Methods and means for metabolic engineering and improved product formation by micro-organisms
09834773 · 2017-12-05
Assignee
- Universiteit Leiden (Leiden, NL)
- University of Liège (Angleur, BE)
- FRIEDRICH-ALEXANDER-UNIVERSITÄT ERLANGEN-NÜRNBERG (Erlangen, DE)
Inventors
- Sebastien Rigali (Angleur, BE)
- Mannfred Friedrich Bruno Titgemeyer (Möhrendorf, DE)
- Gilles Philippus Van Wezel (Oegstgeest, NL)
Cpc classification
C12N1/38
CHEMISTRY; METALLURGY
International classification
C12N1/38
CHEMISTRY; METALLURGY
Abstract
Described are methods and means for metabolic engineering and improved product formation by a filamentous micro-organism or a low G+C gram-positive bacterium. Disclosed is that DasR and DasR binding sites play an important and universal role in the control of gene expression in micro-organisms. Based on this finding, provided are multiple useful applications, such as a method for regulating the expression of a gene of interest, a method for controlling metabolism, a method for decreasing undesired expression and many more. Moreover, provided are means that can be used to establish said methods: for example a micro-organism in which the DasR binding site in operable linkage with a particular gene has been modified to obtain increased or decreased expression of a protein (being a desired or undesired protein) encoded by said gene.
Claims
1. A method for regulating expression of a gene of interest in a micro-organism comprising a DasR protein, wherein the gene of interest is in operable linkage to a DasR-binding site, the method comprising: providing the micro-organism with a compound selected from the group consisting of glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylglucosamine, and multimers thereof, such that the expression of the gene of interest is regulated; screening for the production of a secondary metabolite; and wherein the DasR-binding site is selected from the group consisting of: NN(T/A)GG(T/A)(C/G)T(A/G)N(A/T)C(C/A)(A/C)N(SEQ ID NO:1); (A/G)N(T/A)(G/T)(G/A)T(C/A)TA(G/T)A(C/T)(C/A)(A/T)N(T/C) (SEQ ID NO: 4); A(T/A)(T/C)(G/A)(G/A)TATATA(C/T)(C/T)(A/G)(A/T)T (SEQ ID NO: 5); A(T/C)(T/C)(G/T)(G/A)T(A/C)TA(T/G)A(C/T)(C/A)(A/G)(A/G)T (SEQ ID NO: 6); and (A/T)(T/A)T(G/A)(G/T)(A/C)TA(T/G)N(C/A)(C/T)A(A/T)(T/A) (SEQ ID NO: 7).
2. The method according to claim 1 wherein the compound is selected from the group consisting of glucosamine-6-phosphate, N-acetylglucosamine, and multimers thereof.
3. The method according to claim 1, further comprising providing the micro-organism with a polynucleotide encoding a DasR protein.
4. The method according to claim 1, wherein the microorganism is a Streptomyces, a Nocardia, a Thermobifido, an Amycolatopsis, a Planobispora, a Streptoverticillium, a Rhodococcus, or a Corynebacterium.
5. The method according to claim 1, wherein the micro-organism is a low G+C gram-positive bacterium.
6. The method according to claim 1, further comprising introducing into the micro-organism the DasR-binding site in operable linkage with the gene of interest.
7. The method according to claim 1, wherein the micro-organism is a Streptomyces, a Nocardia, a Thermobifido, a Amycolatopsis, a Planobispora, a Streptoverticillium, a Rhodococcus, a Corynebacterium, or a low G+C gram-positive bacterium.
8. The method according to claim 1, further comprising purifying the secondary metabolite.
Description
(1) The invention will be explained in more detail in the following description, which is not limiting the invention.
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DESCRIPTION OF TABLES
(26) Table 1 Experimentally validated DasR binding sites used to build the matrix for consensus sequence.
(27) Table 2 Non-limiting list of genes controlled by a DasR binding site in Streptomyces coelicolor.
(28) Table 3 List of putative binding sites for DasR relating to secondary metabolism (cut-off score 5). A. Antibiotics and metabolites of known function produced by actinomycetes B. Known and cryptic biosynthesis clusters of Streptomyces coelicolor.
(29) Table 4 DasR target genes related to glutamate and glutamine metabolism.
(30) Table 5 DasR binding sites in Bacillus species A. B. subtilis B. B. halodurans.
(31) Table 6 DasR binding sites in Lactococcus lactis.
(32) Table 7 DasR binding sites in Streptococcus species A. S. pneumoniae B. S. pyogenes C. S mutans D. S. agalactiae.
(33) Table 8 DasR binding sites in Listeria species. A. Listeria innocua B. Listeria monocytogenes.
(34) Table 9 DasR binding sites in Thermobifido fusca. Metabolic genes corresponding to
DETAILED DESCRIPTION OF THE INVENTION
(35) Experimental Part
(36) Materials and Methods
(37) Bacterial Strains.
(38) E. coli DH5α, and BL21(DE3) were used for subcloning and DasR overexpression experiments. S. coelicolor M145, M510 (M145 ΔredD), M511 (M145 ΔactII-IV) and M512 (M145 ΔactII-IV ΔredD) (Floriano and Bibb, 1996) and Streptomzyces lividans 1326 were all obtained from the John Innes Centre strain collection, Streptomyces avermitilis NRRL 8165 (MA-4680), Streptonzyces hygroscopicus ATCC27438, Streptomyces limosus ATCC 19778, Streptomyces rinzosus ATCC 10970, Streptonzyces roseosporus ATCC 31568 and Streptonzyces venezuelae ATCC15439 were obtained from the ATCC strain collection and Streptomyces acrinzycini DSM 40540, Streptomyces cinnanzonensis DSM 40467, Streptomyces clavuligerus NRRL 3585, Streptomyces collinus DSM 40733 and Streptomyces griseus NRRL B2682 from the DSMZ strain collection. The dasR mutant BAP29 (ΔdasR::accC4) was created by replacing the coding region of the gene by the apramycin resistance gene cassette, using plasmid pWHM3, according to a routine procedure (Nothaft et al., 2003). The same strategy was used to create the knock-out mutants for ptsH (BAP1), crr (BAP2), ptsI (BAP3), nagE1 (BAP4), nagE2 (BAP5), and nagE1/E2 (BAP6). BAP1-3 were published previously (Nothaft et al., 2003). S. coelicolor strains were grown at 28° C. using tryptic soy broth without dextrose as complex medium (TSB, Difco) or minimal medium (van Wezel et al., 2005). E. coli cultures were grown in Luria-Bertani broth (LB) at 37° C. Phenotypic characterization of mutants was done on minimal medium agar plates with various carbon sources as indicated in the text (Kieser et al., 2000). Quantification of Act and Red was performed as described previously (Martinez-Costa et al., 1996).
(39) DNase I Footprinting.
(40) A 222-bp DNA fragment corresponding to the −202/+8 region relative to the start of S. coelicolor crr gene (SCO1390) was chosen for DNase I footprinting. The DNA fragment was amplified from chromosomal DNA by PCR. 50 fmoles of .sup.32P end-labelled probe were incubated with the relevant proteins (DasR-(His).sub.6 and/or BSA) and DNaseI (0.4 μg/ml) as described (Sambrook et al., 1989).
(41) Computational prediction. Multiple alignments and position weight matrices were generated as described previously (Rigali et al., 2004) by the Target Explorer automated tool on the world wide web at trantor.bioc.columbia.edu/Target Explorer!) (Sosinsky et al., 2003). The weight matrix was deposited as “DasR4”.
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(43) The minimum score obtained by a sequence scanned by matrix DasR4 is −38.55 and the maximum score is 17.25. According to the current experimental validations, a DasR-binding site could be defined as a sequence of 16 nucleotides that, when scanned by the DasR4 matrix, obtains a score comprised between higher than 6 (and up to 17.25). Illustratively, a truly and experimentally validated DasR-binding site with a score of only −2.97 has been found upstream of gdhA encoding a NADP-specific glutamate dehydrogenase.
(44) Microscopy.
(45) Transmission electron microscopy (TEM) for the analysis of thin sections of hyphae and spores was performed with a Philips EM410 transmission electron microscope (Mahr et al., 2000). Phase contrast micrographs were produced using a Zeiss standard 25 phase-contrast microscope, and a 5 megapixel digital camera.
(46) Sugar Uptake.
(47) Uptake assays with 20 μM N-[.sup.14C]acetyl-D-glucosamine (6.2 mCi mmol.sup.−1) into mycelia were performed as described (Nothaft et al., 2003).
(48) Protein Purification and Western Blot.
(49) Purification of recombinant histidine-tagged DasR (Rigali et al., 2004) and Western blot analysis with antibodies raised against HPr and IIA.sup.Crr have been described elsewhere (Nothaft et al., 2003).
(50) RT-PCR.
(51) RNA was isolated from mycelium of S. coelicolor M145 and BAP29. Minimal medium cultures containing 50 mM glycerol were inoculated with spores and grown until OD.sub.550 of 0.6 (exponential growth). N-acetylglucosamine was added at 0.5% and samples were taken after 0, 15, 30 and 60 minutes. RT-PCR analyses were conducted with the Superscript III one-step RT-PCR Kit (Invitrogen). RT-PCRs without reverse transcription were used as control for absence of residual DNA. For semi-quantitative analysis, samples were taken at three-cycle intervals between cycles 18 to 35 to compare non-saturated PCR product formation (van Wezel et al., 2005). Data were verified in three independent experiments.
(52) Oligonucleotides used for the RT-PCR experiments described in
(53) For redZ (5′-CGACATGAAAGTGCAGGTGG-3′ (SEQ ID NO:518) and 5′-TCGGGCTFGGTCAGCAAAAGC-3′ (SEQ ID NO:519)), for actII-ORF4 (5′-GCTGCAGACGTACGTGTACCACAC-3′ (SEQ ID NO:520) and 5′-GCGTCGATACGGAGCTGCATTCC-3′ (SEQ ID NO:521)), for redD (5′-TCATGGGAGTGCGGAGAACGCG-3′ (SEQ ID NO:522) and 5′-CGCCCCACAGTTCGTCCACCAG-3′ (SEQ ID NO:523)), SCO6273 (5′-CGGGGGCGAACTCGTCAAGGTC-3′ (SEQ ID NO:524) and 5′-GCCGAGATGTCGATGAGGACGCGG-3′ (SEQ ID NO:525)), for kasO (5′-GCGGGATGCTCAGTGAGCACGG-3′ (SEQ ID NO:526) and 5′-GACGAGGTCGGCGAGGACGGG-3′ (SEQ ID NO:527)) and for rpsI (5′-GAGACCACTCCCGAGCAGCCGC-3′ (SEQ ID NO:528) and 5′-GTAGCGGTTGTCCAGCTCGAGCA-3′(SEQ ID NO:529)).
(54) Two-Dimensional Gel Electrophoresis and Protein Spot Identification.
(55) Mycelia of S. coelicolor M145 and BAP29 were grown in minimal medium with 50 mM glycerol, harvested at different time points within exponential phase, washed, resuspended in 20 mM HEPES, pH7.5 and 50 mM MgSO.sub.4 and sonicated; cell debris was removed after centrifugation. DNA and RNA were eliminated by DNase and RNase treatment. The proteins extracts were dialysed twice at 4° C. against water, followed by addition of 6 M solid urea and 2M thiourea, TritonX-100 (2.5% (v/v)) IPG buffer (0.5% (v/v)), DTT (25 mM) and bromophenolblue. Membrane proteins were removed by ultracentrifugation for 1 h at 65.000 g. 1.5 mg of the cytoplasmic protein fraction was applied on 24-cm IPG strips (pH range 4-7) on an IPGPhor unit (Amersham/Pharmacia). The IPG strips were subjected to 12.5% polyacrylamide gels that were run on the Ettan DALT II system (Amersham/Pharmacia). The gels were stained with PhastGel Blue R and scanned. Proteome patterns were compared using two gel sets derived from independent experiments. Protein intensities were analysed by densitometric gray scale analysis with TINA software (Raytest). Protein spots were excised, subjected to in-gel digestion with trypsin and analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (Marvin-Guy et al., 2005).
(56) Electromobility Gel Shift Assay (EMSA).
(57) EMSAs were performed with fluorescent probes (10 nM) with an ALF express sequencer (Filee et al., 2001). Purified DasR (3 μM) and 1000-fold excess of non-specific DNA were used in the reaction mixture. Predicted cis-acting elements were taken from the promoter regions of actII-ORF4 (SCO5085; 5′-CACATTGAAATCTGTTGAGTAGGCCTGTTATTGTCGCCCC-3′ (SEQ ID NO:530)), and redZ (SCO5881; 5′-ACAAGATCTTCTTGAGGTGGAAACCACTTTCGTATCAGTCT-3′ (SEQ ID NO:531)). Known cis-acting elements upstream of crr (SCO1390; CCGTGAGGAGTGTGGTCTAGACCTCTAATCGGAACA-3′ (SEQ ID NO:532)), and crp (SCO3571; 5′-TGCGGCATCCTTGTGACAGATCACACTGTTTGGACT-3′ (SEQ ID NO:533)) were used as positive and negative controls, respectively. The 16 nt dre sites are underlined.
(58) Enzymatic Activities.
