WO1996035804A1 - A novel biochemical interaction in bacteria - Google Patents
A novel biochemical interaction in bacteria Download PDFInfo
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- WO1996035804A1 WO1996035804A1 PCT/US1996/006224 US9606224W WO9635804A1 WO 1996035804 A1 WO1996035804 A1 WO 1996035804A1 US 9606224 W US9606224 W US 9606224W WO 9635804 A1 WO9635804 A1 WO 9635804A1
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Definitions
- the FtsZ protein is highly conserved in bacteria and appears to play a cytoskeletal role during cell division. FtsZ is located in the bacterial cytoplasm during most of the cell cycle. As division begins, FtsZ relocalizes to a ring at the inner surface of the inner membrane at the division site, and remains at the leading edge of the inward growth of the septum until division is completed.
- PBP3 the septum-specific, membrane-spanning penicillin binding protein is encoded by the cell division gene FtsI, and is essential for septum formation.
- One hydrophobic helix of PBP3 spans the inner membrane connecting a 23 amino acid cytoplasmic domain to the transglycosylase and transpeptidase domains located in the periplasm.
- FtsZ is a cytoplasmic protein and PBP3 is a transmembrane protein with its N- terminus residing in the cytoplasm, the only way these proteins could interact in the cell would be through the N-terminal amino acid residues of PBP3.
- FIG. 2 represents theoretically the FtsA-FtsZ-PBP3 complex spanning the inner membrane.
- the present invention relates to a novel interaction between bacterial (E. coli) cell division proteins.
- the present invention also relates to a novel target for antibiotic development. Additionally, the present invention relates to the use of the yeast two-hybrid screen, as well as an in vitro flourescence assay, to screen for agents which would inhibit the novel interaction.
- the essential Escherichia coli cell division protein FtsZ shares biochemical properties and amino acid similarities with tubulins, suggesting a cytoskeletal role for FtsZ. Both have GTPase activity and undergo GTP-dependent polymerization to form structures comprised of protofilaments.
- the yeast two-hybrid protein interaction screen was used to identify associations between FtsZ (the "bait") and other cell division proteins.
- the yeast two-hybrid system is an in vivo method for genetically identifying interacting proteins using reconstitution of activity of a transcriptional activator (GAL4). This reconstitution makes use of chimeric genes which express hybrid proteins.
- GAL4 transcriptional activator
- Two types of hybrid proteins are prepared. The first hybrid contains the DNA- binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. If the two test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which contains a binding site for the DNA-binding domain.
- FtsZ polymerization was measured at optimal and sub- optimal levels of GTP, to determine whether the PBP3 peptide stabilized or destabilized the FtsZ polymer. No effect on polymerization was observed. Neither was there an effect on the GTPase activity of FtsZ in the presence of the PBP3 peptide. Polymerization of FtsZ did not significantly alter the PBP3-FtsZ interaction.
- PBP3 cytoplasmic domain peptide (MKAAAKTQKPKRQEEHANFISWR-NH 2 ) (SEQ ID NO:— -) was prepared by standard Fmoc synthesis.
- the N-dansylated derivative was synthesized by reacting the peptide with dansyl chloride prior to deprotection and removal from the support.
- Peptides were purified by reverse phase C18 HPLC and their structures confirmed by mass spectrometry.
- FtsZ was prepared as described in Bramhill et al., Proc. Nat'l Acad. Sci. 91 , 5813-7 (1994). Ligand binding experiments were conducted at room temperature (19-21 °C).
- Pairs of plasmids each pair consisting of a DNA binding domain and an activation domain fusion, were introduced into the Saccharomyces cerevisiae reporter strain SFY526 (Bartel et al., Bio Techniques 14, 920-924 (1993) and tested for their ability to activate lacZ expression driven from a GAL4 responsive promoter .
- the results of the assay are shown below i-j Table 1.
