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WO2004056996A2 - Polynucleotides de csrc et utilisations associees pour la modulation de film biologique - Google Patents

Polynucleotides de csrc et utilisations associees pour la modulation de film biologique Download PDF

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WO2004056996A2
WO2004056996A2 PCT/CA2003/001998 CA0301998W WO2004056996A2 WO 2004056996 A2 WO2004056996 A2 WO 2004056996A2 CA 0301998 W CA0301998 W CA 0301998W WO 2004056996 A2 WO2004056996 A2 WO 2004056996A2
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csrc
csra
rna
csrb
expression
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WO2004056996A3 (fr
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Tony Romeo
Thomas Weilbacher
Kazushi Suzuki
Xin Wang
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University of North Texas Health Science Center
University of North Texas
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University of North Texas Health Science Center
University of North Texas
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention relates to the modulation of bacterial functions and particularly to modulators of function in biofilm producing bacteria.
  • Bacterial survival and competition under the feast or famine conditions of the natural environment requires remarkable phenotypic plasticity.
  • Escherichia coli the transition from exponential to stationary growth phase leads to increased stress resistance, decreased anabolic metabolism, altered cellular and subcellular morphology, and enhanced ability to scavenge nutrients.
  • Acquisition of the stationary phase phenotype is brought about through changes in gene expression, which are coordinated by global regulatory systems.
  • the present invention provides an isolated csrC polynucleotide.
  • the csrC polynucleotide has at least 70% identity to that depicted in Fig.1 , and comprises at least one binding site or a complement of a binding site for CsrA. ln another embodiment of the invention, the csrC polynucleotide has the nucleotide sequence depicted in Fig. 1.
  • the present invention provides a method of altering the metabolism or structural or functional properties of a bacterial cell comprising altering the genetic expression or CsrA binding activity of csrC.
  • CsrA binding activity of csrC is altered by mutating csrC.
  • a result of altered genetic expression csrC is a change in the level of a metabolic compound, where the level of production is at least partially regulated by CsrA and
  • a result of altered genetic expression of csrC is a change in glycogen biosynthesis or glugoneogenesis.
  • expression of the csrC gene is increased, decreased or under inducible control.
  • the present invention provides a method of reducing biofilm formation by decreasing csrC transcription in a biofilm forming bacterial cell.
  • the present invention also provides a method of inhibiting motility of biofilm producing bacteria comprising increasing csrC expression.
  • the present invention further provides a method of modulating csrC expression in biofilm forming bacteria by altering UvrY levels.
  • the biofilm forming bacterial strain is selected from a group consisting of an E. coli strain, a Salmonella strain, a Klebsiella strain, and a related gamma proteobacteria.
  • the present invention provides a method of reducing the symptoms of a bacterial infection by biofilm producing bacteria in a mammalian patient comprising administering an antibacterial agent and decreasing biofilm formation through modulation of csrC.
  • the antibacterial agent is selected from a group consisting of penicillin related antibiotics and sulfa-drug related antibiotics.
  • csrC is modulated by disrupting csrC-CsrA binding or decreasing csrC transcription.
  • Figure 1 is the csrC gene nucleotide sequence.
  • Figure 2 is a Northern Blot showing CsrC RNA during the growth curve.
  • Figure 3 is a radioautograph illustrating the primer extension analysis of CsrC RNA.
  • Figure 4 illustrates the effects of csrC on glycogen levels in MG1655 (WT), and isogenic csrA (TRMG1655) csrB (RGMG1655), csrC (TWMG1655) and csrB csrC (RGTWMG1655) mutants. Also shown is the csrC mutant containing plasmid pCSRCI (csrC+++), which over expresses csrC.
  • Figure 5 is a line graph illustrating the effects of csrB and csrC on expression of a chromosomal glgCA -'/acZ translational fusion.
  • Figure 6 is a bar graph illustrating the effects of csr genes on biofilm formation.
  • Figure 7A is a Northern blot probed for CsrB RNA.
  • Figure 7B is a Northern blot probed for CsrC RNA.
  • Figure 7C is a bar graph illustrating the phosphorimage analysis of the CsrB Northern blot.
  • Figure 7D is a bar graph illustrating the phosphorimage analysis of the CsrC
  • Figure 8 is a line graph illustrating CsrC RNA chemical decay in MG1655 (triangles) and its isogenic csrA mutant (squares).
  • Figure 9A is a line graph illustrating the effects of mutations in csrA (TRKSB837), csrB (RGKSB837), csrC (TWKSB837), both csrB and csrC (csrBC; RGTWKSB837), or uvrY (UYKSB837) on csrB-lacZ expression in the KSB837 (WT) genetic background.
  • Figure 9B is a line graph illustrating the effects of mutations in csrA (TRGS1114), csrB (RGGS1114), csrC (TWGS1114), both csrB and csrC (csrBC; RGTWGS1114) or uvrY (UYGS1114) on csrC-lacZ expression in the GS1114 (WT) genetic background.
  • Figure 10 is a bar graph illustrating the effects of ectopic expression of csrA (pCRA16) and uvrY (pUY14) on expression of a csrC-/acZ transcriptional fusion in isogenic csrA (TRGS1114) or uvrY (UYRGS1114) mutants.
  • Figure 11 illustrates the predicted secondary structure of CsrC RNA.