(59) Chitinase activity was determined as described previously (Zhang et al., 2002) using a colorimetric assay with carboxymethylchitin-Remazol Brilliant Violet 5R (Loewe Biochemica GmbH, Germany) as substrate. D-Ala-D-Ala aminopeptidase measurements were performed with D-Ala-paranitroanilide as substrate (Cheggour et al., 2000). BCA protein assay (Pierce) was used for determining protein concentrations.
(60) Global Antibiotic Activity Assays.
(61) Samples (1 μl) of diluted spore suspensions were spotted on minimal medium plates containing 0.5% mannitol with or without 1% GlcNAc and incubated three days at 28° C. For the bioassay, we inoculated 10 ml of molten soft nutrient agar (SNA) with 500 μl of a Bacillus subtilis overnight culture (0D.sub.600˜1), and poured the mixture into square 12-cm-side Petri dishes. Plates were kept 2 h at 4° C. to solidify SNA and to allow diffusion of antibiotics produced, then incubated overnight at 30° C.
(62) Experimental Part
(63) Results
(64) Prediction of the DasR Regulon.
(65) To allow the S. coelicolor genome to be scrutinized for the occurrence of the DasR operator site, we performed DNaseI footprinting on the dre of the crr-ptsI operon, encoding the PTS enzyme IIA (IIA.sup.Crr) and enzyme I (EI) (
(66) A genome scan revealed 160 dre sites for 131 transcription units, representing over 200 candidate genes. About 40% of the target genes are related to sugar or aminosugar metabolism, with N-acetylglucosamine (GlcNAc) as the central saccharidic component (
(67) Phenotype of the dasR Mutant
(68) A dasR null mutant (BAP29) was constructed by replacing almost the complete coding region (nt 14-635 out of 765) by the apramycin resistance cassette (aacC4), to study the role of DasR in vivo.
(69) The dasR mutant of S. coelicolor showed medium-dependent development: on some media development was enhanced, while on others it was completely abolished. This is summarised in
(70) Surprisingly, the dasR mutant showed strongly enhanced antibiotic production (
(71) DasR and the Control of Development
(72) How does DasR control the switch to development? Inclusion of pts genes in the dasR regulon allowed us to propose that at least one link is through the DasR-mediated control of the PTS. In a previous publication (Nothaft et al., 2003), we described normal but retarded development for the individual pts knock-out mutants, namely BAP1 (ΔptsH, the gene for HPr), BAP2 (Δcrr, the gene for IIA.sup.Crr), and BAP3 (ΔptsI, the gene for enzyme I (EI)). More detailed phenotypic analysis of the PTS mutants revealed that while eventually all of the pts mutants were on some media able to produce spores, morphogenesis was significantly delayed on diverse complex and minimal media agar plates with the strongest differences when grown in the presence of mannitol and arabinose. Strains were grown for 5 days on SFM or R2YE agar plates. As shown in
(73) Interestingly, on R2YE agar plates all three PTS mutants show vegetative arrest (so-called bald or bld phenotype).
(74) We recently discovered in a proteomics screen of these mutants that the expression patterns (in BAP2 and BAP3) or the modification patterns (in BAP1) of the WhiG protein, a key developmental σ factor for early aerial growth (Chater et al., 1989), strongly differed from those in the parental strain M145. To establish the expression of whiG, its transcription was analysed in all three mutants and in M145. Interestingly, whiG transcription was strongly reduced in the BAP2 and BAP3 mutants (
(75) Hence, we propose that DasR acts as the nutrient sensor, and translates this through control of the PTS, which in turn controls whiG and—in view of the developmental arrest of the pts mutants—most likely at least one or more other early developmental genes. It might be noteworthy that the phosphotransferases EI, HPr, and IIA.sup.Crr provide a perfect signalling system through reversible metabolite-dependent phosphorylation, which in other bacteria is used for diverse but always carbon-related responses (Brückner and Titgemeyer, 2002).
(76) Proteome Analysis
(77) To obtain an assessment of the effect of DasR, we compared the protein profiles of BAP29 and its parent M145. Protein extracts were prepared from mycelia grown in the presence of glycerol (a neutral carbon source) and analyzed by two-dimensional gel electrophoresis. About 4% of the protein spots on the Coomassie-Brilliant-Blue-stained gels were altered in intensity by more than two-fold and eight were identified by mass spectrometry (
(78) The lack of a dre upstream of the other seven genes suggests indirect control of these genes by DasR. Visualising the metabolic pathways related to proteins identified by proteome analysis revealed that most gravitate around GlcNAc and glutamate metabolism, fitting well with the in silico and in vitro data presented above (
(79) DasR Controls Secondary Metabolism and Antibiotic Production in Actinomycetes
(80) Interestingly, two of the targets identified in our proteomics screen, namely ActVA4 and GpsI, which are both up-regulated in the dasR mutant, are involved in the production of the antibiotic γ-actinorhodin (Bibb, 2005) which correlated well with the early an activated production of blue γ-actinorhodin in the dasR mutant (
(81) Excitingly, comparison of the secreted proteins in abstracts of the dasR mutant and its parent S. coelicolor M145 by one-dimensional gel electrophoresis showed that one single protein was extraordinarily highly over-expressed in the dasR mutant (
(82) Also highly interesting is that using the novel bioinformatics techniques described above, we identified dre sites upstream of many more genes involved in the regulation and/or production of antibiotics. These targets are summarised in Table 3.
(83) Control of Enzyme Production
(84) With 16 predicted genes, chitin-related (chi) genes constitute a large subset of potential DasR targets, including chitinases, chitin binding proteins, extracellular β-N-acetylglucosaminidases (convert chito-oligosaccharides into GlcNAc and chitobiose), and intracellular β-N-acetylglucosaminidases (hydrolyse chitobiose to GlcNAc). To substantiate this, we determined the overall chitinolytic activity of BAP29 and M145 grown under inducing (chitin) or repressing (glycerol, glycerol plus GlcNAc, and glucose plus chitin) conditions. As depicted in
(85) Seven examples were selected to validate the predicted cis-trans relationship between DasR and chi genes, for the chitinolytic system that is required for the utilization of chitin, a polymer of N-acetylglucosamine that is the one but most abundant carbon source on earth. Positive DNA-DasR interactions were observed for all tested promoters (
(86) Other extracellular enzymes are also controlled by DasR (
(87) DasR-Mediated Control of Sugar Transport
(88) Since many of the predicted DasR targets were involved in the fate of carbon sources (Table 2), we analyzed the effects of the dasR mutation on sugar import. Transport assays revealed that PTS-mediated internalization of GlcNAc had become constitutive in BAP29, while in the parent M145 uptake was induced by GlcNAc (
(89) As shown above, in silico prediction and proteome analysis identified msiK (SCO4240), encoding the universal ATPase MsiK, as a target for DasR (
(90) DasR-Dependent Peptidoglycan-Associated Proteins
(91) The observed cell-wall anomalies in the dasR mutant (
(92) To assess whether DasR controls the expression of the dppA operon, we performed DNA binding studies with purified His-tagged DasR and a fragment corresponding to 193 by upstream of dppA. Analysis using EMSAs established a weak but significant interaction of DasR with the dppA promoter (
(93) DasR and Central Metabolism
(94) Considering the large number of N-acetylglucosamine-related genes in the list of predicted dre sites, we investigated the impact of DasR on the regulation of the nag metabolic genes. EMSAs were conducted using purified DasR protein and DNA fragments encompassing the predicted dre sites for nagB (Glucosamine-6-P isomerase) and the nagKA operon (GlcNAc kinase, and GlcNAc-6-P deacetylase). In both cases a DasR-dre complex could be demonstrated (
(95) Hence, a picture emerges of a hyper-controlled core network of the DasR regulon, crucial for the cell's energy balance and revolving around the triangle GlcNAc-Gln/Glu-Acetyl-CoA, with almost complete control of all metabolic steps involved. Thus, DasR plays a particularly prominent role in the control of central metabolism and is a very attractive target for metabolic engineering. All targets relating to N-acetylglucosamine and glutamate metabolism are highlighted in Table 4.
(96) Amazingly, our analysis of the Thermobifido fusca genome showed that in fact every single step of glycolysis is controlled by DasR, with highly reliable dre sites located upstream of the respective enzyme-encoding genes (Table 9 and
(97) Glucosamine-6-P is an Effector of DasR
(98) A pivotal question is what is the effector molecule that modulates DasR? As shown in here, many of the targets for DasR relate to the generation (chitinolytic system), transport (PTS.sup.GlcNAc), and metabolism (glycolysis via fructose 6-P) of N-acetylglucosamine. We therefore looked for the inducer among the intermediate molecules that gravitate around aminosugar metabolism. A binding interference experiment was set up where the ability of compounds to interfere with binding of dasR to the nagB and crr promoters was tested. These compounds were: N-acetylglucosamine, N-acetylglucosamine-6-P, glucosamine-6-P, glutamate, glutamine, acetyl-CoA, and fructose 6-P. These binding interference experiments identified glucosamine-6-P as the inducer/effector molecule, as it was the only of the compounds tested that prevented the formation of a complex of DasR with the nagB (
(99) N-Acetylglucosamine is Transported by the NagE2 Transporter
(100) Two possible transporters for N-acetylglucosamine were identified on the S. coelicolor genome, namely the adjacent genes nagE1 (SCO2906) and nagE2 (SCO2907). Mutants were created for both genes by replacing the entire gene by the apramycin resistance cassette aacC4. A double mutant was also produced (BAP6). The method used to do this was by using pWHM3, as described previously (Nothaft et al., 2003). Mutants deleted for the transport genes nagE1 (BAP4), nagE2 (BAP5), or both (BAP6), were plated on R2YE agar plates with or without N-acetylglucosamine (1% w/v). Other strains on the agar plates are: S.coelicolor M145 (parent of all mutants), the dasR mutant BAP29 and the pts mutants ptsH (BAP1), crr (BAP2), and ptsI (BAP3). For phenotypes of the pts mutants see also
(101) Clearly, influencing the activity of NagE2 (positively or negatively) will have a strong effect on the amount of inducer molecules introduced into the Streptomyces cell, and therefore will strongly effect the DasR regulatory system.
(102) The Full Scale of the DasR Regulon
(103) While we describe here around 200 targets (Table 2), the true number is without doubt much larger; for example, we used a highly restrictive dre position weight matrix to avoid false positives, but we have evidence that by doing so many true dre sites have been obscured. Additionally, we identified at least eight transcription factor genes in the list of predicted DasR targets. DasR controls many sensing/transport elements and the expression of Glu- and Gln-tRNAs. This suggests that DasR may be receptive to diverse environmental changes and governs many other regulons, and most likely at both the transcriptional and at the translational level. This multi-level control by DasR is summarized in
(104) Besides the absolute size of the regulon, a prerequisite for a global-acting regulator is further that it should act in concert with single-acting transcription factors (Moreno et al., 2001). Indeed, DasR controls chi-related genes, which are also regulated by the ChiS/ChiR two-component system (Kormanec et al., 2000) and by a third unknown DNA-binding protein identified recently (Fujii et al., 2005), suggesting a multi-partner control of the chitinolytic system. Another example arises from our studies on the regulation of the PTS, where we observed that besides DasR also SCO6008, encoding a ROK-family regulator (Titgemeyer et al., 1994), is required for activation of pts genes.
(105) The Wide-Spread DasR Regulon is a Target for Novel Screening Procedures
(106) Excitingly, the DasR regulatory network is highly conserved in S. avermitilis and S. scabies, with more than 75% of the dre sites predicted in S. coelicolor also found upstream of the orthologous genes in S. avermitilis, providing a strong phylogenetic argument for the presented predictions. The strong conservation of the dasR regulon in other actinomycetes also suggests that DasR may control many genes for natural products and enzymes in this class of bacteria. The conservation of the DasR regulon is underlined by the high conservation of DasR proteins (
(107) Considering the predicted control of clavulanic acid production in S. clavuligerus (Table 3), we cloned the dasR gene by PCR using oligonucleotides matching the −50/−30 and +900/+920 regions of S. coelicolor dasR, with nt positions relative to the start of the gene. The clone was sequenced, and the predicted gene product differed in a single amino acid position, namely an Asn55 in S. clavuligerus DasR and Asp55 in S. coelicolor DasR. On this basis it is obvious that the DasR binding site in S. clavuligerus is highly similar to that in S. coelicolor. The corresponding nucleic acid and amino acid sequence are disclosed in
(108) Surprisingly, we recently discovered that the core gene cluster nagA-nagB-dasR (and the dre elements) is also widespread among low G+C Gram-positives, including Bacillus, Lactococcus, Listeria and Streptococcus. Not only the organization is conserved, but also also the sequence of the dre sites, even though the G+C content of the DNA of Bacillus (43%) is around 30% lower than that of streptomycetes (72-73%). The dre sites of Bacillus subtilis and Bacillus halodurans are summarised in Table 5, those of Streptococcus species in Table 7, of Lactococcus lactis in Table 6, and of Listeria innocua and Listeria monocytogenes in Table 8. The derived consensus sequences for DasR binding sites in these species are summarised in cartoons in
(109) From this we conclude that the DasR core regulon is a very important concept, as its presence in such divergent micro-organisms means that the DasR control system has survived at least half a billion years of evolution. Finding a tool to manipulate the activity of DasR is therefore very important, as it will allow controlling the expression of many industrially and medically relevant compounds (enzymes, antibiotics, anti-tumor agents, agricultural compounds, and other secondary metabolites) from the outside rather than by genetic engineering. This is for example a prerequisite for setting up novel screening strategies, as individual strain manipulation is not an option. Addition of inducer (notably N-acetylglucosamine and derivatives) will trigger or at least enhance the expression of a wide range of natural products, allowing more ready screening. An obvious example is the control of cryptic clusters, which are silenced and therefore cannot be identified by activity-based screening assays. We show that antibiotic biosynthesis clusters are activated by the removal or reduced activity of DasR, and we anticipate that addition of inducer will relieve these clusters and thus boost the potential of novel screening procedures.