- RNA division genes were cloned by PCR using DNA of the Escherichia coli K12 strain R477 as template. PCR primers (CGG AGA GAA ACT ATG TTT GGA TCC ATG GAA CTT ACC (SEQ ID NO:— -)and CGA AAC CCA AAC TGC AGT CAA TTC TTA ATC AGC (SEQ ID NO:—) for FtsZ, GGC ACA GGC
- AGA ACA ACA ATG ATG AAT TCG ACG G (SEQ ID NO: — ) and CCG TAA CAT CGT CGG CTG CAG AAA AAT TAA AAC TC (SEQ ID NO: — ) for FtsA
- CAA AAA TAA GGA TAA ACG GAA TTC ATG AAA GCA GCG GC (SEQ ID NO: — ) and CCC ACG GAG CAA GAA GGA TCC GCA AAT TAC GAT CTG CC (SEQ ID NO — ) for PBP3) introduced appropriate restriction endonuclease sites for in- frame fusion of each gene to the GAL4 activator or DNA-binding domain in the vectors pGAD424 and pGBT9, respectively.
- Plasmid DNAs were prepared from Escherichia coli K12 strain DH5 ⁇ , (Hanahan, D., J. Mol. Biol., 166 557-580 (1983), and transformed into yeast strain SFY526 in the combinations indicated. Yeast transformants were selected on media lacking leucine and tryptophan. ⁇ -galactosidase activity on two independent transformants was determined as previously described, and is expressed as Miller units. Activation domain plasmids containing either no fusion protein or fused with T-antigen were used as controls.
- FIG. 2 diagrams the interaction linking the cytoplasmic and periplasmic components of the division apparatus. If the FtsZ protein is serving as a guide for septum synthesis, and if it is a cytoskeleton acting to overcome the outward osmotic pressure of about four atmospheres, then the fewest number of links connecting FtsZ to the peptidoglycan synthesis complex will provide the strongest structure.
- FtsZ and PBP3 are relatively low. However, this may not be a limiting parameter given the extremely high local concentration of FtsZ at a division site. Also, there may be other components in the cell which stabilize the FtsZ- PBP3 complex. At least four other Fts proteins appear to span the inner membrane and may also interact with FtsZ.
- MOLECULE TYPE peptide
- HYPOTHETICAL NO
- ANTI-SENSE NO
Abstract
A novel biochemical interaction between bacterial cell division proteins has been identified. This interaction may be a potential therapeutic target for a novel class of antibiotics which inhibit the interaction.
Description
TITLE OF THE INVENTION
A NOVEL BIOCHEMICAL INTERACTION IN BACTERIA
BACKGROUND OF THE INVENTION The FtsZ protein is highly conserved in bacteria and appears to play a cytoskeletal role during cell division. FtsZ is located in the bacterial cytoplasm during most of the cell cycle. As division begins, FtsZ relocalizes to a ring at the inner surface of the inner membrane at the division site, and remains at the leading edge of the inward growth of the septum until division is completed. PBP3, the septum-specific, membrane-spanning penicillin binding protein is encoded by the cell division gene FtsI, and is essential for septum formation. One hydrophobic helix of PBP3 spans the inner membrane connecting a 23 amino acid cytoplasmic domain to the transglycosylase and transpeptidase domains located in the periplasm.
Interaction between PBP3 and FtsZ would provide a direct link between bacterial cytoskeleton and cell wall synthesis. While this interaction has been mentioned as a hypothetical possibility in earlier journal articles; Ayala et al., J. Bacteriol. 170, 3333-3341 (1988); Pla, Dopazo and Vicente, J. Bacteriol. 172, 5097-5102 (1990); Bi and
Lutkenhaus, Molecular Microbiol., 9, 403-409 (1993); Shapiro, Cell 73, 841-855 (1993) and Bramhill and Thompson, PNAS 91 , 5813-5817 91994), no one has shown any data to support the hypothesis.
SUMMARY OF THE INVENTION
A novel interaction between two essential E. coli cell division proteins has been identified through the use of the yeast two- hybrid interaction screen. The screen is described in U.S. Patent No. 5,283,173 to Fields et al., which is incorporated by reference. Cell division protein FtsZ has been found to interact with a number of other cell division proteins. In particular, it was found that FtsZ interacts with PBP3, a septum-specific transglycosylase/transpeptidase that is the target of the known antibiotic, aztreonam. Because FtsZ is a
cytoplasmic protein and PBP3 is a transmembrane protein with its N- terminus residing in the cytoplasm, the only way these proteins could interact in the cell would be through the N-terminal amino acid residues of PBP3.