  • Figure 12 is a DNA sequence alignment for apparent homologues of E. coli K-12 csrC found in E. coli O157:H7, Salmonella entehca serovar Typhimurium, Salmonella typhi, Salmonella, paratyphi, and Klebsiella pneumoniae performed by BLAST analysis at NCBI. Residues that are identical to those of E. coli K-12 are highlighted.
  • RNA-binding protein CsrA of E. coli is a key component of a global regulatory system that represses several stationary phase processes, while it activates certain exponential phase functions. Glycogen synthesis and catabolism, gluconeogenesis, and biofilm formation are repressed by CsrA, while glycolysis, motility and flagellum synthesis, and acetate metabolism are activated by this protein.
  • the mechanism by which CsrA represses glycogen metabolism involves the binding of CsrA to the untranslated leader of the glgCAP transcript, which blocks translation and causes this transcript to be rapidly degraded.
  • Positive control of flhDC expression by CsrA involves a similar post- transcriptional mechanism, whereby CsrA binding to the untranslated leader ultimately stabilizes this mRNA.
  • CsrB 366 nt untranslated RNA molecule
  • CsrB 366 nt untranslated RNA molecule
  • An imperfect repeat sequence (CAGGAUG) that is located primarily in the loops of predicted RNA hairpins is believed to permit CsrA to bind to CsrB.
  • CsrB functions as an antagonist of CsrA.
  • CsrA also binds to the glgC Shine- Dalgarno sequence and to a site further upstream in the untranslated glgC leader transcript, both of which are related in sequence to the repeated elements of CsrB.
  • CsrA and CsrB levels accumulate as the culture approaches the stationary phase of growth. Although CsrA binds to CsrB, it does not appear to alter CsrB stability. Instead, CsrA activates csrB transcription, providing an autoregulatory mechanism for intracellular CsrA activity. Activation of csrB transcription by CsrA is mediated indirectly, through the BarA/UvrY two component signal transduction system. Purified UvrY protein stimulates csrB-lacZ expression in vitro, revealing that UvrY resides immediately upstream from csrB in the signalling pathway between CsrA and csrB.
  • sRNAs in E. coli regulate translation by base-pairing with complementary segments of mRNAs, with assistance from Hfq protein.
  • CsrB and 6S RNA regulate gene expression by binding to proteins, CsrA and ⁇ 70 -RNA polymerase, respectively.
  • RNA binding reversibly inhibits the activity of the target protein and results in global changes in gene expression.
  • CsrB or its homologues has evolved a mechanism for binding to a large number of subunits of a target protein, providing an efficient means of sequestering CsrA during conditions of nutrient limitation.
  • the Csr system of E. coli is a global regulatory system that has profound effects on metabolism, physiology and multicellular behaviour.
  • the RNA binding protein CsrA of this system regulates gene expression by binding to specific RNA transcripts, leading to mRNA stabilization or translation inhibition and transcript decay.
  • the sRNA CsrB sequesters ⁇ 18
  • CsrA subunits in a globular complex by exploiting the binding specificity of CsrA.
  • CsrB apparently accomplishes this by displaying 18 repeated sequence elements primarily within single stranded loops of the CsrB structure.
  • a second sRNA molecule, CsrC also interacts with CsrA, albeit at lower affinity than CsrB, and antagonizes the regulatory effects of CsrA in the cell (e.g. Figs. 4-6).
  • CsrC appears to utilize a similar mechanism for antagonizing CsrA activity, and contains 9 related repeat sequences distributed mainly in predicted single stranded loops and bulges.
  • CsrB and CsrC sequences exhibit no striking similarity.
  • the riboprobe for each RNA was completely specific (Fig. 7) and BLASTTM analyses with each
  • RNA failed to identify the other. Unlike many sRNAs, notably those that are known to regulate gene expression by base-pairing mechanisms, CsrC RNA does not bind to Hfq protein.
  • CsrA, CsrB and CsrC accumulate as the culture enters the stationary phase of growth (Fig. 2). While the sigma factor RpoS ( ⁇ s ) activates many genes that are induced in the stationary phase, it does not affect the levels of any of the E. coli Csr components (Fig. 2). In contrast, transcription of csrB and csrC indirectly depends upon CsrA (Figs.1 , 9). Complementation studies revealed that the response regulator UvrY mediates the effect of CsrA on csrB and csrC (Fig. 10).
  • CsrC levels are somewhat less dependent upon functional csrA or uvrY genes than those of CsrB, and csrC-/acZ expression in vivo and in vitro exhibits weaker activation by UvrY.
  • the significance of these minor regulatory distinctions between CsrB and CsrC is uncertain.
  • the physiological stimulus for UvrY phosphorylati ⁇ n remains to be determined.
  • CsrB and CsrC synthesis by CsrA defines an autoregulatory mechanism for CsrA, since each sRNA also antagonizes CsrA activity.
  • This mechanism is that CsrB and CsrC exhibit compensatory effects on each other.
  • a csrB null mutation results in a ⁇ 20% increase in CsrC levels and a csrC null mutation similarly increases CsrB levels (Fig. 7).
  • CsrC effects on glycogen levels and glgCA'-'lacZ expression were only observed in a csrB mutant background and were modest (Fig. 5).
  • csrC overexpression had more dramatic effects on both processes.