(110) Detailed Analysis of Effect of DasR on Act and Red Production
(111) As shown above, on media that do allow development (e.g. mannitol-containing solid media) the dasR mutant showed enhanced production of the pigmented antibiotics actinorhodin (Act) and undecylprodigiosin (Red). The relative increase in antibiotic production in the dasR mutant was quantified by determining Act and Red concentrations in the spent agar of solid-grown cultures (MM without any added carbon source). Under these conditions S. coelicolor grows solely on agar, enabled by induction of the DagA agarase (Buttner et al., 1987). Spectroscopic measurements showed that Act and Red production were consistently enhanced in BAP29 by factors of 3.2 (±0.2) and 3.9 (±0.3), respectively (averages of three independent experiments). As shown in
(112) We observed putative dre sites upstream of actII-ORF4 and redZ encoding transcriptional activators of the act and red gene clusters, respectively (for dre sites see Table 2). The dre site upstream of actII-ORF4 (nt positions −59/−44 relative to the translational start site) lies precisely between the canonical −35 and −10 sequences of the promoter, a position strongly suggesting that DasR should function as a transcriptional repressor. The dre site upstream of redZ (nt positions −201/186 relative to the translational start site) lies around 50 bp upstream of the −35 sequence of the redZ promoter.
(113) Electrophoretic mobility gel shift assays (EMSAs) with purified His.sub.6-tagged DasR and double-stranded oligonucleotide probes showed direct binding to the predicted dre sites of redZ and actII-ORF4 and to the positive control (dre site of crr-ptsI), while DasR did not bind to the cis-acting element of crp, which lacks similarity to the dre element and was therefore used as the negative control (
(114) The role of DasR in the control of actII-ORF4, redZ and redD was further assessed by semi-quantitative RT-PCR on RNA samples collected from the parental strain (M145) and the dasR mutant (BAP29) grown on MM mannitol agar plates for 30 h (vegetative growth), 42 h (initiation of aerial growth), and 72 h (aerial growth and spores). RT-PCR analysis revealed strongly enhanced transcription of actII-ORF4 at all time points in the dasR mutant and a discrete but significantly enhanced transcription of redZ (
(115) More Evidence that N-Acetylglucosamine Targets DasR to Unlock Antibiotic Production
(116) A signalling cascade from initial detection of the nutritional status of the environment to the onset of physiological and chemical differentiation should contain at least the following steps: (1) availability and sensing of an extracellular signal; (2) transport of ‘signalling nutrients’ into the cell; (3) their intracellular modification into an inducer molecule; (4) its binding to a global regulator, which is the checkpoint for (5) signalling the information to pathway-specific activators and (6) the switch to development and antibiotic production. Our experiments suggest that the GlcNAc sensory cascade controlled by DasR is a global system that triggers antibiotic production in direct response to nutrients (
(117) Arguing from the regulatory pathway deduced from the newly extended characterization of the DasR regulon (
(118) Application of GlcNAc for Drug Discovery
(119) Is the GlcNAc-mediated control of antibiotic production a more widespread phenomenon in streptomycetes? To assess this, we evaluated the effect of GlcNAc on total antimicrobial activity (bactericidal and bacteristatic) of several streptomycetes, using Bacillus subtilis as the indicator strain. The tested strains were spotted on minimal medium containing mannitol (0.5%) with or without GlcNAc (1%). Excitingly, growth inhibition zones indicative of antibiotic production were much larger for Streptomyces clavuligerus (a producer of cephamycin), Streptomyces collinus (produces kirromycin), Streptomyces griseus (streptomycin producer), Streptomyces hygroscopicus (produces hygromycin), Streptomyces rimosus (produces oxytetracycline), and Streptomyces venezuelae (chloramphenicol, methymycin) (
(120) In order to assess if DasR plays a role in silencing cryptic secondary metabolite clusters in streptomycetes, we analysed the expression level of a putative antibiotic biosynthetic cluster for a hypothetical type I polyketide (SCO6273-6288), the only cryptic cluster studied so far. Induction of this biosynthetic pathway depends on a pathway-specific activator, KasO (SCO6280), which is in turn repressed by the γ-butyrolactone (SCB1) binding protein ScbR (Takano et al., 2005). Repression of kasO is relieved by production of SCB1. To test the possible “awakening” of this cluster by the absence of dasR we performed semi-quantitative RT-PCR on RNA samples collected from the parental strain (M145) and the dasR mutant (BAP29) grown on MM mannitol agar plates for 30 h (vegetative growth), 42 h (initiation of aerial growth), and 72 h (aerial growth and spores). Excitingly, kasO transcripts were detected in the 30-h and 72-h RNA samples of BAP29, but were not seen in M145 in any of the samples (
(121) Involvement of DasR in the Control of Cell Wall Lysis
(122) Chitin is the main form of storage of GlcNAc and the second most abundant polymer on earth, and as such is of immense importance for soil-dwelling bacteria. GlcNAc is a rich N- and C-source and, with its metabolic products acetate, ammonia and fructose-6-P, stands at the crossroads of the major primary metabolic pathways. This underlines the selective advantage of being able to colonise different types of chitin-containing substrates (Saito et al., 2003; Schrempf, 2001). Our experiments suggest that GlcNAc can provide opposite signals, namely expansion (growth and developmental block) under nutrient-rich conditions and growth cessation followed by development (antibiotic production; sporulation) under nutrient-limited conditions. There are two major sources of GlcNAc: chitin and the bacterium's own cell wall, and they may trigger opposite responses. Bacterial chitinases mainly generate chito-oligosaccharides and N,N′-diacetylchitobiose (GlcNAc).sub.2 from chitin, and little GlcNAc. Also, dasR mutants have a five-fold lower chitinolytic activity than the parental strain (Colson et al., 2007), but overproduce antibiotics, suggesting that chitinases do not produce the signal. The ‘GlcNAc effect’ was observed only at higher concentrations (>5 mM). Perhaps the most likely natural source would be autolysis of the bacterial cell wall. Large amounts of GlcNAc were found to accumulate locally after programmed cell lysis, when general nutrient limitation necessitates development of an aerial mycelium at the expense of the vegetative hyphae (Miguelez et al., 2000). Since we show that nutrient sensing, cell wall lysis and proteolysis and secondary metabolism (in particular antibiotic production) are all linked directly to the function of DasR, there is a highly suggestive clustering within a single regulon of genes involved in the catabolism of peptidoglycan precursors, together with antibiotic pathway-specific activators.
(123) The contrast between the large number of secondary metabolites produced by streptomycetes and the relatively limited knowledge on the global regulatory mechanisms that trigger their production implies that much is to be gained in terms of drug discovery by learning from the organism itself. We propose a signalling cascade from nutrient stress to antibiotic production. Our deduced pathway proposes GlcNAc as an important signalling molecule for streptomycetes, allowing them to determine the nutritional status of the habitat. The signal that is transported by the PTS.sup.GlcNAc is metabolized to glucosamine-6-P, inactivating DasR, which in turn is responsible for suppression of antibiotic production and development under nutrient-rich conditions. Besides the PTS.sup.GlcNAc, DasR controls many more ABC sugar transporters and the functions of several of these are currently under investigation. The observation that antibiotic production can be awakened and/or enhanced by interfering with the DasR-mediated control system opens new perspectives for screening programmes directed at the discovery of novel natural products. Conceivably, the producing potential of thousands of strains could be boosted by addition of GlcNAc, and we have strong evidence that in many cases this makes the difference between a hit and a miss. This will improve the success rate of screening procedures aimed at the discovery of drugs for the treatment of infectious diseases caused by the recurring multi-drug resistant strains (such as MDR- and XDR-Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecalis (VRE)) but also of specific cancers.
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(125) TABLE-US-00001 TABLE 1 Experimentally validated DasR binding sites used to build the matrix for consensus sequences. malX2Sco ACTGGTGTAGACCAGT (SEQ ID NO: 11) score = 16.20 nagE2Sco(1) CAAGGTGTAGACCTCT (SEQ ID NO: 12) score = 11.35 nagE2Sco(2) AGTGGTGTAGACCTGT (SEQ ID NO: 13) score = 16.98 nagE2Sco(3) AGTGGTGTAGACCACC (SEQ ID NO: 14) score = 15.01 ptsHSco(1) AGTTGTCTAGACCAGT (SEQ ID NO: 15) score = 15.29 ptsHSco(2) TCTTGTCTAGACCAGT (SEQ ID NO: 16) score = 13.71 crr-ptslSco(1) TGTGGTCTAGACCTCT (SEQ ID NO: 17) score = 15.61 msiKSco GGTGGTGTAGTCCACA (SEQ ID NO: 18) score = 12.52 nagBSco TGTGGTTTAGACCAAT (SEQ ID NO: 19) score = 13.72 nagKASco(1) GGTGGTGTAGACCTTA (SEQ ID NO: 20) score = 13.05 nagKASco(2) AGTGGACTAGACCTCT (SEQ ID NO: 21) score = 14.45 chiFSco(1) AAGGGTGTAGACCAGT (SEQ ID NO: 22) score = 13.55 chiFSco(2) ACTGGTACAGACCAAA (SEQ ID NO: 23) score = 9.73 actII-4 TGTTGAGTAGGCCTGT (SEQ ID NO: 24) score = 11.22