Thus, it has been found that this interaction may be a novel target for the development of antibiotics which would inhibit the interaction and prevent bacterial cell division.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS, la-c represent the interaction of FtsZ with PBP3 cytoplasmic domain: la) Fluorescence emission scans of the dansylated peptide, FtsZ, and the dansylated peptide and FtsZ mixture, at λex = 350 nm. lb) Titration of the dansyl-PBP3 peptide into reactions containing FtsZ (0.5 μM). lc) Competition of FtsZ binding with non-dansylated peptides.
FIG. 2 represents theoretically the FtsA-FtsZ-PBP3 complex spanning the inner membrane.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel interaction between bacterial (E. coli) cell division proteins. The present invention also relates to a novel target for antibiotic development. Additionally, the present invention relates to the use of the yeast two-hybrid screen, as well as an in vitro flourescence assay, to screen for agents which would inhibit the novel interaction. The essential Escherichia coli cell division protein FtsZ shares biochemical properties and amino acid similarities with tubulins, suggesting a cytoskeletal role for FtsZ. Both have GTPase activity and undergo GTP-dependent polymerization to form structures comprised of protofilaments. To understand how a cytoplasmic FtsZ structure at
the division site might direct cell wall synthesis in the periplasm, we searched for proteins that associate with FtsZ. Interaction between FtsZ and the septum-specific, membrane -spanning penicillin binding protein, PBP3 (the ft si gene product), was detected by the yeast two-hybrid system. The transglycosylase and transpeptidase domains of PBP3, which catalyze the synthesis of the cell wall peptidoglycan, reside in the periplasm, across the membrane from the N-terminal 23 amino acid cytoplasmic domain. In vitro, an N-dansylated derivative of the PBP3 cytoplasmic domain showed saturable binding to purified FtsZ and was displaced by a cognate peptide. Interaction between PBP3 and FtsZ provides a direct link between a bacterial cytoskeleton and cell wall synthesis.
The yeast two-hybrid protein interaction screen was used to identify associations between FtsZ (the "bait") and other cell division proteins. The yeast two-hybrid system is an in vivo method for genetically identifying interacting proteins using reconstitution of activity of a transcriptional activator (GAL4). This reconstitution makes use of chimeric genes which express hybrid proteins. Two types of hybrid proteins are prepared. The first hybrid contains the DNA- binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. If the two test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which contains a binding site for the DNA-binding domain.
In order to determine if an interaction existed between essential bacterial proteins, the following experimental procedures were conducted. The following is provided for the purpose of illustrating, but not limiting, the invention.
IN VITRO ASSAY To test directly whether FtsZ and the N-terminal domain of PBP3 form a specific complex, a fluorescence binding assay was devised. The binding assay is based on the enhancement of fluorescence and the accompanying shift to lower wavelength emission maximum of certain fluorophores, like dansyl (5-dimethylaminonaphthalene-l - sulfonyl), upon change from a polar to non-polar molecular environment. An N-dansylated peptide corresponding to the N- terminal 23 amino acids of PBP3 (SEQ ID NO: — ) was synthesized as a fluorescent reporter ligand to monitor ligation to FtsZ protein.
Addition of purified FtsZ to a solution of the dansylated peptide resulted in a large increase in fluorescence intensity together with a shift in emission wavelength maximum [λem(max)] when the excitation wavelength was 350 nm (Fig la). The λem(max) for the dansylated peptide alone was near 550 nm. In the presence of FtsZ, two new emission peaks were seen, one at 490 nm and a second at 515 nm. Both of these new emission peaks showed a several-fold greater quantum yield than for the 550 nm peak of the peptide alone. The 490 nm peak was used to quantify the interaction, since the unbound peptide showed low emission at this wavelength. Titration of dansylated peptide against a fixed concentration of FtsZ (Fig. lb) yielded D(peptide) = 5 μM, calculated from a non-linear least squares fit of the data to the equation describing the equilibrium.