  • Biofilm formation also exhibited greater effects of csrC overexpression versus disruption (Fig. 6). At least two factors likely contribute to these quantitative discrepancies. First, the compensatory effects CsrB and CsrC on each other would tend to minimize the effects of either single mutation on CsrA regulated processes. Second, under the culture conditions used for these experiments, there is sufficient CsrB in the cell to sequester only ⁇ 30% of the CsrA protein. CsrB is believed to be the major RNA species of this complex. Thus, intracellular CsrC is expected to bind ⁇ 30% of the CsrA in the cell, and on this basis, a single disruption of csrC should be expected to have minimal effects on CsrA-regulated gene expression. Of course, it is possible that csrC disruption might exhibit more pronounced regulatory effects under other growth conditions.
  • CsrB and/or CsrC may possess a CsrA-independent function.
  • the apparent CsrA binding sites of CsrC RNA tend to be clustered toward the 5' segment of CsrC RNA (Fig. 1). This suggests that the 3' segment of CsrC may possess a regulatory function that is distinct from sequestration of CsrA.
  • RNAs of related gram negative bacteria are the only known examples of RNA molecules that bind to multiple copies of a regulatory protein and function as its antagonist.
  • LB medium (Miller, 1972) with 0.2% glucose was used for routine cultures.
  • SOC medium (Miller, 1972) was used for recovery of transformed cells.
  • Kornberg medium (1.1% K 2 HPO 4 , 0.85% KH 2 PO 4 , 0.6% yeast extract containing 0.5% glucose for liquid or 1% glucose for agar) was used for gene fusion assays, northern blot and RNA stabililty studies, and assessment of the glycogen phenotype by iodine staining.
  • Semisolid tryptone medium (pH 7.4) containing 1% tryptone, 0.5% NaCI and 0.35% agar was used for motility studies (Wei et al., 2001).
  • Colonization factor antigen (CFA) medium (pH 7.4) (Evans et al., 1997) contained 1 % casamino acids, 0.15% yeast extract, 0.005% MgSO 4 , and 0.0005% MnCI 2 , and was used to grow cultures for biofilm studies.
  • Antibiotics were added at the following concentrations: chloramphenicol, 20 ⁇ g/ml; kanamycin, 100 ⁇ g/ml; ampicillin, 100 ⁇ g/ml; tetracycline, 10 ⁇ g/ml; rifampicin, 200 ⁇ g/ml, except that ampicillin and kanamycin were used at 50 ⁇ g/ml and 40 ⁇ g/ml during the construction of the chromosomal csrC-/acZ fusion. Liquid cultures were grown at 37°C with rapid shaking, unless otherwise noted.
  • Total cellular RNA was isolated using the MasterpureTM RNA purification kit (Epicentre), quantified by UV absorbance, and suspended in 70% ethanol at -80°C.
  • the oligonucleotide primer D1prB (Table 2) was labelled using T4 polynucleotide kinase and [ ⁇ - 32 P]ATP (3,000 Ci mmol "1 ; NEN Life Science Products Inc.) according to standard procedures (Ausubel et al., 1989). Approximately 15 ng of labelled primer was annealed to 10 ⁇ g of total RNA.
  • the same labelled primer was used to prepare a DNA sequencing ladder, with pMR2113-D1 as the template, which served as a standard for the reverse transcription product. Products were analyzed on 6% polyacrylamide gels containing 6 M urea (e.g. Preiss and Romeo, 1989).
  • the csrB riboprobe was produced from plasmid pSPT18-CsrB as described elsewhere (Gudapaty, 2001).
  • the plasmid for the production of csrC riboprobe was generated by subcloning a 209 bp Nsil/Kpnll fragment from pMR2113-D1 into the multiple cloning site of pSPT18.
  • the resulting plasmid, pSPT18-D1 was used to generate digoxigenin-labelled (DIG) riboprobe, using T7 RNA polymerase and the DIG-RNA Labeling Kit (SP6/T7), according to the manufacturer's instructions (Roche Diagnistics Corp., Indianapolis, IN).
  • the synthesis reaction was carried out for 2 h at 37°C, followed by 15 min incubation with 2 ⁇ l RNase-free DNasel. The reaction was subsequently terminated with the addition of 2 ⁇ l of 0.2 M EDTA. Probes were stored at -80°C.
  • Quantitative biofilm assay Overnight cultures were inoculated 1 :100 into fresh medium. In the microtiter plate assay, inoculated cultures were grown in a 96-well polystyrene microtiter plate. Growth of planktonic cells was determined by absorbance at 600 nm or total protein assay. Biofilm was measured by discarding the medium, rinsing the wells with water (three times), and staining bound cells with crystal violet (BBL) (O'Toole, Mol. Microbiol., 30: 295 (1998)).
  • BBL crystal violet
  • the dye was solubilized with 33% acetic acid (EM Science, Gibbstown, N.J.), and absorbance at 630 nm was determined using a microtiter plate reader (DynaTech, Chantilly, Va.). For each experiment, background staining was corrected by subtracting the crystal violet bound to uninoculated controls. All comparative analyses were conducted by incubating strains within the same microtiter plate to minimize variability. To confirm that observed effects on biofilm formation in microtiter wells were not surface specific, cultures were grown and tested simultaneously in new borosilicate glass test tubes (18 mm).