(126) TABLE-US-00002 TABLE 2 Known or predicted co-transcribed # Evidence.sup.1 Target gene function dre Pos.sup.2 Score.sup.3 gene.sup.4 Known or predicted function 1 ERA− SCO2907, PTS EIIC component, ACAGGTCTACACCACT −49 16.98 15.01 — — nagE2 N-acetylglucosamine AGTGGTGTAGACCACC −32 11.35 uptake CAAGGTGTAGACCTCT −236 (SEQ ID NO: 25) 2 SCO2906, PTS EIIC component, not ACTGGTCTACACCAGT −41 16.2 — — nagE1 functional (SEQ ID NO: 26) 3 ERA− SCO2905c, PTS EIIB component, −134 16.2 — — malX2 N-acetylglucosamine uptake 4 E SCO5232, ABC sugar transporter, ACTGGTCTACACCATT −106 15.79 8.15 SCO5233, ABC sugar transporter membrane dasA sugar binding protein CTTGGTCTAGTCCATA −322 dasB/SCO5234, protein/ABC sugar transporter (SEQ ID NO: 27) dasC/SCO5235 membrane protein/putative intracellular Beta-N-acetylglucosaminidase 5 ERA− SCO1390, crr PTS EIIA.sup.crr component, TGTGGTCTAGACCTCT −130 15.61 SCO1391, ptsI PTS EI component, N-acetylglucosamine (SEQ ID NO: 28) phosphoenolpyruvate-protein uptake phosphatase 6 E SCO5842 putative acetyl-coenzyme AGTTGTCTAGACCAGT −168 15.29 13.71 — — A synthetase TCTTGTCTAGACCAGT −153 7 ERA− SCO5841c PTS Hpr, (SEQ ID NO: 29) −51, −66 — — N-acetylglucosamine and fructose uptake 8 E SCO4286 ABC sugar transporter, AGAGGTCTAGTCCACT −81. −63 14.45 hypothetical hypothetical protein, unknown sugar binding protein GGTGGTGTAGACCTTA 13.05 function 9 ER− SCO4285c, NagK, (SEQ ID NO: 30) −83, −101 SCO4284c, nagA NagA, nagK N-acetylglucosamine N-acetylglucosamine-6-phosphate kinase deacetylase 10 E SCO5239 Two-component sensor AGTGGTCTAGTCCACA −335 14.19 — — histidine kinase (SEQ ID NO: 31) 11 ER− SCO5236c, NagB, probable TGTGGTTTAGACCAAT −68 13.72 — — nagB glucosamine phosphate (SEQ ID NO: 32) isomerase 12 E SCO3563, acetoacetyl-coenzyme A ACAGGTCTAAACCATT −102 13.59 — — acsA synthetase (SEQ ID NO: 33) 13 ER+ SCO7263, chiF ChiF chitinase ACTGGTCTACACCCTT −172 13.55 9.73 SCO7264 probable NADPH dependent ACTGGTACAGACCAAA −155 oxidoreductase, (SEQ ID NO: 34) Aldo/keto reductase 14 E SCO7225 secreted chitinase TATGGTCTAGACCTGA −55 13.1812.4611.46 — — TCAGGTCTAGACCTGT −34 CCTTGTCTAGACCAAT −168 15 E SCO7224c possible integral (SEQ ID NO: 35) −272, −293 — — membrane protein, DoxX −159 family, unknown function 16 E SCO1444, chiI ChiI chitinase ACTGGTCTAGTCCTCT −53 12.81 5.22 — — ATTGGTCCATACCTAT −75 (SEQ ID NO: 36) 17 EP− SCO4240c, MsiK, multiple sugar GGTGGTGTAGTCCACA −75 12.52 — — msiK import protein, ABC (SEQ ID NO: 37) transporter ATP-binding protein 18 E SCO5004 hypothetical protein, GGTGGTCCAGACCAAT −258 12.08 — — unknown function (SEQ ID NO: 38) 19 E SCO5003c, ChiA chitinase −77 — — chiA 20 E SCO7056c GntR-family transcriptional ATTGGTCTAAACCAGC −79 12.08 6.85 — — regulator (new subfamily) GCAGGTCTGGTCCTCC −282 (SEQ ID NO: 39) 21 EA− SCO6486, DppA, AGTGGTCCAGACCACC −71 12.03 SCO6487, SCO6488, possible aminoacylase/putative dppA D-alanyl-aminopeptidase (SEQ ID NO: 40) SCO6489, SCO6490 acyl-peptide hydrolase/LD-carboxypeptidase/ putative alanine acetyltransferase 22 E SCO2672 membrane protein, ABC AGAGGTCTGGACAACA −32 11.99 — — transporter, FtsX cell (SEQ ID NO: 41) division permease family, unknown function 23 E SCO2503, chiJ ChiJ putative chitinase AAAGGTCTGGACCACA −78 11.818.47 5.73 — — CTTGGTCCAGACCTCT −99 TCTGGACCACAGCACT −73 (SEQ ID NO: 42) 24 E SCO1429, chiD ChiD chitinase ACTGGTCTAGTCCTCC −96 11.5 5.86 — — AATGGTCCGAACCATT −118 25 E SCO1428c, acd acyl-CoA dehydrogenase (SEQ ID NO: 43) −312 — — −290 26 SCO3679 hypothetical protein, sigma TGTTGTCTAGTCCAAT −314 11.41 — — factor PP2C-like (SEQ ID NO: 44) phosphatase 27 ER− SCO5085, actinorhodin cluster TGTTGAGTAGGCCTGT −59 11.22 — — actII-4 activator protein (SEQ ID NO: 45) 28 E SCO6013 probable AATGGTCTGGACCAGA −274 11.03 8.63 7.95 — — 1-deoxyxylulose-5-phosphate GGTGGACTGGACCACC −201 synthase ATGGGACTAGACCAAT −258 29 E SCO6012c, ChiH chitinase (SEQ ID NO: 46) −111, −184, — — chiH −127 30 SCO4315 possible copper ATTGGACTAGACCTGT −39 10.99 — — homeostasis protein, CutC (SEQ ID NO: 47) family 31 E SCO4671c LysR-family regulatory GCTGGTACAGACCAGT −55 10.83 — — protein (SEQ ID NO: 48) 32 E SCO6300c probable secreted AGAGGTCTAGACAAAA −116 10.67 9.78 — — Beta-N-acetylglucosaminidase ATAGGTCTAGACAAAA −131 (SEQ ID NO: 49) 33 E SCO6005, ABC sugar transporter, AGTGGACTATACCTGT −334 10.56 SCO6006, ABC sugar transporter membrane ngcE sugar binding protein, (SEQ ID NO: 50) ngcF/SCO6007, ngcG protein, NgcF/ABC sugar NgcE transporter membrane protein, NgcG 34 E SCO6004c putative AGTGGACTATACCTGT −244 10.56 — — alpha-1,2-mannosidase (SEQ ID NO: 51) 35 E SCO5376c, ChiC chitinase AAAGGTCTGGACCATA −88 10.35 9.29 SCO5375c possible secreted protein, chic ATAGGTCTGGACCAAT −109 unknown function (SEQ ID NO: 52) 36 E SCO6345 chitinase TAAGGTCTAGACCTGC −114, −94 9.99 8.74 — — 37 E SCO6344c putative secreted amidase GTAGGTCTAGACCTGC −133, −153 — — (SEQ ID NO: 53) 38 SCO1212 putative Mur_like ligase TGAGGTCCACACCACG −76 9.92 SCO1213 conserved hypothetical protein 39 SCO1211c putative polypeptide (SEQ ID NO: 54) −5 — — deformylase 40 SCO1083c putative flavin-dependent TGTGGAGAAGACCTCA −129 9.48 — — reductase (SEQ ID NO: 55) 41 E SCO1433 hypothetical protein, ATTGGTGTCGACCACT −205 9.41 — — unknown function (SEQ ID NO: 56) 42 E SCO1432c possible membrane ATTGGTGTCGACCACT −86 SCO1431c possible membrane protein, protein, unknown function (SEQ ID NO: 57) unknown function 43 SCO5266 putative membrane protein CATGGTGCAGACCTCC −139 9.25 — — (SEQ ID NO: 58) 44 SCO5265c hypothetical protein CATGGTGCAGACCTCC 38 9.25 SCO5264c hypothetical protein SC7G11.26c (SEQ ID NO: 59) 45 E SCO0481, putative secreted chitin TATGGTCTAGTCCAAC −201 9.19 — — chb3 binding protein (SEQ ID NO: 60) 46 E SCO2753 Lacl-family transcriptional GGTGGTCTGGACAAGA −120 9.15 — — regulator, NagR (SEQ ID NO: 61) 47 E SCO2752c possible oxidoreductase, −127 SCO2751c, hypothetical protein/putative unknown SCO2750c isomerase, unknown function 48 E SCO7250c putative AGTGGCGTACACCTGT −213 9.04 — — N-acetylmuramoyl-L-alanine (SEQ ID NO: 62) amidase 49 E SCO5673, chiB ChiB chitinase ATTGGTCTGGACCAAA −63 9.03 — — (SEQ ID NO: 63) 50 SCO7699 putative nucleotide-binding GAGGGTCCAGACCTCT −245 9.0 SCO7700/SCO7701 putative cyclase/putative protein, (SEQ ID NO: 64) methyltransferase sporulation-specific protein p3 (S. griseus) 51 SCO7698c putative merR-family −19 — — transcriptional regulator 52 E SCO2833c, chb secreted chitin binding GCAGGTCTAGACCAAG −70 8.91 — — protein (SEQ ID NO: 65) 53 E SCO2946c ABC sugar transporter, AGAGGTCTGAACCAAT −112 8.91 SCO2945c, ABC sugar transporter membrane sugar binding protein (SEQ ID NO: 66) SCO2944c, protein, ABC sugar transporter SCO2943c membrane protein, putative intracellular beta-N-acetylglucosaminidase 54 E SCO1117c putative CGCGGTCTAGACCAAA −131 8.79 — — 3-carboxymuconate (SEQ ID NO: 67) cyclase 55 E SCO5230c integral membrane protein, TCTGGTCTAGTCCTGG −118 8.77 SCO5229c probable permease, putative sensory protein (SEQ ID NO: 68) sodium:solute symporter family 56 SCO6149 putative ATP GTP-binding GGAGGTGTCGACCAAT −140 8.76 SCO6150, CO6151 putative ADA-like regulatory protein (SEQ ID NO: 69) protein/putative methylated-DNA-protein-cysteine methyltransferase 57 SCO6319 putative lipoprotein ATTGGTCTGAACCATG −30 8.76 — — (SEQ ID NO: 70) 58 SCO6033 hypothetical protein CTTGGTCTAGTCCATT −278 8.68 — — SC1C3.21 (SEQ ID NO: 71) 59 SCO6032c beta-N-acetylglucosaminidase −154 — — 60 E SCOEc, chiE ChiE chitinase and CTTGGTCCAGACCTGT −188 8.65 — — metallopeptidase (SEQ ID NO: 72) 61 SCO4394, iron repressor TGCGGTCTGGACCAGT −184, +9 8.42 7.45 — — desR ACTGATCGACACCACG 62 SCO4393c Possible phosphosugar (SEQ ID NO: 73) −247, −55, — — isomerase 63 SCO6084 putative DNA polymerase GAGGGTGGAGACCACT −292, −49 8.3 8.13 — — GGTGGTGCAGTCCTAC (SEQ ID NO: 74) 64 SCO5046, wb/l hypothetical protein TCAGGAGTAGACCCGT −14 8.23 — — (SEQ ID NO: 75) 65 SCO1954 hypothetical protein GGATGTGAAGACCTCT −101 8.15 — — 66 SCO1953c ABC excision nuclease (SEQ ID NO: 76) −268 SCO1952c, hypothetical protein/conserved subunit C SCO1951c, hypothetical protein/ SCO1950c hypothetical protein 67 SCO5231c, DasR, gntR-family CTTGGTCTAGTCCATA −150 8.15 — — dasR transcriptional regulator (SEQ ID NO: 77) 68 SCO4506 conserved hypothetical AGAGGTCAAGATCACT −103 8.05 SCO4507 putative serine/threonine protein (SEQ ID NO: 78) protein kinase 69 SCO4505c, cold shock protein −206 — — scoF2 70 SCO3152c hypothetical protein AGTGGACTCCTCCACC −50 8.03 — — SCE87.03c (SEQ ID NO: 79) 71 SCO6232 putative beta-mannosidase TCAGGACTAGACCGGT −86 7.97 SCO6233 putative transcriptional regulator 72 SCO6231c probable sugar transport (SEQ ID NO: 80) −202 — — system sugar-binding lipoprotein SC2H4.13c 73 E SCO1906c putative secreted protein, ACTGGCGGAGACCTCT −128 7.93 — — unknown function (SEQ ID NO: 81) 74 SCO2119c, 6-phosphofructokinase GGTGGTTGAGGCCACT −40 7.83 — — pfkA (SEQ ID NO: 82) 75 SCO1971 conserved hypothetical TGTGGTCGAGACGTGT −172 7.77 SCO1972 putative sugar kinase protein (SEQ ID NO: 83) 76 SCO1970c putative dioxygenase 30 — — 77 SCO1289 putative gntR-family CGTGGTGCAGACGTGA −36 7.73 — — regulatory protein (SEQ ID NO: 84) 78 SCO1288c putative integral −143 SCO1287c hypothetical protein membrane protein 79 SCO0073 hypothetical protein CCAGGTTCAGACCTGT −219 7.69 SCO0074, SCO0075 hypothetical protein/ (SEQ ID NO: 85) hypothetical protein 80 SCO0072c putative wall associated −309 — — protein 81 SCO5463 putative MerR-family ACTGGCCCGCACCACC 39 7.55 SCO5464 — transcriptional regulator (SEQ ID NO: 86) 82 SCO5462c hypothetical protein −103 — — SC3D11.19c 83 SCO5016c putative integral GGTGGAGCAGACCGGA −280 7.5 — — membrane protein (SEQ ID NO: 87) 84 E SCO3975c putative regulator TGTGGTCGAGACCGGA −86 7.49 — — (SEQ ID NO: 88) 85 SCO5366, atpl ATP synthase protein I AGAGGTAAAGACCTCA −172 7.49 — — (SEQ ID NO: 89) 86 SCO2787 conserved hypothetical ACGGGTGCGGACCACT −61 7.44 SCO2788, SCO2789, hypothetical protein protein SCC105.18 (SEQ ID NO: 90) glmS2 SCC105.19/glucosamine- fructose-6-phosphate aminotransferase 87 SCO2786c beta-N-acetylhexosaminidase −70 — — 88 SCO4442 hypothetical protein ATTGGCGTAAACCACA −41 7.41 — — SCD6.20 (SEQ ID NO: 91) 89 SCO1752 putative integral TGTGGCATGCACCACT −80 7.29 — — membrane protein (SEQ ID NO: 92) 90 SCO1751c putative transmembrane −198 — — transport protein 91 SCO6003c putative DNA-binding GCCGGTGAAGACCAGT −235 7.26 — — protein (SEQ ID NO: 93) 92 E SCO5716c putative peptide transport ATTGGCGCAGACCACT −197 7.24 — — system secreted peptide (SEQ ID NO: 94) binding protein 93 SCO5330 hypothetical protein GCTGGCGTAGCCCACT −54 7.16 — — SC6G9.03c (SEQ ID NO: 95) 93 SCO5430c putative extracellular AATGGTCTAGTCAGGT −81 7.02 SCO5429c, putative integral membrane solute-binding lipoprotein (SEQ ID NO: 96) SCO5428c transport protein/putative integral membrane transport protein 94 SCO2685c putative ATP-binding AGTGGACAACACCCGA −142 6.95 SCO2684c putative ATP-binding protein SCC61A.06c (SEQ ID NO: 97) membrane protein 96 E SCO4516c hypothetical protein ACTGGTCTGGATCCGT −20 6.91 SCO4515c putative membrane protein SCD35.23c (SEQ ID NO: 98) 97 SCO7054 conserved hypothetical TGTGGAGTAGAGTAGT −47 6.89 SCO7055 putative methyltransferase protein (SEQ ID NO: 99) 98 SCO7053c hypothetical protein −50 — — 99 E SCO4735 30S ribosomal protein S9 CGTGGCCGAGACCACT −1 6.88 — — (SEQ ID NO: 100) 100 SCO4722, preprotein translocase GCTCGTCTGAACCACT −266 6.87 SCO4723 adenylate kinase secY SecY subunit (SEQ ID NO: 101) 101 SCO7509c conserved hypothetical GCGGGTGAAGACCAGC 20 6.83 — — protein (SEQ ID NO: 102) 102 SCO4646, preprotein translocase ACTGGTCTCCAAAACC −156 6.81 SCO4647 transcription antitermination secE SecE subunit (SEQ ID NO: 103) protein 103 SCO4645c aspartate ACTGGTCTCCAAAACC −281 6.81 — — aminotransferase (SEQ ID NO: 104) 104 SCO4562 NuoA, NADH GGTGGTGGAGATCACA −206 6.78 SCO4563-SCO4575 NADH dehydrogenase subunits dehydrogenase subunit (SEQ ID NO: 105) (nuoBCDEFGHIJKLM NuoA-NuoN N) 105 SCO1692c putative oxidoreductase CGCGGTCTACTCCATT −116 6.76 SCO1691c putative tetR transcriptional (SEQ ID NO: 106) regulator 105 SCO4904c putative integral TGAGGCCTGCGCCACA −72 6.69 — — membrane protein (SEQ ID NO: 107) 107 SCO5954 chitinase (putative ATTGGTCCAGACCTTC −95 6.64 — — secreted protein) (SEQ ID NO: 108) 108 