Competitive binding experiments confirmed the specificity of the interaction (Fig. lc). The non-dansylated peptide displaced the fluorescent peptide from FtsZ. The dansyl group appeared to increase the affinity by about 10-fold over the unmodified peptide, as judged by this competition assay. In contrast to the cognate peptide, non-specific peptides showed little effect in competition. A number of purified proteins were used in place of FtsZ to assess the specificity of this component, including bovine serum albumin, single strand DNA binding protein and tubulin. None of these showed significant changes of dansylated PBP3 peptide fluorescence.
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FtsZ polymerization was measured at optimal and sub- optimal levels of GTP, to determine whether the PBP3 peptide stabilized or destabilized the FtsZ polymer. No effect on polymerization was observed. Neither was there an effect on the GTPase activity of FtsZ in the presence of the PBP3 peptide. Polymerization of FtsZ did not significantly alter the PBP3-FtsZ interaction.
METHODS. PBP3 cytoplasmic domain peptide (MKAAAKTQKPKRQEEHANFISWR-NH2) (SEQ ID NO:— -)was prepared by standard Fmoc synthesis. The N-dansylated derivative was synthesized by reacting the peptide with dansyl chloride prior to deprotection and removal from the support. Peptides were purified by reverse phase C18 HPLC and their structures confirmed by mass spectrometry. FtsZ was prepared as described in Bramhill et al., Proc. Nat'l Acad. Sci. 91 , 5813-7 (1994). Ligand binding experiments were conducted at room temperature (19-21 °C). Fluorescence data collected at a right angle to the excitation source using the photon counting mode were obtained using an SLM/AMNICO Model 8100D spectrofluorometer fitted with a standard quartz cuvette holder. Fluorescence enhancements upon ligand binding were measured using λex = 350 nm (slit width = 8 nm) and λem = 490 nm (slit width = 8 nm). Unless otherwise specified, binding assay mixtures contained FtsZ (0.5 μM), dansylated PBP3 peptide (1 μM), non-fluorescent peptides as indicated in 10 mM Tris-Cl, pH 7.5 in a total volume of 1.0 mL. Concentrations of stock solutions of dansyl peptide (in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were calculated from the extinction coefficient of the dansyl moiety at 340 nm (ε = 4250 M^cπr1). Blank control runs consisted of buffer and (for panels C and D) dansylated peptide at the experimental concentrations, and the resulting signal was subtracted from the experimental data. Fluorescence is expressed in arbitrary units, normalized to the maximum signal measured in that experiment.
YEAST TWO-HYBRID ASSAY The ftsZ gene was cloned into vector pGBT9 behind the GAL4 DNA binding domain gene, forming the C-terminal FtsZ fusion protein coding sequence. Likewise, various cell division genes from E. coli were cloned into the pGAD424 vector, creating C-terminal translational fusions of each cell division protein to the GAL4 activator domain. Both the pGBT9 and pGAD424 vectors are described in Bartel, et al., Cellular Interactions in Development: A Practical Approach (Hartley, D.A., ed.) pp. 153-179, Oxford Univ. Press (1993). Pairs of plasmids, each pair consisting of a DNA binding domain and an activation domain fusion, were introduced into the Saccharomyces cerevisiae reporter strain SFY526 (Bartel et al., Bio Techniques 14, 920-924 (1993) and tested for their ability to activate lacZ expression driven from a GAL4 responsive promoter . The results of the assay are shown below i-j Table 1.