  • Prehybridization hybridization to DIG-labelled riboprobes (2 ⁇ l probe per 10 ml of prehybridization buffer), and membrane washing were conducted using the DIG Luminescent Detection Kit for Nucleic Acids (Roche Diagnostics Corp.), according to the manufacturer's instructions, except that the membrane was incubated for 10 h in blocking solution.
  • the resulting chemiluminescent signals were detected using Kodak X-OMAT-AR film and were quantified by phosphorimaging using a GS-525 Phosphor Imager (Biorad, Hercules, CA) with a chemiluminescent screen. Phosphorimaging data were analyzed using Molecular Analyst software and Microsoft Excel. The 23S rRNA signal was used to normalize for minor loading differences between samples.
  • a linear DNA fragment was amplified by PCR, which contained the tetR gene from pBR322 flanked on either side by 40 nucleotides homologous to the upstream and downstream regions of csrC.
  • Primers used in generating the fragment were D1 KOF and D1 KOR (Table 2). Linear recombination of this fragment into the E. coli genome was performed using the protocol described elsewhere (Datsenko, Wanner 2000). Cells were then plated on selective Kornberg medium containing tetracycline. Overnight colonies were chosen for confirmation by PCR and Northern Blot. PCR was performed using 15 ⁇ l whole cells harvested from overnight cultures.
  • csrC The transcribed region of csrC was amplified by PCR using primers D1 Pr1 and D1 Pr2 (Table 2). This PCR product was T-A cloned into the pCR-XL-TOPO cloning vector (Invitrogen). The csrC region was excised from this clone using EcoRI and subcloned into the EcoRI site of pUC18. Clones containing csrC in the forward or reverse orientation with respect to the lacZ promoter of pUC18 were designated as pCSRCI and pCSRCrel , respectively. Plasmid DNA inserts were sequenced. at the University of Arizona core facility using primers D1 Pr1 and D1 Pr2.
  • a 243-bp PCR product was prepared, which contained the 3'-end of the yihA gene, the upstream untranscribed region of csrC, and the first 4 transcribed nt of csrC.
  • the primers csrC-UP and csrC-DN were used for generating this PCR product.
  • the PCR product was gel-purified, treated with T4 DNA polymerase to create blunt-ends, and cloned into Smal-treated and dephosphorylated pGE593 plasmid.
  • the resulting plasmid, pCCZ1 was partially sequenced and found to be free of PCR-generated mutations.
  • the csrC-/acZ fusion in pCCZ1 was moved into the E coli CF7789 chromosome using the ⁇ lnChl system, as described elsewhere (Boyd et al., 2000).
  • the presence of the csrC-/acZ transcriptional fusion in this strain was confirmed by PCR analysis, as recommended (Boyd et al., 2000).
  • Plasmid pEL1 which contains nucleotides +1 to +209 relative to the start of csrC transcription, was constructed by cloning a chromosomally generated PCR product into the Ecof?l and SamHI sites of the pTZ18U polylinker. Quantitative gel shift assays were performed as described below. RNA was synthesized in vitro using the Ambion MEGAscript kit and linearized plasmids pEL1 , plM5 (containing csrB (+1 to +338)) or pPB77 (Babitzke et al., 1994) as templates (Table 1).
  • RNA suspended in TE was renatured by heating to 85°C followed by slow cooling.
  • Binding reactions (10 ⁇ l) contained 10 mM Tris-HCI, pH 7.5, 10 mM MgCI 2 , 100 mM KCI, 32.5 ng yeast RNA, 7.5% glycerol, 20 mM DTT, 4 U RNase inhibitor (Ambion), 10 pM CsrB RNA or 0.2 nM CsrC RNA, purified CsrA (various concentrations) and 0.1 ⁇ g/ml xylene cyanol.
  • the transcription inhibitor rifampicin was added to cultures at 2 h post-exponential phase. The culture was harvested at regular intervals thereafter, and total cellular RNA was isolated. CsrC RNA was analyzed by northern blot and phosphorimage analysis (Gudapaty, 2001). Glycogen, ⁇ -galactosidase, total protein, and motility assays
  • Plasmid pCSB27 contains csrA under the control of an IPTG-inducible promoter
  • pAltC4 contains portions of the WT glgCAP operon leader and glgC coding regions (+46 to +179 relative to the start of transcription) (Liu and Romeo, 1997).
  • plM1 contains a deletion of G94, G95 and A96 ( ⁇ GGA) from the glgCAP leader, whereas plM2 also contains a deletion of G99 and T100 ( ⁇ GAA-GU).
  • plM4 contains the flhDC leader region (+1 to +276)
  • plM5 contains csrB (+1 to +338)
  • pPB77 contains the B.
  • subtilis trp leader Bacillis trp leader (Babitzke et al., 1994).
  • pCZ3-3 contains the WT glgCAP operon promoter, leader region and a glgC- VacZ translational fusion (Romeo et al., 1990).
  • Plasmids pCSB25 and plM3 are identical to pCZ3-3 except that they contain the ⁇ GGA or the ⁇ GGA-GU gig leaders respectively.
  • E. coli strains used for ⁇ -galactosidase assays were constructed to create single-copy gene insertions of glgC'-'lacZ translational fusions as described previously (Boyd et al., 2000).