SCO5805, nrdJ ribonucleotide reductase AGTGAACAAGACCTGT −117 6.61 — — (SEQ ID NO: 109) 109 SCO4609 putative peptidase CCTGGCGTTGACCAGT −136 6.6 SCO4610/SCO4611 putative integral membrane (SEQ ID NO: 110) protein/hypothetical protein SCD39.11 110 SCO2547c putative hydrolase GGTGGTCCGGTCCTGT −24 6.58 SCO2546c probable adenosine deaminase (SEQ ID NO: 111) 111 SCO4963 putative ABC transporter CCTGGTGAAGACCTTC 27 6.55 SCO4964 putative integral membrane ATP-binding protein (SEQ ID NO: 112) transport protein 112 SCO2037c, tryptophan synthase beta GGTGGATCAGACCGCT −69 6.54 SCO2036c tryptophan synthase alpha trpB subunit (SEQ ID NO: 113) subunit 113 SCO0915 hypothetical protein TTTGGTATGGACCATT −98 6.52 — — SCM10.03 (SEQ ID NO: 114) 114 SCO0914c putative lipoprotein −127 — — 115 SCO2802 putative secreted protein AGAGGACTCGTCCACG 16 6.51 — — (SEQ ID NO: 115) 116 SCO6914 hypothetical protein GGTGCTGGAGACCTCA −133 6.51 SCO6915 hypothetical protein SC1B2.21 SC1B2.20 (SEQ ID NO: 116) 117 E SCO5609c hypothetical protein ACTGGTCAGGACCGCT −133 6.38 — — SC2E1.26c (SEQ ID NO: 117) 118 SCO3261 putative ATP-binding ACCGGGCTACACACCT −284 6.37 SCO3262 hypothetical protein protein (SEQ ID NO: 118) 119 SCO5636c, transcriptional regulator ACTGGACGAGACCCCG −187 6.34 — — korSA (SEQ ID NO: 119) 120 E SCO7070 chitosanase ACAGGTCCGGACCAAT −50 6.34 — — (SEQ ID NO: 120) 121 E SCO7069c chitinase −61 — — 122 SCO1558c putative ABC transporter GGAGGCCTGCTCCAGC 11 6.32 — — permease protein (SEQ ID NO: 121) 123 SCO5717c conserved hypothetical CCGGGTGTAGCCCAGC 31 6.32 — — protein SC3C3.03c (SEQ ID NO: 122) 124 SCO3510c putative DNA methylase CGGGGTCTGGACCTGC −42 6.29 — — (SEQ ID NO: 123) 125 SCO4811 putative integral CGTGATCCAGACCACC 9 6.25 — — membrane protein (SEQ ID NO: 124) 126 SCO3560 putative ATP-binding AGTGGTCTTCTTCACC −61 6.24 — — protein (SEQ ID NO: 125) 127 SCO3559c putative oxidoreductase −58 — — 128 SCO5276 conserved hypothetical ACGGGTCCACTCAACA −56 6.24 SCO5277, SCO5278, putative magnesium protein (SEQ ID NO: 126) SCO5279, SCO5280 chelatase/putative magnesium chelatase/hypothetical protein SCCB12.03/putative ATP-binding protein 129 R− SCO5881c, Undecylprodigiosine AGTGGTTTCCACCTCA −201 6.24 — — redZ activator (SEQ ID NO: 127) 130 SCO1262c putative gntR-family CTTGGTCAAGACCAAT −113 6.22 — — transcriptional regulator (SEQ ID NO: 128) 131 SCO2141 putative small secreted ATCGGCCTGGACAACT −167 6.19 SCO2142, SCO2143, putative two component sensor hydrophilic protein (SEQ ID NO: 129) SCO2144, SCO2145 kinase/putative two component system response regulator/ putative integral membrane transporter/putative glycerate kinase .sup.1If confirmed target, method is indicated: E, EMSA (target site bound by DasR), A, enzyme Assay, R, RT PCR. “+” means shown to be activated by DasR, “−”, shown to be repressed by DasR. .sup.2Position is last nucleotide position of the dre target site relative to the translational start site of the gene. .sup.3Weighed matrix score of the dre site. Cut-off of 6.00 was taken as bottom limit. Higher score means better fit to the consensus sequence. .sup.4If genes are known or predicted to be in an operon (generally because there is no or almost no intergenic region), the presumed co-transcribed genes are shown.
(127) TABLE-US-00003 TABLE 3 List of putative binding sites for DasR relating to secondary metabolism (cut-off score 5) A. Antibiotics and metabolites of known function produced by actinomycetes Secondary metabolite Streptomyces strain dre sequence Target Function score Clavulanic acid S. clavuligerus attggagtagacctct (SEQ ID NO: 130) pcbR PBP; β-lactam resistance 11.47 ggagggctggaccagc (SEQ ID NO: 131) pcbR PBP; β-lactam resistance 5.72 Actinorhodin S. coelicolor tgttgagtaggcctgt (SEQ ID NO: 132) actII-ORF4 pathway-specific activator 11.22 Undecylprodigiosin S. coelicolor gcaggtggagaccacc (SEQ ID NO: 133) redZ pathway-specific activator 6.61 tgaggtggaaaccact (SEQ ID NO: 134) redZ pathway-specific activator 6.24 Valanimycin S. viridifaciens ctctgagtaggcctgt (SEQ ID NO: 135) vlmM Valanimycin transferase 8.01 Daptomycin S. filamentosus actggtgtcgaccagc (SEQ ID NO: 136) dptD peptide synthetase 3 10.12 ggaggtcgagaccagt (SEQ ID NO: 137) dptP hypothetical 9.24 tgaggtgtacgccacc (SEQ ID NO: 138) AAX31564 Putative phosphatase 6.59 tgaggtcgaggccacc (SEQ ID NO: 139) dptD peptide synthetase 3 5.98 agtggtgctgcccaat (SEQ ID NO: 140) dptP hypothetical 5.97 cgtgatctacacctcc (SEQ ID NO: 141) dptBC peptide synthetase 2 5.91 cgtgatctacacctcc (SEQ ID NO: 142) dptBC peptide synthetase 2 5.91 acaagtccacaccccc (SEQ ID NO: 143) dptA peptide synthetase 1 5.53 cgaggcgtagacctgg (SEQ ID NO: 144) AAX31565 metalloprotease 5.43 cgtggcctggacctca (SEQ ID NO: 145) dptBC peptide synthetase 2 5.39 ggtggtcctcaccacg (SEQ ID NO: 146) dptBC peptide synthetase 2 5.33 actggagtccacctga (SEQ ID NO: 147) AAX31520 ATP-dependent helicase 5.00 cgaggcctacaccctc (SEQ ID NO: 148) AAX31577 hypothetical 6.05 Novobiocin S. spheroides ggaggtgtagatcaca (SEQ ID NO: 149) novH Peptide synthase 8.45 Actinomycin S. anulatus ggaggtgtagatcaca (SEQ ID NO: 150) acmC peptide synthase III 8.45 acmC peptide synthase III A47934 S. toyocaensis ggaggtgtagatcaca (SEQ ID NO: 151) staC peptide synthetase 8.45 (teichoplanin) tctggtggagaccttc (SEQ ID NO: 152) staP membrane protein 7.73 actggtctgctcgatg (SEQ ID NO: 153) staN ion transporter 6.23 gctggtccaggcccct (SEQ ID NO: 154) dpgB enhancer of DihydroxyphenylAcCoA synthetase activity 6.21 ggtggtctccagcacc (SEQ ID NO: 155) vanSst histidine kinase 5.87 gctggtctcgaccctc (SEQ ID NO: 156) vanSst histidine kinase 5.76 catggtctcgtccagc (SEQ ID NO: 157) staF P450-related oxidase 5.06 Streptomycin S. griseus gggggtgtcgaccagc (SEQ ID NO: 158) CAH94374 hypothetical 7.81 cgtggcgcagtccaca (SEQ ID NO: 159) CAH94409 methyltransferase 5.74 cctggtcttcaccccg (SEQ ID NO: 160) strZ transmembrane protein 5.66 agtggtctgcgcatgc (SEQ ID NO: 161) apbE thiamine biosynthesis lipoprotein 5.13 tgacgtctactccttc (SEQ ID NO: 162) CAH94395 PqrB-type multidrug efflux protein 5.03 ggtggggcagaccatc (SEQ ID NO: 163) strB1 Amidinotransferase I 6.22 agttgtgcagacgggt (SEQ ID NO: 164) strZ transmembrane protein 6.09 attggcctgcaccgcg (SEQ ID NO: 165) strU NAD(P) dependent oxidoreductase 6.04 Chloramphenicol S. venezuelae acgggtctacacctcc (SEQ ID NO: 166) papD p-aminobenzoic acid synthase ORFIV 10.38 gctggtgtcgaccatc (SEQ ID NO: 167) papA 4-amino-4-deoxychorismate 7.34 synthase cgaggtggagacctac (SEQ ID NO: 168) papA 4-amino-4-deoxychorismate 5.91 synthase cgaggtggagacctac (SEQ ID NO: 169) papAB p-aminobenzoate synthase Butyrolactone S. virginiae actggtgtcgaccag (SEQ ID NO: 170) barB hormone-like γ-butyrolactone 10.12 biosynthesis B. Known and cryptic biosynthesis clusters of Streptomyces coelicolor metabolite gene function dre position score co-transcribed function actinorhodin SCO5085 actinorhodin cluster TGTTGAGTAGGCCTGT −59 11.22 — — activator protein (SEQ ID NO: 171) actinorhodin SCO5090 actinorhodin GGTGGTCCACACCCTG 24 5.58 SCO5091/SCO5092 cyclase/actinorhodin polyketide putative polyketide synthase (SEQ ID NO: 172) dimerase bifunctional cyclase/ dehydratase Prodigiosin SCO5879 acyl-coa dehydrogenase ACAGGTCTACGGCACG −324 7.33 SCO5880 RedY protein RedW (SEQ ID NO: 173) Prodigiosin SCO5881c RedZ response AGTGGTTTCCACCTCA −201 6.24 — — regulator (SEQ ID NO: 174) Prodigiosin SCO5883c hypothetical protein GTTGGCCTGCTCCAGG −252 5.11 — — SC3F7.03c (SEQ ID NO: 175) CDA SCO3237c conserved hypothetical GTAGGTCTCGACCTCC −151 5.84 — — (calcium-dependent protein (SEQ ID NO: 176) antibiotic) CDA SCO3226 two component system AGCGGTCTGCTCGACT −99 5.75 — — (calcium-dependent response regulator (SEQ ID NO: 177) antibiotic) CDA SCO3234 putative CGATGTCCAGACCGGT 15 5.51 — — (calcium-dependent phosphotransferase (SEQ ID NO: 178) antibiotic) Isorenieratine SCO0188 putative GCAGGACTACACCGTG −168 5.6 — — methylesterase (SEQ ID NO: 179) Tetrahydroxynaphtalene SCO1206 putative polyketide GCTGGTGGAGACCGGC −274 5.6 SCO1207/SCO1208 putative cytochrome P450/hypothetical protein (melanine) synthase (SEQ ID NO: 180) Unknown SCO0387 putative bi-domain GGTTGTGCAGAACTAC −4 5.04 SCO0388/SCO0389/ hypothetical protein SCF62.14/putative oxidoreductase (SEQ ID NO: 181) SCO0390/SCO0391/ lipoprotein/putative membrane protein/putative SCO0392/SCO0393/ transferase/putative methyltransferase/ SCO0394/SCO0395/ putative transferase/hypothetical protein SCO0396/SCO0397/ SCF62.20/putative epimerase/dehydratase/ SCO0398/SCO0399/ hypothetical protein SCF62.22/putative SCO0400/SCO0401 integral membrane protein/putative glycosyl transferase/putative membrane protein/ putative epimerase/putative aminotransferase Unknown SCO0388 hypothetical protein GCTGGTCGCCACCACG −87 5.51 SCO0389/SCO0390/ putative lipoprotein/putative membrane SCF62.14 (SEQ ID NO: 182) SCO0391/SCO0392/ protein/putative transferase/putative SCO0393/SCO0394/ methyltransferase/putative transferase/ SCO0395/SCO0396/ hypothetical protein SCF62.20/putative SCO0397/SCO0398/ epimerase/dehydratase/hypothetical protein SCO0399/SCO0400/ SCF62.22/putative integral membrane SCO0401 protein/putative glycosyl transferase/putative membrane protein/putative epimerase/ putative aminotransferase Unknown SCO6282c putative GCTGGACGAGTCCACC −262 5.42 — — 3-oxoacyl-[acyl- (SEQ ID NO: 183) carrier protein] reductase
(128) TABLE-US-00004 TABLE 4 DasR target genes related to glutamate metabolism. Targets, validation (experimental or in silico) and gene function are presented. For meaning and deduction of the score, see text. # Gene Known or predicted gene product Evidence dre Score 1 tRNA Gln tRNA Gln anticodon CTG. S ACTGGTCTAAACCACA (SEQ ID NO: 184) 14.43 2 tRNA Glu tRNA Glu anticodon CTC. S ACTGGTCTAAACCACA (SEQ ID NO: 185) 14.43 3 SCO4285c, N-acetylglucosamine kinase. S, E AGAGGTCTAGTCCACT GGTGGTGTAGACCTTA (SEQ ID NO: 186) 12.82 8.00 nagK ATP + N-acetyl-D-glucosamine = ADP + N-acetyl-D-glucosamine- 6-phosphate 4 SCO4284c, N-acetylglucosamine-6-phosphate deacetylase. S, E, P AGAGGTCTAGTCCACT GGTGGTGTAGACCTTA (SEQ ID NO: 187) 12.82 8.00 nagA CoA + N-acetyl-D-glucosamine 6-phosphate = acetyl-CoA + D-glucosamine-6-phosphate 5 SCO5236c, Glucosamine-6 phosphate isomerase. S, E TGTGGTTTAGACCAAT (SEQ ID NO: 188) 11.36 nagB L-glutamine + D-fructose 6-phosphate = L-glutamate + D-glucosamine-6-phosphate 6 SCO6344 Glu-tRNAGln amidotransferase A subunit. S TAAGGTCTAGACCTGC (SEQ ID NO: 189) 9.99 ATP + glutamyl-tRNA(Gln) + L-glutamine = ADP + phosphate + glutaminyl-tRNA(Gln) + L-glutamate 7 SCO5520 pyrroline-5-carboxylate dehydrogenase. P — — 1-pyrroline-5-carboxylate + NAD+ + H2O = L-glutamate + NADH + H+ 8 SCO4683 GdhA, NADP-specific glutamate dehydrogenase. P — — L-glutamate + H2O + NAD+ = 2-oxoglutarate + NH3 + NADH + H+ 9 SCO4366 phosphoserine aminotransferase. P — — O-phospho-L-serine + 2-oxoglutarate = 3-phosphonooxypyruvate + L-glutamate S, predicted in silico; E, experimentally validated in vitro; P, deduced from proteomics experiments; dre, DasR responsive element. Scores are expressed in unit of bits.
