 nm because methylene blue absorbs light between 609-668 nm. The differential rate of β-galactosidase synthesis was determined by calculating the slope from plots of enzyme activity (units/ml of culture) against optical density. All experiments were repeated a minimum of three times with differential rates of β-galactosidase synthesis typically deviating less than two-fold between experiments.
Identification of σE target genes: Triplicate cultures of WT and ΔChrR were grown aerobically to ˜2-3×108 CFU/ml. RNA was isolated and cDNA was synthesized, labeled and hybridized to R. sphaeroides GeneChip Custom Express microarrays (Affymetrix). After data extraction using Affymetrix MAS 5.0 software, data sets were imported into GeneSpring software (Silicon Genetics; Redwood City, Calif.) for normalization and analysis (Gene Expression Omnibus (GEO) accession number GSE2219).
Candidate σE promoters (extending ˜200 bp upstream of the predicted start of translation, Table 1) were amplified from 20 ng of WT chromosomal DNA in EasyStart PCR tubes (Molecular BioProducts; San Diego, Calif.) with 2.5 units Pfu Turbo (Stratagene; La Jolla, Calif.). PCR products were cloned into a plasmid (pRKK96) containing a known transcriptional terminator for in vitro assays or into a lacZ reporter plasmid (pRKK200) for determining activity in vivo. In vitro transcription assays with reconstituted R. sphaeroides σE (EσE) were performed with 20 nM of plasmid DNA.
1ΔChrR: R. sphaeroides WT with trimethoprim cartridge inserted into ChrR.
2Increase in RNA abundance from comparing transcript levels in WT and ΔChrR cells. Data has been deposited at GEO under accession number GSE2219.
3Function known or predicted by genome annotation.
Results
Conditions that generate 1O2 within the photosynthetic apparatus increase R. sphaeroides σE activity: Mutations that inactivate an early enzyme in carotenoid biosynthesis, CrtB, cause a small increase in σE activity (data not shown). Since carotenoids play a protective role against 1O2, it was determined whether 1O2 directly affected σE activity.
To determine if R. sphaeroides σE activity responds to 1O2, we examined the differential rate of β-galactosidase synthesis from a σE-dependent rpoE P1::lacZ reporter fusion after anaerobic, photosynthetic cells were exposed to O2 in the presence of light. After exposure to O2, the cells maintain approximately the same doubling rate, since O2 is used as a respiratory electron acceptor. However, after exposure to O2, the differential rate of β-galactosidase synthesis from the σE-dependent promoter increased ˜10-fold (from 6 to 65) when compared to a control culture grown under either a steady state photosynthetic condition (light in the absence of O2) or a respiring condition (30% O2) (
1Aero = cells grown by aerobic respiration (30% O2),
2PS = cells grown photosynthetically.
This transcriptional response was maintained throughout the experiment, suggesting that σE activity was sustained. There was less than a two-fold increase in the differential rate of β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion when photosynthetic cells were shifted to aerobic conditions in the dark (Table 2). This was expected since little 1O2 is made under this condition due to lack of light needed to produce triplet state chlorophyll molecules. From these results, one can conclude that the combination of light and O2, conditions known to generate 1O2 within the photosynthetic apparatus, are required for this transcriptional response.
Control experiments indicated that this response was dependent on σE since the differential rate of a β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion in ΔσE cells (<1 unit) did not increase upon exposure to 1O2. ΔσE cells grow under these conditions, presumably because the carotenoids within the photosynthetic apparatus quench 1O2. In addition, it appears that 1O2 does not fully induce σE activity since the differential rate of β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion in WT cells exposed to 1O2 was 10-fold less than that seen in ΔChrR cells (65 versus 650).
Wavelengths of light that excite chlorophyll pigments are sufficient to increase σE activity: If production of 1O2 by the photosynthetic apparatus was responsible for this transcriptional response, then wavelengths of light known to generate triplet state chlorophyll molecules within the light harvesting complexes should increase σE activity. R. sphaeroides contains two light harvesting complexes, B800-850 and B875, named for their absorption maxima in the near infrared. To determine if light absorbed by the light harvesting complexes could cause this response, we looked at the action spectrum of this transcriptional response. Under photosynthetic conditions with light that was filtered to remove wavelengths <830 nm, the differential rate of β-galactosidase synthesis from the σE-dependent promoter was an ˜4-fold lower than that observed with cells grown in white light (Table 2), presumably because the cells grow slower when light <830 nm is removed. However, there was an ˜17-fold increase in the differential rate of β-galactosidase synthesis when cultures illuminated with >830 nm light were exposed to O2 (Table 2). The magnitude of this response was similar to that observed when photosynthetic cells were exposed to O2 and white light (˜17-fold versus ˜10-fold, Table 2). Thus, wavelengths of light that excite the light harvesting complexes are sufficient to increase σE activity.