  • Strains KSC365, PLB660 and KSC121 contain fusions in strain CF7789 ( ⁇ lacl-Y) derived from plasmids pCZ3-3 (WT), pCSB25 ( ⁇ GGA) and plM3 ( ⁇ GGA-GU) respectively.
  • CsrA was overproduced by inducing the expression of csrA carried on pCSB27 by the addition of IPTG.
  • Cell pellets were suspended in 10 mM sodium phosphate, pH 6.0, 50 mM NaCI and 10% glycerol (w/v).
  • S100 extracts were prepared from cells that were lysed by sonication.
  • CsrA was then precipitated with 21 % ammonium sulphate.
  • the protein pellet was dissolved in 10 mM sodium phosphate, pH 6.0, until the conductivity of the solution was equivalent to 50 mM NaCI.
  • This protein solution was mixed with pre-equilibrated phosphocellulose (10 mM sodium phosphate, pH 5.0, 50 mM NaCI, 10% glycerol) and then packed into a column. CsrA eluted from the column between 250 and 750 mM NaCI. These fractions were combined and brought to pH 9.0 by the addition of 1 M NaOH before dialysis against 10 mM Tris-HCI, pH 9.0, 50 mM KCI and 4% glycerol. The dialysate was loaded onto a MonoQ column (Pharmacia HR 5/5) that was pre-equilibrated with 10 mM Tris-HCI, pH 9.0 and 50 mM KCI.
  • CsrA was eluted with a linear KCI gradient.
  • the peak fraction (200 mM KCI) was brought to pH 7.5 by the addition of 100 mM MOPS and subsequently dialysed against 10 mM Tris-HCI, pH 8.0, 50 mM KCI and 25% glycerol.
  • CsrA purity was assessed by electrophoresis through 15% polyacrylamide-SDS gels. Protein concentrations were determined using the Bio-Rad protein assay. Gel shift assay
  • Binding reactions (10 ⁇ l) contained 10 mM Tris- HCI, pH 7.5, 10 mM MgCI 2 , 100 mM KCI, 32.5 ng of yeast RNA, 7.5% glycerol, 20 mM dithiothreitol (DTT), 4 U of RNase inhibitor (Ambion), 0.5 nM WT or mutant glgCAP leader RNA, purified CsrA (various concentrations) and 0.1 ⁇ g ml "1 xylene cyanol.
  • Competition assays also contained unlabelled RNA competitor (see text for details). Reaction mixtures were incubated for 30 min at 37°C to allow CsrA-RNA complex formation.
  • glgCAP leader transcripts used in this analysis were synthesized using pAltC4 as template.
  • Gel-purified glgCAP leader RNA (250 nM) in TE was renatured and hybridized to a 32 P end-labelled DNA olfgonucleotide (500 nM) complementary to the 3' end of the transcript by heating to 80°C followed by slow cooling.
  • Toeprint assays were carried out with 200 nM CsrA and/or 100 nM 30S ribosomal subunits and 500 nM tRNATM 6 '.
  • Toeprint reactions (20 ⁇ l) contained 2 ⁇ l of the hybridization mixture, 375 ⁇ M each dNTP and 10 mM DTT in toeprint buffer (10 mM Tris-HCI, pH 7.4, 10 mM MgCI 2l 60 M NH 4 OAc, 6 mM 2-mercaptoethanol) (Hartz et al., 1988).
  • Mixtures containing CsrA were incubated for 30 min at 37°C to allow CsrA-mRNA complex formation.
  • 30S ribosomal subunit toeprint reactions were performed by incubating RNA, 30S ribosomal subunits and tRNATM 6 ' in toeprint buffer as described previously (Hartz et al., 1988). After the addition of 0.6U of avian myelobiastosis virus reverse transcriptase (Roche), incubation was continued at 37°C for 15 min. Reactions were terminated by the addition of 12 ⁇ l of stop solution (70 mM EDTA, 95% formamide, 0.1x TBE, 0.025% xylene cyanol, 0.025% bromophenol blue). Samples were fractionated through a 6% sequencing gel. Sequencing reactions were performed using pAltC4 as the template and the same end-labelled DNA oligonucleotide as a primer.
  • a polymerase chain reaction (PCR) product containing a T7 RNA polymerase promoter, the glgC leader and coding sequence and the trpT terminator sequence was used as a template for transcription.
  • the 28 base E. coli trpT terminator sequence was included after the glgC stop codon to inhibit exonucleolytic degradation of the mRNA in S30 extracts (Wu and Platt, 1978).
  • the glgC-trpT transcript was generated in vitro using 1 ⁇ g of the PCR product and the Ambion MEGAscript kit.
  • CsrA protein The effects of CsrA protein on cell-free translation of glgC mRNA were assessed in S-30 extracts prepared from TR1-5BW3414 (csrA::kanR) as described previously (Romeo and Preiss, 1989; Liu and Romeo, 1997), except that reaction volumes were scaled down to 17.5 ⁇ l and in v/ ' tro-generated mRNA replaced DNA in the reaction. Protein was labelled by incorporation of [ 35 S]-methionine (1175 Ci mmol "1 ; NEN Life Science Products), denatured, and equal volumes of each reaction were subjected to electrophoresis on 9.5% SDS-PAGE gels.