(129) TABLE-US-00005 TABLE 5 DasR binding sites in Bacillus species. Score Position Site Gene Synonym COG Product A. B. subtilis 4.70572 −121 AGTGATCTATACCAAT (SEQ ID NO: 190) yflG Bsu0769 COG0024 similar to methionine aminopeptidase 4.70572 −67 ATTGGTATAGATCACT (SEQ ID NO: 191) yflF Bsu0770 COG1264 similar to phosphotransferase system enzyme II 4.54507 −60 AACGGTCTAGACAAAT (SEQ ID NO: 192) yxaG Bsu3995 — yxaG 4.47401 −127 AGTGATCTAGACCAGC (SEQ ID NO: 193) yvoB Bsu3497 COG1493 similar to hypothetical proteins 4.47401 −71 GCTGGTCTAGATCACT (SEQ ID NO: 194) nagA Bsu3498 COG1820 N-acetylglucosamine-6-phosphate deacetylase 4.41253 −44 AGTTGTATATACAAGT (SEQ ID NO: 195) treP Bsu0780 COG1264 phosphotransferase system (PTS) trehalose-specific enzyme IIBC component 4.41253 −165 ACTTGTATATACAACT (SEQ ID NO: 196) yfkQ Bsu0779 — similar to spore germination response 4.3857 −174 ATATGTATAGACCTGT (SEQ ID NO: 197) yqjU Bsu2373 — yqjU 4.3246 −326 ATCTGTCTATACCTAT (SEQ ID NO: 198) yomE Bsu2140 — yomE 4.28564 −82 AATAGTATAGACTATT (SEQ ID NO: 199) pckA Bsu3051 COG1866 phosphoenolpyruvate carboxykinase 4.25776 −320 TTTTGTATATACCATT (SEQ ID NO: 200) ydbO Bsu0454 COG0053 similar to hypothetical proteins 4.25776 −61 AATGGTATATACAAAA (SEQ ID NO: 201) ydbN Bsu0453 — ydbN 4.25487 −68 AGTGGTCTAAACTCCT (SEQ ID NO: 202) bofA Bsu0023 — integral membrane protein 4.24264 −226 AATGGTATATATCATA (SEQ ID NO: 203) yodL Bsu1963 — yodL 4.23975 −346 ATGGCTCTACACCATT (SEQ ID NO: 204) ylaD Bsu1476 — ylaD 4.23813 −2 AATGGAATATACCAGT (SEQ ID NO: 205) yhdN Bsu0953 COG0667 similar to aldo/keto reductase 4.23813 −211 ACTGGTATATTCCATT (SEQ ID NO: 206) yhdM Bsu0952 COG1595 similar to RNA polymerase ECF-type sigma factor 4.23131 −190 ATTGGTTTAGACAACA (SEQ ID NO: 207) tenI Bsu1167 COG0352 transcriptional regulator 4.23065 −91 ACATTTCTATACCATT (SEQ ID NO: 208) hemE Bsu1012 COG0407 uroporphyrinogen III decarboxylase 4.20249 −113 AATTATATATACAATT (SEQ ID NO: 209) rbsR Bsu3589 COG1609 transcriptional regulator (LacI family) 4.20249 −46 AATGTTATATAACATT (SEQ ID NO: 210) yurO Bsu3257 COG1653 similar to multiple sugar-binding protein 4.20249 −115 AATGGTATATAATATT (SEQ ID NO: 211) yndL Bsu1783 — similar to phage-related replication protein 4.19036 −170 ATTCGTATAAACAAGT (SEQ ID NO: 212) yeeA Bsu0677 COG1002 similar to hypothetical proteins 4.19036 −10 AATGGTTTATATGAAT (SEQ ID NO: 213) yqeK Bsu2559 COG1713 similar to hypothetical proteins 4.19036 −237 ACTTGTTTATACGAAT (SEQ ID NO: 214) yefB Bsu0675 — similar to site-specific recombinase B. B. halodurans 4.62842 −168 ATTTGTATATACCAAT (SEQ ID NO: 215) BH0422 BH0422 COG1263 PTS system, N-acetylglucosamine-specific enzyme II, ABC component 4.62842 −88 ATTGATATATACCAAT (SEQ ID NO: 216) BH3323 BH3323 COG2188 transcriptional regulator (GntR family) 4.62842 −155 ATTGGTATATACAAAT (SEQ ID NO: 217) nagA BH0421 COG1820 N-acetylglucosamine-6-phosphate deacetylase 4.62842 −231 ATTGGTATATATCAAT (SEQ ID NO: 218) BH3324 BH3324 COG1208 glucose-1-phosphate thymidylyltransferase 4.54325 −152 ATTGGTATAGACATTT (SEQ ID NO: 219) BH0419 BH0419 COG2188 transcriptional regulator (GntR family) 4.54325 −103 AAATGTCTATACCAAT (SEQ ID NO: 220) BH0418 BH0418 COG0500 BH0418~unknown conserved protein in others 4.5403 −155 TATGGTATAGACCACT (SEQ ID NO: 221) BH2230 BH2230 — BH2230~unknown 4.50391 −214 ATTGGTATAAACAAAT (SEQ ID NO: 222) BH1924 BH1924 COG1653 sugar transport system (sugar-binding protein) 4.48969 −175 ATTCGTTTAGACCAAT (SEQ ID NO: 223) BH0593 BH0593 — BH0593~unknown 4.45248 −41 ATTGTTCTAGACCCTT (SEQ ID NO: 224) BH2561 BH2561 COG3879 BH2561~unknown conserved protein in bacilli 4.41889 −63 ACTTGTATATACAAAT (SEQ ID NO: 225) BH2216 BH2216 COG1264 PTS system, trehalose-specific enzyme II, BC component 4.41579 −316 AATGGTCTACACCAAG (SEQ ID NO: 226) BH1484 BH1484 COG3595 BH1484~unknown conserved protein in others 4.41579 −302 CTTGGTGTAGACCATT (SEQ ID NO: 227) BH1482 BH1482 COG0517 BH1482~unknown conserved protein in B. subtilis 4.40381 −99 ACTTGTATATACAAGT (SEQ ID NO: 228) treA BH0872 COG0366 alpha,alpha-phosphotrehalase 4.40367 −81 AAAGGTGTAGATCATT (SEQ ID NO: 229) BH0661 BH0661 — response regulator aspartate phosphatase 4.37739 −104 AATGATTTAGATCAAT (SEQ ID NO: 230) BH0786 BH0786 COG1940 transcriptional regulator 4.33078 −136 TATGGTCTATATCATT (SEQ ID NO: 231) BH0464 BH0464 COG1968 bacitracin resistance protein (undecaprenol kinase) 4.29237 −81 AATAGTATAGACTATT (SEQ ID NO: 232) pckA BH3302 COG1866 phosphoenolpyruvate carboxykinase 4.25057 −344 ACAGGTGTAGACATTT (SEQ ID NO: 233) BH3024 BH3024 COG0745 BH3024~unknown 4.22134 −148 AGTTGTTTAGACCAGA (SEQ ID NO: 234) BH2314 BH2314 COG0191 fructose bisphosphate aldolase 4.21655 −71 ATTGATCTATACAAAC (SEQ ID NO: 235) BH3244 BH3244 — general stress protein 4.21022 −216 ATTGATATAGATGAGT (SEQ ID NO: 236) BH2450 BH2450 — BH2450~unknown 4.19716 −314 AATGGTGTATAGAAAT (SEQ ID NO: 237) BH3530 BH3530 — BH3530~unknown 4.19716 −145 ATTGATTTATAGCATT (SEQ ID NO: 238) BH4039 BH4039 COG0582 BH4039~unknown conserved protein 4.1957 −3 ACATGTCTATACATCT (SEQ ID NO: 239) BH3678 BH3678 COG2972 two-component sensor histidine kinase 4.16786 −302 AATGGTGTAGAGGATT (SEQ ID NO: 240) rpoB BH0126 COG0085 DNA-directed RNA polymerase beta subunit 4.15636 −186 ATTGGTTTATATATAT (SEQ ID NO: 241) BH2699 BH2699 COG1136 ABC transporter (ATP-binding protein) 4.13718 −216 ATTGATCTAGAGCATA (SEQ ID NO: 242) spoVFA BH2403 — dipicolinate synthase subunit A 4.13468 −241 ATCGGTTTACACAATT (SEQ ID NO: 243) rplC BH0134 COG0087 50S ribosomal protein L3
(130) TABLE-US-00006 TABLE 6 DasR binding sites in Lactococcus lactis Score Position Site Gene Synonym COG Product 5.86694 −36 ATTGATATATACCAAT (SEQ ID NO: 244) nagB L14408 COG0363 glucosamine-6-P isomerase (EC 5.3.1.10) 5.58801 −72 ATTGGTATATACTGTT (SEQ ID NO: 245) nagA L173068 COG1820 N-acetylglucosamine-6- phosphate deacetylase (EC 3.5.1.25) 5.4239 −40 ATTGGTATATAAAAAT (SEQ ID NO: 246) yxfB L141634 COG0500 HYPOTHETICAL PROTEIN 5.3542 −215 AACGGTATATACGATT (SEQ ID NO: 247) yveE L127921 — UNKNOWN PROTEIN 5.24344 −173 AACAGTATATATCATT (SEQ ID NO: 248) pi228 L51784 — prophage pi2 protein 28 5.18192 −37 AGTGGTATATATTGTT (SEQ ID NO: 249) rgrB L0151 COG2188 GntR family transcriptional regulator 5.16551 −279 AATGATATATATCTTT (SEQ ID NO: 250) ymgC L61341 COG2936 conserved hypothetical protein 5.11627 −170 ACTTGTATATACTTAT (SEQ ID NO: 251) rplJ L0407 COG0244 50S ribosomal protein L10 5.03205 −164 ATGGGTAGATAACAAT (SEQ ID NO: 252) pi234 L57508 — prophage pi2 protein 34 5.03205 −1 AATGAGATATATCAAT (SEQ ID NO: 253) zitQ L166512 COG1121 zinc ABC transporter ATP binding protein 4.95193 −261 ATTGGTTTATACCGAC (SEQ ID NO: 254) dexC L128694 COG0366 neopullulanase (EC 3.2.1.135) 4.95193 −123 GTCGGTATAAACCAAT (SEQ ID NO: 255) malE L128695 COG2182 maltose ABC transporter substrate binding protein 4.92129 −265 ATTGGTATACAATATT (SEQ ID NO: 256) yviA L163025 COG2323 HYPOTHETICAL PROTEIN 4.92129 −118 ATTAGTCTATATCTAT (SEQ ID NO: 257) tra983B L0444 COG2826 transposase of IS983B 4.92129 −189 ATAAATAAATACCAAT (SEQ ID NO: 258) yrjB L174321 COG0247 oxidoreductase 4.91311 −348 AATGGGATATACTGGT (SEQ ID NO: 259) yqeL L22900 COG1161 GTP-binding protein 4.89476 −286 ATTGATATATATGTCT (SEQ ID NO: 260) ycbC L11986 — HYPOTHETICAL PROTEIN 4.89476 −314 ACGAGTATATATAAAT (SEQ ID NO: 261) yliA L179789 — positive transcriptional regulator 4.88629 −220 ATTGGTATAGGTCAAT (SEQ ID NO: 262) ybhE L176316 COG3589 HYPOTHETICAL PROTEIN 4.88246 −50 ATAAGTATATACATCT (SEQ ID NO: 263) yfhF L174076 — HYPOTHETICAL PROTEIN 4.8617 −262 AATGGAAGATACCATT (SEQ ID NO: 264) ywaH L191704 — UNKNOWN PROTEIN 4.85976 −241 ACTTGTATTTATCAAT (SEQ ID NO: 265) ps208 L106731 — prophage ps2 protein 08