METHODS. Cell division genes were cloned by PCR using DNA of the Escherichia coli K12 strain R477 as template. PCR primers (CGG AGA GAA ACT ATG TTT GGA TCC ATG GAA CTT ACC (SEQ ID NO:— -)and CGA AAC CCA AAC TGC AGT CAA TTC TTA ATC AGC (SEQ ID NO:—) for FtsZ, GGC ACA GGC
AGA ACA ACA ATG ATG AAT TCG ACG G (SEQ ID NO: — ) and CCG TAA CAT CGT CGG CTG CAG AAA AAT TAA AAC TC (SEQ ID NO: — ) for FtsA, and CAA AAA TAA GGA TAA ACG GAA TTC ATG AAA GCA GCG GC (SEQ ID NO: — ) and CCC ACG GAG CAA GAA GGA TCC GCA AAT TAC GAT CTG CC (SEQ ID NO — ) for PBP3) introduced appropriate restriction endonuclease sites for in- frame fusion of each gene to the GAL4 activator or DNA-binding domain in the vectors pGAD424 and pGBT9, respectively. The Pstl site within the PBP3 coding sequence was used to generate the deletions of the first 233 codons or the last 344 codons. Plasmid DNAs were prepared from Escherichia coli K12 strain DH5α, (Hanahan, D., J. Mol. Biol., 166 557-580 (1983), and transformed into yeast strain SFY526 in the combinations indicated. Yeast transformants were selected on media lacking leucine and tryptophan. β-galactosidase
activity on two independent transformants was determined as previously described, and is expressed as Miller units. Activation domain plasmids containing either no fusion protein or fused with T-antigen were used as controls.
Table 1. Interactions between PBP3, FtsZ and FtsA detected by activation of transcription in the yeast two-hybrid system .
Fusion to GAL4 DNA- Fusion to GAL4 β-Galactosidase
Binding Domain Activator Domain Activity (Miller Units)
FtsZ FtsZ 90
FtsZ * FtsA 47
FtsZ PBP3 (wt) 12
FtsZ PBP3Δaa(l-233) 0.030
FtsZ PBP3Δaa(234-577) 1 1 no fusion FtsZ 0.030 no fusion FtsA 0.030 no fusion PBP3 (wt) 0.030
FtsZ T-antigen 0.028 no fusion no fusion 0.030
Cells co-transformed with plasmids carrying the FtsZ protein fused to each GAL4 domain showed activation of lacZ expression, consistant with the various FtsZ-FtsZ interactions reported Interaction between FtsA and FtsZ was also detected by the two-hybrid system (Table 1 ). Although this association was anticipated from physiological studies, this is the first experimental evidence of a direct interaction between FtsA and FtsZ. However, the interaction between
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PBP3 and FtsZ was less anticipated and previously unconfirmed. The fact that only the N-terminal 233 (of 577) amino acids of PBP3 were required for the interaction (Table 1 ) is suggestive as this portion of PBP3 contains a 23 amino acid domain predicted to be located in the cytoplasm where it could readily interact with FtsZ.
Although there has been speculation that such an association might occur, this is the first evidence, both genetic and biochemical, of direct interaction between the cell division proteins PBP3 and FtsZ. Fig. 2 diagrams the interaction linking the cytoplasmic and periplasmic components of the division apparatus. If the FtsZ protein is serving as a guide for septum synthesis, and if it is a cytoskeleton acting to overcome the outward osmotic pressure of about four atmospheres, then the fewest number of links connecting FtsZ to the peptidoglycan synthesis complex will provide the strongest structure. The lack of effect of the PBP3 peptide on FtsZ polymerization and its interaction with polymerized FtsZ is also consistent with a cytoskeletal role, since PBP3 presumably interacts with the polymerized form of FtsZ in vivo.
It should be noted that the affinity between FtsZ and PBP3 is relatively low. However, this may not be a limiting parameter given the extremely high local concentration of FtsZ at a division site. Also, there may be other components in the cell which stabilize the FtsZ- PBP3 complex. At least four other Fts proteins appear to span the inner membrane and may also interact with FtsZ.