  • Radiolabelled proteins were detected by fluorography using sodium salicylate (Chamberlain, 1979), and methionine incorporated into full-length GlgC polypetide was quantified by liquid scintillation counting of H 2 O 2 -solubilized gel sections (Romeo and Preiss, 1989).
  • WT and mutant 5' end-labelled glgCAP leader transcripts used in this analysis were generated as described for the gel shift analysis. Titrations of RNase T1 (Roche) and lead acetae were performed to optimize the amount of each reagent to prevent multiple cleavages in any one transcript. Binding reactions (10 ⁇ l) containing various concentrations of CsrA and 2 nM WT or mutant glgCAP leader RNA were identical to those described for the gel shift assay. RNase T1 (0.02 U) was added to the binding reaction, and incubation was continued for 15 min at 37°C. Reactions were terminated by the addition of 5 ⁇ l of gel loading buffer II (Ambion).
  • Pb 2+ -mediated cleavage of glgCAP leader RNA was achieved by the addition of 2 ⁇ l of 5 mM lead acetate to the binding reaction, and incubation was continued for 10 min at 37°C (Ciesiolka et al. 1998). Parial alkaline hydrolysis and RNase T1 digestion ladders of each transcript were prepared as described previously (Bevilacqua and Bevilacqua, 1998). Samples were fractionated through 6% sequencing gels. Growth studies and ⁇ -galactosidase assay
  • Bacterial growth in LB at 37°C was monitored using a Klett-Summerson colorimeter (No. 52 green filter). Culture samples (4 ml) were harvested at various times, suspended in 0.5 ml of Z buffer (Miller, 1972) containing 0.2 mg ml "1 lysozyme and lysed by the freeze- thaw/deoxycholate method (Ron et al., 1966). B-Galactosidase assays were performed as described previously (Miller, 1972). The specific activity of the enzyme was determined by normalizing the activity to the protein concentration of the supernatant. Protein concentrations were determined using the Bio-Rad protein assay.
  • Example 1 The csrC gene specifies a -245 nucleotide regulatory RNA
  • pMR2113 The original csrC clone, pMR2113 (Romeo et al., Gene 108: 23, 1991), was isolated due to its stimulatory effects on glycogen accumulation, and was shown to activate glgC'-'lacZ expression. Subclones and deletions from this insert revealed a minimal functional region of 0.36 Kb, which lacked an apparent open reading frame. This suggested that pMR2113 might either contain a cis-acting element that sequesters a transcriptional repressor or may express a regulatory RNA. Furthermore, the nucleotide sequence of the functional region contains an apparent Rho-independent transcriptional terminator at one end, consistent with the latter hypothesis (Fig. 1).
  • the csrC gene is located in the intergenic region between the divergent open reading frames yihA and yihl.
  • the 5'-end of CsrC RNA (+1) and the -10 and -35 elements of an apparent promoter and repeat sequences representing putative CsrA binding sites are underlined.
  • Inverted repeats of the apparent Rho-independent terminator are underlined with arrows.
  • Asterisks flank a minimal functional region, deduced by subcloning and deletion analyses.
  • the location of the initiating nucleotide of csrC on the E coli genome (Blattner et al., 1997) is indicated.
  • RNA which accumulates as the culture approaches the stationary phase of growth
  • Fig. 2 A DIG-labelled riboprobe was used for Northern analysis of CsrC RNA harvested throughout the growth curve of MG1655 (WT) or isogenic csrA or rpoS strains.
  • the positions of RNA standards are marked in panel B of Fig. 2.
  • Stationary phase occurred at ⁇ 6 h.
  • the levels of this RNA were affected by csrA, but not by rpoS, which encodes a sigma factor needed for the expression of a variety of stationary phase genes.
  • Primer extension analysis revealed a single 5'-terminus for this small RNA molecule (Fig.
  • the radiolabeled oligonucleotide primer D1 PrB was annealed to total RNA from MG1655, and extended using reverse transcriptase.
  • the single product of this reaction (lane 1) was analyzed with a Sanger sequencing ladder prepared using the same primer and plasmid pMR2113-D1.
  • An asterisk (*) marks the 3'-terminus of the extension product.
  • Example 2 Effects of csrC on glycogen levels and glgA'-'lacZ expression
  • a csrC mutant TWMG1655 (Table 2), was generated using a linear transformation protocol (Datsenko and Wanner, P.N.A.S. USA 97: 6640-6645, 2000), wherein the transcribed region of csrC was precisely replaced with a tetR marker.
  • the transcribed region of the csrC gene lacking the apparent promoter sequence, was amplified and subcloned into the multiple cloning site of pUC18 in both orientations. Glycogen levels in the csrC mutant were similar to those of the parent strain (Fig. 4).
  • glycogen was observably decreased in a csrB csrC double mutant, relative to the wild type strain or to either the csrB or csrC single mutant.
  • a csrA mutant strain background no change in glycogen was observed when csrC or both csrC and csrB were disrupted, indicating that CsrC effects on glycogen are mediated through CsrA.
  • the plasmid pCSRCI in which csrC is positioned downstream from the lac promoter, caused a substantial increase in glycogen levels (Fig. 4), while no effect was observed for pCSRCrel , in which csrC lacks a promoter. This result indicates that csrC must be transcribed to be functional and provides genetic evidence that CsrC is a regulatory RNA molecule.