(131) TABLE-US-00007 TABLE 7 DasR binding sites in Streptococcus species Score Position Site Gene Synonym COG Product A. S. pneumoniae TIGR4 4.56012 −264 AGTGGTGTATGCCAAT (SEQ ID NO: 266) — SP0571 COG2184 cell filamentation protein Fic-related protein 4.52678 −49 ATTGGTCTATACCATA (SEQ ID NO: 267) — SP1415 COG0363 glucosamine-6-phosphate isomerase 4.52678 −118 TATGGTATAGACCAAT (SEQ ID NO: 268) — SP1416 COG0809 S-adenosylmethionine:tRNA ribosyltransferase-isomerase 4.50546 −56 ATTAGACTATACCAAT (SEQ ID NO: 269) — SP0266 COG0449 glucosamine--fructose-6-phosphate aminotransferase, isomerizing 4.45088 −9 AGTGGAATATGACAGT (SEQ ID NO: 270) — SP0856 COG0115 branched-chain amino acid aminotransferase 4.444 −69 ATTATAATATTCCAAT (SEQ ID NO: 271) — SP1211 — hypothetical protein 4.444 −296 ATTATAATATTCCAAT (SEQ ID NO: 272) — SP1210 — hypothetical protein 4.41439 −134 ATTATTATATAGCAAT (SEQ ID NO: 273) — SP2103 COG0500 rRNA (guanine-N1-)-methyltransferase 4.41439 −1 ATTGCTATATAATAAT (SEQ ID NO: 274) — SP2102 — hypothetical protein 4.41439 −314 ATTGCTATATAATAAT (SEQ ID NO: 275) — SP2101 COG2217 cation-transporting ATPase, EI-E2 family 4.3461 −68 ACTGTTATATAATACT (SEQ ID NO: 276) — SP0088 COG0840 hypothetical protein 4.3461 −19 AGTATTATATAACAGT (SEQ ID NO: 277) — SP0087 — hypothetical protein 4.32609 −290 AGTGGTCTATTCGAAT (SEQ ID NO: 278) — SP1249 COG0516 conserved hypothetical protein 4.27568 −345 CTTGGGATAAACCACT (SEQ ID NO: 279) — SP1702 COG0653 preprotein translocase, SecA subunit 4.27445 −316 ATTAGAATATAAAAAT (SEQ ID NO: 280) — SP1956 — hypothetical protein 4.27389 −93 ATAGGTCTATACCATT (SEQ ID NO: 281) — SP2056 COG1820 N-acetylglucosamine-6-phosphate deacetylase 4.25853 −232 AGTGTTGTATGCCAGT (SEQ ID NO: 282) — SP0056 COG0015 adenylosuccinate lyase 4.25658 −35 TTTGGAGTATTCCAAT (SEQ ID NO: 283) — SP1319 COG1527 v-type sodium ATP synthase, subunit C 4.24921 −212 AAGGATATATACCAAT (SEQ ID NO: 284) — SP1431 COG2189 type II DNA modification methyltransferase, putative 4.24425 −46 AGTGGTATATTTAATT (SEQ ID NO: 285) — SP0474 COG1455 PTS system, cellobiose-specific IIC component 4.24425 −138 AATTAAATATACCACT (SEQ ID NO: 286) — SP0473 COG1940 ROK family protein 4.24051 −42 AATGGTATAATTCATT (SEQ ID NO: 287) — SP1810 — hypothetical protein 4.23862 −94 TATACTATATACCATT (SEQ ID NO: 288) — SP0839 COG1072 pantothenate kinase 4.23862 −112 AATGGTATATAGTATA (SEQ ID NO: 289) — SP0840 — hypothetical protein 4.23664 −76 ATTGGCATATCAGACT (SEQ ID NO: 290) — SP1264 COG1808 conserved domain protein 4.2252 −87 AATGTGATATAATAGT (SEQ ID NO: 291) — SP1421 COG1488 conserved hypothetical protein 4.21304 −40 CTTTGTATATACTAGT (SEQ ID NO: 292) — SP0394 COG2213 PTS system, mannitol-specific IIBC components 4.21243 −149 ATGGGGATATAACATT (SEQ ID NO: 293) — SP2159 — fucolectin-related protein 4.20615 −50 AGTGTGATATAATAGT (SEQ ID NO: 294) — SP0499 COG0126 phosphoglycerate kinase 4.20387 −42 ACTAGTATAGCACAAT (SEQ ID NO: 295) — SP1011 — GtrA family protein B. S. pyogenes 4.95709 −66 AGTGGTATATACCATT (SEQ ID NO: 296) nagA SPy1694 COG1820 putative N-acetylglucosamine-6-phosphate deacetylase 4.76896 −156 TATGGTATATACCAAT (SEQ ID NO: 297) queA SPy1400 COG0809 putative S-adenosylmethionine-tRNA ribosyltransferase-isomerase 4.76896 −62 ATTGGTATATACCATA (SEQ ID NO: 298) nagB SPy1399 COG0363 putative N-acetylglucosamine-6-phosphate isomerase 4.57524 −163 ATTAGCATATCCCAAT (SEQ ID NO: 299) — SPy0433 — hypothetical protein 4.45835 −148 ATTAGACTATACCAAT (SEQ ID NO: 300) glmS SPy1280 COG0449 putative L-glutamine-D-fructose-6-phosphate amidotransferase 4.31846 −123 ATTGTGATATAATAAT (SEQ ID NO: 301) gatC SPy1772 COG0721 putative Glu-tRNA Gln amidotransferase subunit C 4.29471 −2 AATGATATATAATAAT (SEQ ID NO: 302) — SPy0045 COG0534 conserved hypothetical protein 4.2322 −131 AATTGGATATCACAAT (SEQ ID NO: 303) — SPy0593 — conserved hypothetical protein 4.21387 −155 AGTTTAATATCCCAAT (SEQ ID NO: 304) lysS SPy0595 COG1190 putative lysyl-tRNA synthetase 4.21387 −35 ATTGGGATATTAAACT (SEQ ID NO: 305) — SPy0596 COG1011 conserved hypothetical protein 4.21271 −144 TATGGAATATTACACT (SEQ ID NO: 306) hasB SPy2201 COG1004 UDP-glucose 6-dehydrogenase 4.20978 −301 ACTTGTATATGCCAAG (SEQ ID NO: 307) accD SPy1744 COG0777 putative acetyl-CoA carboxylase beta subunit 4.20304 −170 ACTGTTATATAGTATT (SEQ ID NO: 308) acoA SPy1026 COG1071 putative acetoin dehydrogenase (TPP-dependent) alpha chain 4.2014 −69 ATTGTATTATAACAAT (SEQ ID NO: 309) — SPy1884 COG0330 similar to several eukaryotic hypersensitive-induced response proteins 4.17464 −139 AGTGGCATAACACAAT (SEQ ID NO: 310) fabG SPy1749 COG1028 putative beta-ketoacyl-ACP reductase 4.16261 −165 TGTTGGATATTCCAAT (SEQ ID NO: 311) — SPy1253 — conserved hypothetical protein 4.1621 −17 ATTCGGATATAACAAA (SEQ ID NO: 312) — SPy1297 COG1609 putative transcription regulator (LacI family) 4.14754 −111 ATTAGTATAGGCTACT (SEQ ID NO: 313) — SPy1437 — hypothetical protein 4.13534 −279 ATTGGGATATGCAACA (SEQ ID NO: 314) pyrR SPy0830 COG2065 putative pyrimidine regulatory protein 4.12956 −148 AATTGTATAGACCAAC (SEQ ID NO: 315) — SPy0539 — hypothetical gene 4.1271 −161 AATGAAATATTCAAAT (SEQ ID NO: 316) — SPy2099 COG2188 putative transcriptional regulator (GntR family) 4.1271 −66 ATTTGAATATTTCATT (SEQ ID NO: 317) — SPy2097 COG1264 putative PTS system enzyme II 4.12284 −105 ATTGTGCTAGACCATT (SEQ ID NO: 318) — SPy1494 — hypothetical protein 4.10357 −100 ATTGGAATATGATAAA (SEQ ID NO: 319) — SPy1249 COG1393 conserved hypothetical protein 4.10336 −42 AATAGTATATTAGATT (SEQ ID NO: 320) — SPy0338 COG1327 conserved hypothetical protein 4.09424 −271 ATTGGTACATGTCAAT (SEQ ID NO: 321) glpF.2 SPy1854 COG0580 putative glycerol uptake facilitator protein C. S. mutans 4.71268 −42 TTTGGTATATACCATT (SEQ ID NO: 322) — SMU.435 — putative N-acetylglucosamine-6-phosphate deacetylase 4.56555 −17 ATTGGAATACACCAAT (SEQ ID NO: 323) — SMU.284 — hypothetical protein 4.39528 −69 ATTAGACTATACCAAT (SEQ ID NO: 324) glmS SMU.1187 — glucosamine-fructose-6-phosphate aminotransferase 4.35759 −36 ACTGGTATAAACCAAA (SEQ ID NO: 325) gtfA SMU.881 — sucrose phosphorylase, GtfA 4.31505 −125 AATGTTATATTACAGT (SEQ ID NO: 326 — SMU.995 — putative ABC transporter, permease protein; possible ferrichrome transport system 4.28134 −295 AATGGGAAATACCATT (SEQ ID NO: 327) rexA SMU.1499 — putative exonuclease RexA 4.27644 −101 ATTGGAATATAAGACT (SEQ ID NO: 328) — SMU.458 — putative ATP-dependent RNA helicase 4.26492 −195 ATTAGTATAAAACAAT (SEQ ID NO: 329) — SMU.1912c — hypothetical protein 4.23783 −238 ATTGATATATTTCAAT (SEQ ID NO: 330) mleS SMU.137 — malolactic enzyme 4.21286 −67 AATAGTTTATACTAAT (SEQ ID NO: 331) — SMU.753 — conserved hypothetical protein 4.19185 −199 AGTTTTATATAACAAT (SEQ ID NO: 332) — SMU.1145c — putative histidine kinase; homolog of RumK and ScnK 4.14917 −331 TATGGAATATAATAAT (SEQ ID NO: 333) parC SMU.1204 — topoisomerase IV, subunit A 4.14346 −39 AATAGTTTATACTACT (SEQ ID NO: 334) — SMU.1349 — hypothetical protein 4.14346 −330 AGTAGTATAAACTATT (SEQ ID NO: 335) — SMU.1348c — putative ABC transporter, ATP-binding protein 4.11591 −270 ATTGATATAGAACAGT (SEQ ID NO: 336) pstS SMU.1138 — putative ABC transporter, phosphate-binding protein 4.11572 −161 GGTGGAATAGTCCAAT (SEQ ID NO: 337) glgB SMU.1539 — putative 1,4-alpha-glucan branching enzyme 4.10525 −298 TGTGGCCTATGCCAAT (SEQ ID NO: 338) — SMU.166 — hypothetical protein 4.10485 3 AATGGTATAAAAAAAT (SEQ ID NO: 339) msmE SMU.878 — multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE 4.10136 −341 ATTAGAATATGGCAGT (SEQ ID NO: 340) hprT SMU.14 — putative hypoxanthine-guanine phosphoribosyltransferase 4.10001 −125 ATTAGAATATACCTCT (SEQ ID NO: 341) — SMU.1908c — hypothetical protein 4.0932 −38 ATTGGTATATTAAAAA (SEQ ID NO: 342) — SMU.1764c — conserved hypothetical protein 4.0924 −252 AAAGGTATAAACCATT (SEQ ID NO: 343) — SMU.2162c — conserved hypothetical protein 4.09059 −161 TTTAGAATAGACCATT (SEQ ID NO: 344) guaB SMU.2157 — inosine monophosphate dehydrogenase 4.07559 −65 AATTTGATATTCCAGT (SEQ ID NO: 345) rmlC SMU.1460 — putative dTDP-4-keto-L-rhamnose reductase 4.06457 −113 TTTATTATATACTATT (SEQ ID NO: 346) — SMU.624 — putative 1-acylglycerol-3-phosphate O-acyltransferase 4.06457 −42 AATAGTATATAATAAA (SEQ ID NO: 347) — SMU.623c — putative deacetylase 4.05513 −256 AATAGCTTATACTAAT (SEQ ID NO: 348) — SMU.40 — conserved hypothetical protein 4.05348 −42 AGTGTTATATGCTATA (SEQ ID NO: 349) scnR SMU.1815 — putative response regulator; ScnR homolog 4.0529 −215 TGTGGTTTATACCACA (SEQ ID NO: 350) asd SMU.989 — aspartate-semialdehyde dehydrogenase D. S. agalactiae 4.67663 −61 ATTGGTATATACCATA (SEQ ID NO: 351) nagB SAG0799 — glucosamine-6-phosphate isomerase 4.57476 −306 AATGGAATATACTAAT (SEQ ID NO: 352) — SAG0698 — beta-glucuronidase 4.43079 −66 ATAGGTATATACCATT (SEQ ID NO: 353) nagA SAG0266 — N-acetylglucosamine-6-phosphate deacetylase 4.42251 −54 ATTGGTATATATTAAT (SEQ ID NO: 354) — SAG0943 — hypothetical protein 4.42251 −74 ATTAATATATACCAAT (SEQ ID NO: 355) glmS SAG0944 — glucosamine--fructose-6-phosphate aminotransferase, isomerizing 4.37914 −81 AGTGGTATAATCCAGT (SEQ ID NO: 356) ksgA SAG1779 — dimethyladenosine transferase 4.37243 −332 ATTGGTATATATTATT (SEQ ID NO: 357) — SAG1033 — FtsK/SpoIIIE family protein 4.33644 −229 ATTGGAATATCCGATT (SEQ ID NO: 358) — SAG2003 — IS1381, transposase OrfA 4.28704 −61 AATGGTATATCACAAG (SEQ ID NO: 359) — SAG2008 — conserved hypothetical protein 4.20258 −210 TCTATTATATACCAAT (SEQ ID NO: 360) — SAG2170 — conserved hypothetical protein 4.20258 −50 ATTGGTATATAATAGA (SEQ ID NO: 361) — SAG2169 — membrane protein, putative 4.13411 −232 AATATGATATACTAAT (SEQ ID NO: 362) — SAG1186 — metallo-beta-lactamase superfamily protein 4.13259 −292 CTTGGAATATTCCATA (SEQ ID NO: 363) — SAG0699 — transcriptional regulator, GntR family 4.10916 −124 AGTAGAATAGTCCATT (SEQ ID NO: 364) — SAG1951 — PTS system, IIA component, putative 4.09376 −202 AGTGGAATAGACAAGT (SEQ ID NO: 365) cglB SAG0164 — competence protein CglB 4.09142 −270 AGTGGTATAATCCAGG (SEQ ID NO: 366) — SAG1307 — hypothetical protein 4.09022 −9 ATTGGGCTATGCGAAT (SEQ ID NO: 367) — SAG0277 — conserved hypothetical protein 4.08873 −44 ATTAGGATAAACTAAT (SEQ ID NO: 368) — SAG0021 — protease, putative 4.08273 −289 ACTTGAATATCCTAAT (SEQ ID NO: 369) — SAG0626 — MutT/nudix family protein 4.07572 −69 TATAGTATATAGCATT (SEQ ID NO: 370) neuB SAG1161 — N-acetyl neuramic acid synthetase NeuB 4.07402 −31 ATTTTAATATAACAAT (SEQ ID NO: 371) pepX SAG1736 — X-pro dipeptidyl-peptidase 4.07379 −257 AGTTGAATATGCTAAT (SEQ ID NO: 372) — SAG1571 — hypothetical protein 4.06691 −122 ATTGGTATTTACGAGT (SEQ ID NO: 373) — SAG1711 — magnesium transporter, CorA family 4.06603 −120 AATGGAATATTTTATT (SEQ ID NO: 374) cylF SAG0670 — cylF protein 4.04872 −169 CATGGGATATTCAAAT (SEQ ID NO: 375) — SAG1260 — hypothetical protein 4.04872 −324 GTTGGAATATCGCATT (SEQ ID NO: 376) tkt SAG0278 — transketolase 4.04511 −103 ATTGGCTTATTCAAAT (SEQ ID NO: 377) — SAG0231 — hypothetical protein 4.0417 −180 AATGATATATGCAACT (SEQ ID NO: 378) asd SAG1051 — aspartate-semialdehyde dehydrogenase 4.02438 −141 ATTGTCATATAACACC (SEQ ID NO: 379) — SAG1569 — copper homeostasis protein CutC, putative 4.0221 −8 ATTAGTATATGTCAAA (SEQ ID NO: 380) — SAG1683 — immunogenic secreted protein, putative 4.00725 −283 AGTACAATATAACAAT (SEQ ID NO: 381) — SAG1982 — transcriptional regulator, Cro/CI family
(132) TABLE-US-00008 TABLE 8 DasR binding sites in Listeria species. Score Position Site Gene Synonym COG Product A. Listeria innocua 5.36566 −121 ATTGGTCTATATCAAT (SEQ ID NO: 382) — lin1996 COG3469 similar to chitinases 5.36389 −38 ATTGGTATAGACCGAT (SEQ ID NO: 383) — lin0955 COG1820 similar to N-acetylglucosamine-6P-phosphate deacetylase (EC 3.5.1.25) 5.26731 −173 AATGGTCTAGACAAAT (SEQ ID NO: 384) codV lin1316 COG0582 similar to integrase/recombinase 5.24253 −34 ACTTGTATATACAAGT (SEQ ID NO: 385) — lin1223 COG1264 similar to PTS system trehalose specific enzyme IIBC 5.24253 −98 ACTTGTATATACAAGT (SEQ ID NO: 386) — lin1224 COG0494 lin1224 5.16643 −44 ACTGGTATAAACAAGT (SEQ ID NO: 387) — lin0296 COG0366 lin0296 5.0365 −143 AACTGTCTAGACCAAT (SEQ ID NO: 388) — lin0780 COG1113 similar to amino acid transporter 4.98056 −87 ATTGGTATAAAGCAGT (SEQ ID NO: 389) — lin2570 — similar to Orf51 [bacteriophage bIL285] 4.97549 −138 ATCGGTTTATACCGGT (SEQ ID NO: 390) — lin1779 COG0803 similar to ABC transporter and adhesion proteins 4.96433 −93 TTTTGTATAGACCAAT (SEQ ID NO: 391) fbp lin0825 COG0639 highly similar to fructose-1,6-bisphosphatase 4.95402 −201 ACAAGTATAGACCAAT (SEQ ID NO: 392) — lin1606 COG0205 lin1606 4.90623 −72 ATTTGTCTATAATAAT (SEQ ID NO: 393) pheT lin1648 COG0073 similar phenylalanyl-tRNA synthetase (B subunit) 4.89907 −315 ACTGTTTTATACAAAT (SEQ ID NO: 394) pflB lin1443 COG1882 pyruvate formate-lyase 4.86345 −1 AATGGTCAATACAAAT (SEQ ID NO: 395) aroA lin1641 COG1605 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 4.83652 −129 AGTGGTCTATATAATT (SEQ ID NO: 396) — lin1069 — lin1069 4.82408 −299 ATCGGTATATACTTGT (SEQ ID NO: 397) — lin1328 — internalin like protein (LPXTG motif) 4.80458 −342 AATGGTTTATATCACT (SEQ ID NO: 398) — lin0432 COG0574 similar to phosphoenolpyruvate synthase 4.79819 −178 AATGCTTTATACAAAT (SEQ ID NO: 399) — lin0169 — similar to phage proteins 4.78736 −349 AATGGAATACACCAAT (SEQ ID NO: 400) — lin0406 — lin0406 4.77453 −294 ACAGATCTAGACCAGT (SEQ ID NO: 401) — lin1754 COG1235 similar to hypothetical protein 44 - Staphylococcus aureus phage phi PVL 4.77453 −295 ACAGATCTAGACCAGT (SEQ ID NO: 402) — lin1243 COG1235 similar to hypothetical protein 44 - Staphylococcus aureus phage phi PVL 4.71781 −330 ATTGGTGTAGATCCGT (SEQ ID NO: 403) — lin1990 COG2759 similar to formyl-tetrahydrofolate synthetase 4.70658 −158 ACTGGTATATATAGCT (SEQ ID NO: 404) — lin2737 COG0489 similar to ATP binding proteins 4.70658 −122 AGCTATATATACCAGT (SEQ ID NO: 405) — lin2738 COG1705 surface protein (GW repeat) similar to N-acetylmuramidase 4.70449 −132 ATTAGTATATAGAATT (SEQ ID NO: 406) — lin2295 COG1393 similar to unknown proteins B. Listeria monocytogenes 5.38367 −121 ATTGGTCTATATCAAT (SEQ ID NO: 407) — lmo1883 COG3469 similar to chitinases 5.37731 −38 ATTGGTATAGACCGAT (SEQ ID NO: 408) — lmo0956 COG1820 similar to N-acetylglucosamine-6P-phosphate deacetylase (EC 3.5.1.25) 5.2828 −47 ATTGGTATAAACAAGT (SEQ ID NO: 409) — lmo0270 — lmo0270 5.25914 −34 ACTTGTATATACAAGT (SEQ ID NO: 410) — lmo1255 COG1264 similar to PTS system trehalose specific enzyme IIBC 5.25914 −98 ACTTGTATATACAAGT (SEQ ID NO: 411) — lmo1256 COG0494 lmo1256 5.07626 −156 ACTTGTATATAACAAT (SEQ ID NO: 412) — lmo1393 COG0612 similar to putative protease 5.05059 −173 AATGGTCTAGACAGAT (SEQ ID NO: 413) codV lmo1277 COG0582 similar to integrase/recombinase 4.97633 −93 TTTTGTATAGACCAAT (SEQ ID NO: 414) fbp lmo0830 COG0639 highly similar to fructose-1,6-bisphosphatase 4.96887 −200 ACAAGTATAGACCAAT (SEQ ID NO: 415) pfk lmo1571 COG0205 highly similar to 6-phosphofructokinase 4.92791 −72 ATTTGTCTATAATAAT (SEQ ID NO: 416) pheT lmo1607 COG0073 similar phenylalanyl-tRNA synthetase (beta subunit) 4.8688 −109 ATTGGTATATACCGGA (SEQ ID NO: 417) — lmo1289 — similar to internalin proteins, putative peptidoglycan bound protein (LPXTG motif) 4.82689 −117 ATTCGTATAGAAAAAT (SEQ ID NO: 418) — lmo1139 — lmo1139 4.82474 −282 AACTGTATATATCAAT (SEQ ID NO: 419) — lmo2445 — similar to internalin 4.79049 −14 AGGGGTCTACACAAGT (SEQ ID NO: 420) — lmo1351 COG0607 lmo1351 4.75049 −81 ATTTGTCGATATCAAT (SEQ ID NO: 421) — lmo2691 COG1705 similar to autolysin, N-acetylmuramidase 4.73024 −181 ATTGGTATAAATTATT (SEQ ID NO: 422) — lmo2748 — similar to B. subtilis stress protein YdaG 4.72697 −234 ACTCGTATATCCAAAT (SEQ ID NO: 423) — lmo0475 — lmo0475 4.72372 −143 AACTATCTAGACCAAT (SEQ ID NO: 424) — lmo0787 COG1113 similar to amino acid transporter 4.71951 −62 ATTAGTATATACTTTT (SEQ ID NO: 425) — lmo2110 COG1482 similar to mannnose-6 phospate isomerase 4.71834 −1 AATGGTTAATACAAAT (SEQ ID NO: 426) aroA lmo1600 COG1605 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 4.71724 −160 ATTTCTGTAGACCATT (SEQ ID NO: 427) — lmo0969 COG0564 similar to ribosomal large subunit pseudouridine synthetase 4.70973 −98 ATGGGAATACACCAAT (SEQ ID NO: 428) — lmo0792 COG0388 similar to conserved hypothetical protein 4.7002 −31 ATCAGTATACACAATT (SEQ ID NO: 429) — lmo1125 — lmo1125 4.69794 −1 AATGATTTATACAATT (SEQ ID NO: 430) fruB lmo2336 COG1105 fructose-1-phosphate kinase 4.69157 −275 ACTTGTTTAAACCGTT (SEQ ID NO: 431) — lmo1219 — lmo1219
(133) TABLE-US-00009 TABLE 9 DasR binding sites in Thermobifido fusca. Co-transcribed # Gene Function dre Pos Scr gene Function