Interaction between FtsA and FtsZ has also been detected in the two-hybrid system. Such an interaction was inferred from the observation that the balance of FtsA to FtsZ is critical for successful division. The data demonstrate a direct interaction between these two division proteins. Interactions have been inferred between FtsA and a number of other division factors, particularly FtsN. Also, PBP3 has been found associated with particular periplasmic proteins PBP7 and soluble lytic transglycosylase possibly pointing to a large multiprotein complex, centered around PBP3 and linked by direct and indirect interactions to FtsZ, that is guided (or driven by a motor protein) along an FtsZ cytoskeleton to form the septum and complete cell division.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: POMPLIANO, DAVID L BRAMHILL, DAVID CUNNINGHAM, BARRY R EL-SHERBEINI, MOHAMED JONES, BRIAN TRUSCA, DORINA
(ii) TITLE OF INVENTION: A NOVEL BIOCHEMICAL INTERACTION IN BACTERIA
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ELLIOTT KORSEN
(B) STREET: 126 E. LINCOLN AVENUE, P.O. BOX 2000
(C) CITY: RAHWAY
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07065-0900
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/437,253
(B) FILING DATE: 08-MAY-1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: KORSEN, ELLIOTT
(B) REGISTRATION NUMBER: 32,705
(C) REFERENCE/DOCKET NUMBER: 19455
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908 594-5493
(B) TELEFAX: 908 594-4720
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 -amino acids
(B) -TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Lys Ala Ala Ala Lys Thr Gin Lys Pro Lys Arg Gin Glu Glu His 1 5 10 15
Ala Asn Phe lie Ser Trp Arg 20
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sing1e
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CGGAGAGAAA CTATGTTTGG ATCCATGGAA CTTACC 36
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGAAACCCAA ACTGCAGTCA ATTCTTAATC AGC 33
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGCACAGGCA GAACAACAAT GATGAATTCG ACGG 34
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCGTAACATC GTCGGCTGCA GAAAAATTAA AACTC 35
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CAAAAATAAG GATAAACGGA ATTCATGAAA GCAGCGGC 38
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCCACGGAGC AAGAAGGATC CGCAAATTAC GATCTGCC 38
Claims
1. A novel biochemical interaction between two essential E. coli cell division proteins which comprises an interaction between FtsZ and PBP3.
2. The novel protein-protein interaction of Claim 1 which comprises the interaction between FtsZ and the 23 N-terminal amino acids of PBP3.
3. The novel interaction of Claim 1 which can be determined by use of the yeast two-hybrid system.
4. A method of targeting novel antibiotics against bacteria cell wall division which comprises identifying compounds that inhibit the novel interaction of Claim 1.
5. A method of identifying novel antibiotics that inhibit the novel interaction of Claim 1 which comprises measuring the ability of test compounds to displace the N-dansylated PBP3 peptide reporter ligand from FtsZ protein in an in vitro competitive binding assay.
6. A method of identifying novel antibiotics that inhibit the novel interaction of Claim 1 which comprises measuring the ability of test compounds to disrupt the interaction between FtsZ and PBP3 in vivo as monitored by the yeast two-hybrid system.
7. A method of identifying novel biochemical interactions in bacterial cell division proteins which comprises (a) expressing in yeast the first test cell division protein as a fusion to the DNA binding domain of a transcriptional activator such as GAL1 and expressing the second test cell division protein or library of proteins as a fusion to the transcriptional activation domain; and (b) testing for the ability of the two expressed proteins to interact by monitoring the catalytic activity of a gene product whose gene is under transcriptional control of the transcriptional activator described in (a).
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| US43725395A | 1995-05-08 | 1995-05-08 | |
| US08/437,253 | 1995-05-08 |
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| WO1996035804A1 true WO1996035804A1 (en) | 1996-11-14 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998026088A1 (en) * | 1996-12-12 | 1998-06-18 | Isis Innovation Limited | Bacillus strain and antibiotic screening method |
| EP1043403A1 (en) * | 1999-04-09 | 2000-10-11 | GPC AG, Genome Pharmaceuticals Corporation | Novel method for identifying antibacterial compounds |
| WO2000061793A2 (en) * | 1999-04-09 | 2000-10-19 | Gpc Biotech Ag | Novel method for identifying antibacterial compounds |
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| US5283173A (en) * | 1990-01-24 | 1994-02-01 | The Research Foundation Of State University Of New York | System to detect protein-protein interactions |
| US5409898A (en) * | 1990-02-23 | 1995-04-25 | Bristol-Myers Squibb Company | Compositions and methods for treating infections caused by organisms sensitive to β-lactam antibiotics |
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| US5283173A (en) * | 1990-01-24 | 1994-02-01 | The Research Foundation Of State University Of New York | System to detect protein-protein interactions |
| US5409898A (en) * | 1990-02-23 | 1995-04-25 | Bristol-Myers Squibb Company | Compositions and methods for treating infections caused by organisms sensitive to β-lactam antibiotics |
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| Title |
|---|
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