  • plM5 Partial csrB clone (+1 to +337) in Baker et al., pT218U 2002
  • KSGA18 (WT), RGKSGA18 (csrB), TWKSGA18 (csrC), RGTWKSG18 (csrB csrC) and KSGA18[pCsrC1] were grown in Kornberg medium and assayed for specific ⁇ -galactosidase activity (A420/mg protein) as described in Experimental Procedures. Disruption of csrC in a csrB wild type strain did not affect expression of a glgCA'-'lacZ translational fusion (Fig. 5). This experiment was conducted twice with essentially identical results.
  • CsrA is a repressor of biofilm formation, while CsrB activates this process.
  • the effect of csrC on biofilm formation in static cultures was measured using a microtiter plate assay.
  • Figure 6 illustrates the effects of csr genes on biofilm formation. The bars depict the means and the standard errors from two independent experiments with 8 replicates per strain in each experiment. Double asterisks denote statistically significant differences with respect to the control strains, WT (MG1655) in panel A, or its csrC mutant (TWMG1655) containing the vector pUC18 in panel B (PO.01). The strain designations are as listed in Fig. 4 legend.
  • csrC Disruption of csrC modestly decreased biofilm formation, an effect that was determined to be statistically significant (Fig. 6A). Biofilm formation was reduced in a csrB mutant. A csrB csrC double mutant was further compromised for biofilm formation and produced only 10% of the biofilm of the wild type strain. In contrast, increased gene dosage of csrC led to several-fold greater accumulation of biofilm (Fig. 6B), an effect that was dependent upon csrC transcription. (csrC lacks a promoter for expression from pCSRCre ) Thus, CsrC RNA activates biofilm formation in E. coli, similar to CsrB.
  • CsrA is required for motility of E. coli under a variety of conditions, e.g. in tryptone medium.
  • the effects of csrC disruption and increased copy number on motility were examined in tryptone medium. Similar tp its isogenic parent, the csrC null mutant of MG1655 was fully motile.
  • increasing the copy number of csrC by introducing pCSRCI , which contains csrC downstream from the lac promoter completely inhibited motility, while no effect was observed using pCSRrel , in which csrC lacked a promoter.
  • CsrC and CsrB levels were measured at 2 hours post-exponential phase in a series of isogenic strains varying in csrA, csrB, csrC or uvrY (Fig. 7).
  • This experiment showed that the CsrB and CsrC riboprobes were specific, since no signal was detected from the csrB mutant with the CsrB probe or from the csrC mutant with the CsrC probe.
  • CsrC accumulation was found to depend upon both CsrA and UvrY (Fig. 7B), although CsrC levels were somewhat less sensitive than those of CsrB (Fig. 7A).
  • Example 6 Expression of a csrC-lacZ transcriptional fusion.
  • csrC-/acZ and csrB- lacZ gene fusions were designed to contain the upstream sequences and only four base pairs of the DNA templates of these two genes. Thus, transcripts synthesized from these fusions only contain 4 CsrB or CsrC nucleotides, and therefore lack CsrA-binding elements.
  • the expression of each gene fusion was reduced in csrA and uvrY mutant strains, indicating that expression is activated by CsrA and UvrY.
  • Example 7 S-30 transcription-translation Similarly, Purified CsrA had no effect on the transcription-translation of the pCCZ1- encoded csrC-lacZ transcriptional fusion in S-30 extracts, which contained the same fusion construct that was activated by CsrA from the chromosome (Fig. 9B). In contrast, expression of this plasmid-encoded csrC-lacZ fusion was stimulated 2-fold by purified recombinant UvrY protein. Expression of csrC-lacZ exhibited a linear dose response up to 13 ⁇ M UvrY protein, which was the highest concentration that was tested, due to technical constraints. This response was weaker than that of csrB-lacZ expression, confirming the stronger dependence of csrB expression on UvrY in vivo (Figs. 7, 9).
  • the vector control in each case was pBR322. Liquid cultures were grown to 2 h post-exponential phase and specific ⁇ -galactosidase activity was determined as the average of duplicate samples. The error bars in Fig. 10 indicate standard deviation. This experiment was repeated in entirety, with essentially identical results.
  • CsrB RNA A striking feature of CsrB RNA is the observation that 18 conserved sequences are located primarily in the loops of predicted hairpins or other single stranded regions of CsrB.
  • the CsrA:CsrB complex contains ⁇ 18 subunits of CsrA.
  • the repeated sequences of CsrB resemble a high affinity CsrA-binding site of glgC mRNA, strongly suggesting that these sequences are binding sites for CsrA.
  • CsrC RNA contains 9 similar repeated sequence elements (Fig. 1), which tend to be located in predicted single stranded loops or bulges of the molecule (Fig. 11). Secondary structure of CsrC RNA was predicted using MFOLD, as described in the Methods. Imperfect repeat sequences that resemble the CsrB-type repeats are shown in red, predicted base- paring interactions are indicted by filled circles.
  • CsrC RNA is unusual in containing a 16 nucleotide ("nt") inverted repeat, in which the oligo-U sequence of the terminator is complementary to an oligo-A sequence (Fig. 12), a feature distinct from that of CsrB.
  • nt 16 nucleotide
  • csrC homologues were identified in species representing 3 genera of Enterobacteriaceae.
  • the CsrC homologue of Klebsiella pneumoniae was the most distinct from that of E coli, and exhibited 75% identity.