Continued exposure to conditions that generate 1O2 in the photosynthetic apparatus are needed to sustain this response: The half-life of 1O2 in cells is less than 100 ns and was used to further test if σE activity was responding to 1O2. For example, if increased σE activity required 1O2, then placing photosynthetic cells that had previously been exposed to O2 in the dark should terminate this transcriptional response. When the cells were shifted to aerobic conditions in the presence of light, we observed an expected increase in the differential rate of β-galactosidase synthesis from the σE-dependent promoter (˜10-fold,
R. sphaeroides σ
E activity is increased by formation of 1O2 in the absence of the photosynthetic apparatus: If 1O2 was responsible for the observed σE transcriptional response, then other conditions that generate this ROS should also increase σE activity. To test this hypothesis, one can generate 1O2 by illumination of methylene blue in the presence of O2 to produce a similar response. When aerobically grown WT cells were exposed to 1 μM methylene blue in the presence of light and O2, cell growth continued and the differential rate of β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion increased ˜20-fold compared to aerobic cells grown in the absence of methylene blue (Table 4). Control experiments indicated there was less then a two-fold increase in the rate of β-galactosidase synthesis when aerobic cultures were exposed to methylene blue in the dark (Table 4). The lack of a comparable increase in σE activity in aerobic cells exposed to methylene blue in the dark is expected since both light and O2 are required for this compound to generate 1O2.
1Differential rates of β-galactosidase synthesis from the σE-dependent rpoE::lacZ fusion when WT cells are grown aerobically under conditions that either do or do not generate 1O2.
For these experiments, cells were grown in the presence of 30% O2, a condition where pigment-protein complexes of the photosynthetic apparatus are not detectable. Therefore, the transcriptional response to 1O2 can occur in cells that either contain or lack the photosynthetic apparatus.
Other ROS do not produce a similar increase in σE activity: The damaging effects of 1O2 on many biomolecules could stimulate the formation of other ROS. To test if other ROS could produce an increase in σE activity, the differential rate of β-galactosidase synthesis from a rpoE P1::lacZ reporter fusion was monitored in aerobic cells treated with concentrations of hydrogen peroxide (H2O2), paraquat (to stimulate superoxide (O2−) formation) or diamide (to alter the oxidation-reduction state of the cytoplasmic thiol pool), previously shown to generate an oxidative stress response in R. sphaeroides. Li K, et al., “Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress,” Arch. Microbiol. 180:484-489 (2003). For these experiments, the differential rate of β-galactosidase synthesis was monitored from a control trxA::lacZ reporter fusion, since the trx promoter has previously been shown to respond to oxidative stress in R. sphaeroides.
The addition of paraquat or H2O2 to aerobic cells produced increases in the differential rate of β-galactosidase synthesis from the trxA::lacZ reporter gene that are consistent with changes in abundance of trxA transcripts produced by these compounds in previous studies (Table 5). However, the differential rate of β-galactosidase synthesis from the σE-dependent reporter fusion either decreased (paraquat) or increased no more than 1.2-fold (H2O2) when compared to untreated cells (Table 5). Any observed increase in σE activity in the presence of these ROS was below the 10-fold increase in σE activity seen when cells are exposed to 1O2.
1Differential rates of β-galactosidase synthesis from the indicated promoters when WT cells are grown aerobically under conditions that either do or do not generate indicated ROS.
ND—Not Determined.
σE activity in the presence of diamide was not monitored because previous work has shown that σE activity does not increase upon exposure to this compound. Based on these results, the transcriptional response observed when 1O2 is generated does not occur in the presence of other ROS.
When carotenoids are low, cells require σE to mount response to 1O2: While cells ΔσE cells are unable to mount this transcriptional response to 1O2 (
Exponential growth of aerobically grown WT cells continued after exposure to 1O2 (
Additional members of the σE regulon: To identify genes that are part of this transcriptional response to 1O2, we compared RNA levels in aerobically grown (30% O2) WT cells and in a ΔChrR mutant. Because ChrR inhibits σE activity, one looks for RNA that is more abundant in the ΔChrR mutant. As expected, global gene expression analysis showed an increase (˜12-fold) in rpoE-specific RNA from ΔChrR cells.
RNA from ˜180 genes (˜60 operons) was >3-fold more abundant in cells that contained increased σE activity (Table 1). In contrast, the ˜35-fold increase in cycA P3 activity that occurs in ΔChrR cells in vivo causes only an ˜1.6-fold increase in total cycA-specific RNA (Table 1). The smaller increase in cycA-specific RNA levels reflects the fact that cycA contains additional strong promoters that are recognized by other sigma factors. This suggests that a global gene expression microarray approach might miss other σE-dependent genes that also contain multiple promoters.