  • the nucleotide sequence of the terminal stem-loop of CsrC is highly conserved in these bacteria. See for example Figure
  • CsrA was purified and it was estimated that the protein was >95% pure.
  • the amino acid sequence of purified CsrA was confirmed by tryptic digestion followed by mass spectrometry of the fragments. No formyl group was present on the N-terminal methionine.
  • Gel filtration of purified CsrA suggested that CsrA is multimeric.
  • Preliminary results from mass spectroscopy and analytical ultracentrifugation experiments suggest that CsrA exists as a dimer or a trimer. Owing to the uncertainty of the oligomeric structure of CsrA, the- concentrations reported in this study are for the monomeric CsrA polypeptide.
  • Example 12 CsrA binds specifically to glgCAP leader RNA
  • the major glgCAP transcript contains a 134nt untranslated leader. Previous gel mobility shift and deletion studies localized the presumed CsrA binding site within this leader sequence. To characterize further the interaction of CsrA with the glgCAP leader transcript, quantitative gel shift assays were performed with a gig transcript containing nucleotides +46 to +179 relative to the start of transcription. CsrA binding to this transcript was detected as a distinct band in native gels between 15 and 250 nM CsrA (Fig. 1). Non-linear least-squares analysis of these data yielded an estimated d value of 39 nM CsrA. As the concentration of CsrA was increased further, additional shifted species were observed.
  • CsrA-g/g leader RNA interaction was investigated by performing competition experiments with specific (glgCAP leader, flhDC leader and CsrB RNA) and non-specific (Bacillus subtilis trp leader) uniabelled RNA competitors (Fig. 1).
  • CsrB RNA which contains 18 putative CsrA binding sites, was the most effective competitor followed by glgCAP leader RNA and the flhDC leader RNA.
  • CsrA-g/g RNA complex formation was not competed by B. subtilis trp leader RNA.
  • biofilm-producing bacteria are bacteria capable of producing biofilm under suitable physiological conditions.
  • E. coli are biofilm- producing bacteria.
  • an antibacterial agent may be any pharmaceutically acceptable agent useful in decreasing the extent of infection by the infecting bacteria in the mammalian patient.
  • TRAP the trp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG transcripts. J Biol Chem 269:16597-16604.
  • RNA binding protein CsrA activates csrB transcription in Escherichia coli. J Bacteriol 183:6017-6027.
  • RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli. J Biol Chem 272: 17502-17510.
  • Neidhardt, F., and Savageau, M.A. (1996) Regulation beyond the operon. In Escherichia coli and Salmonella: Cellular and Molecular Biology, vol. 1 , 2nd edn. Neidhardt, F.C., Curtiss, III, R., Ingraham, J.L., Lin, E.C.C., Low, K.L., Magasanik, B., Reznikoff, W.S:, Riley, M., Schaechter, M., and Umbarger, H.E. (eds). Washington, D.C.: American Society for Microbiology, pp.1310-1324.
  • rsmC of the soft- rotting bacterium Erwinia carotovora subsp. carotovora negatively controls extracellular enzyme and harpin (Ecc) production and virulence by modulating levels of regulatory RNA (rsmB) and RNA-binding protein (RsmA). J Bacteriol 181: 6042-6052.
  • Quorum sensing controls the synthesis of virulence factors by modulating rsmA gene expression in Erwinia carotovora subsp. carotovora. Mol Gen Genet 265: 287-292.
  • Bacteriophage regA protein binds to mRNAs and prevents translation initiation. Proc Natl Acad Sci USA 84: 7822-7826.

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L'invention concerne le gène csrC, l'ARN codé par celui-ci et des procédés d'utilisation associés pour la modulation de film biologique.
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WO2008013187A1 (fr) * 2006-07-25 2008-01-31 Kyowa Hakko Bio Co., Ltd. Procédé de production d'un acide aminé
WO2018030578A1 (fr) * 2016-08-11 2018-02-15 한국과학기술원 Composition de régulation de la formation de biofilms, et procédé de régulation de la formation de biofilms au moyen de la composition
WO2021173953A1 (fr) * 2020-02-26 2021-09-02 Pebble Labs, Inc. Nouvelles bactéries endophytiques de végétaux et procédés de régulation de pathogènes et d'organismes nuisibles des végétaux

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JPH07143889A (ja) * 1993-06-16 1995-06-06 Mitsubishi Chem Corp 細胞分裂に関与するｄｎａ断片
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WO2008013187A1 (fr) * 2006-07-25 2008-01-31 Kyowa Hakko Bio Co., Ltd. Procédé de production d'un acide aminé
JP5297804B2 (ja) * 2006-07-25 2013-09-25 協和発酵バイオ株式会社 アミノ酸の製造法
US8759042B2 (en) 2006-07-25 2014-06-24 Kyowa Hakko Bio Co., Ltd. Method for production of amino acid
WO2018030578A1 (fr) * 2016-08-11 2018-02-15 한국과학기술원 Composition de régulation de la formation de biofilms, et procédé de régulation de la formation de biofilms au moyen de la composition
WO2021173953A1 (fr) * 2020-02-26 2021-09-02 Pebble Labs, Inc. Nouvelles bactéries endophytiques de végétaux et procédés de régulation de pathogènes et d'organismes nuisibles des végétaux

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