To test if any of these candidate operons contained a σE-dependent promoter, we tested DNA upstream of the first gene in each of twenty-eight potential operons for transcription by EσE. (Table 6) These operons were chosen either because of their increased levels of expression in cells with elevated σE activity or because of a potential role of their gene products in the photosynthetic apparatus (a source of 1O2). It was observed that rpoHII, which encodes one of two R. sphaeroides heat shock sigma factors (Rsp0601), is transcribed by σE. Production of the rpoHII transcript is inhibited by addition of ChrR, as is the case with other σE-dependent promoters like rpoE P1 and cycA P3 (
1Function known or predicted by genome annotation. Genes were chosen based on increased RNA abundance in cells that have elevated σE activity or for their known role in photosynthetic growth.
2Increase in RNA abundance from comparing transcript levels in WT and ΔChrR cells. Data has been deposited at GEO under accession number GSE2219. NC—no change.
3Coordinates are numbered relative to the start site of translation.
4Based on the ability to detect a σE-dependent transcript in vitro (see
Each gene is predicted to be part of a polycistronic operon that encodes uncharacterized proteins. The level of transcripts produced from the rpoHII, Rsp1087 and Rsp2143 promoters are comparable to that of rpoE P1 (within 1.1 -fold), suggesting that these 4 promoters are of similar strength. In contrast, the abundance of the σE-dependent transcript produced by Rsp1409 in vitro is comparable to the σE-dependent promoter, cycA P3, which has ˜80-fold less activity than rpoE P1.
The same putative rpoHII and Rsp1087 promoter regions were fused to lacZ to test for σE-dependent activity in vivo. Expression was not detectable from these reporter fusions in WT R. sphaeroides cells, but it was comparable to that of rpoE P1 in ΔChrR cells (
Generation of ChrR mutants to irreversibly bind to σE: The N-terminal anti-sigma domain of ChrR (ChrR-ASD) appears important in binding between ChrR and σE (data not shown). The skilled artisan is familiar with methods for delivering genetically engineered antimicrobial agents to microbes by phage therapy. Westwater C, et al., “Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections,” Antimicrob. Agents Chemother. 47:1301-1307 (2003), incorporated herein as if set forth in its entirety. Phage delivery systems are advantageous because they allow for targeting specific bacterial cells at a high frequency. Accordingly, a phage DNA is modified to contain a coding sequence that codes for at least amino acids 1-85 from GenBank Accession No. AAB 17905 (SEQ ID NO:1), which discloses the R. sphaeroides full-length ChrR sequence. The N-terminal portion of ChrR encoded by this construct is sufficient to irreversibly bind zinc and σE. However, cells containing this or similar N-terminal ChrR variants are not able to mount a response to 1O2, resulting in a condition where cells have increased sensitivity to this reactive oxygen species.
Bacterial cells are grown under standard culture conditions. Once an adequate concentration of bacterial cells are present, they are infected with a phage modified to express at least amino acids 1-85 of SEQ ID NO:1. Following exposure to the phage, oxidative stress ensues, but the cells do not express genes regulated by σE. Consequently, the concentration of bacterial cells decreases.
Alternatively, bacterial cells are infected with a phage modified to express at least amino acids 1-85 of SEQ ID NO:1. They are then grown under standard culture conditions; however, the concentration of bacterial cells does not increase upon oxidative stress because the cells do not express genes regulated by σE.
Other methods for reducing availability of σE can include using RNAi directed against σE, mutating the promoter that directs transcription of σE (see Newman et al. (1999), supra), and engineering the cells to put σE under control of a regulatable promoter or repressor.
Bacterial cells with a modified ChrR that cannot bind σE are grown under standard culture conditions. However, growth and consequently production of a commodity chemical are increased because the cells are protected against the deleterious effects of 1O2.
Likewise, bacterial cells with a modified σE that cannot be bound by ChrR are grown under standard culture conditions. However, growth and consequently production of a commodity chemical are increased because the cells are protected against the deleterious effects of 1O2.
Methods of manipulating plant genes are known to the skilled artisan. For example, Constabel C, et al., “Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X,” Plant Mol. Biol. 22:775-782 (1993), incorporated herein by reference as if set forth in its entirety. Accordingly, a plant is modified such that the plant exhibits a high level of cyclopropane-fatty-acyl-phospholipid synthase (CfaS) relative to an unmodified plant. The plant is grown under standard conditions; however, growth is increased because the plant is protected against the deleterious effects of 1O2.
Methods of inserting a gene of interest into a plasmid are known to the skilled artisan. Schilke & Donohue, supra. A gene encoding a product of interest is inserted into a plasmid under regulation of a σE-dependent promoter selected from the consensus sequence (SEQ ID NO:3) in
The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.
This application claims the benefit of U.S. Provisional Patent Application No. 60/674,470, filed Apr. 25, 2005, incorporated herein by reference as if set forth in its entirety.
This invention was made with United States government support awarded by the following agencies: National Institute of General Medical Science, GM37509; and Department of Energy, DE-FG02-05ER15653. The United States has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60674470 | Apr 2005 | US |
