JP2002541819A - New essential bacterial genes and their proteins - Google Patents

Abstract

  (57) [Summary] The present invention relates to nucleic acids having functions essential for the survival of Escherichia coli and protein sequences derived from the nucleic acids. The invention also relates to the vectors and host cells transformed with these vectors to express the essential proteins of the above-described format, and the proteins produced with these vectors. Finally, the invention also relates to the use of the proteins produced using these vectors in a screening method for identifying antibacterial substances and to the antibacterial substances found together with these proteins.

Description

DETAILED DESCRIPTION OF THE INVENTION

The research and development of new and innovative antibiotics has previously been described, inter alia, in the modification and optimization of known types of substances, for example β-lactams (penicillin-cephalosporins IV) and the so-called MIC test Tests of these effects on microorganisms in

In recent years, targets whose biochemical and microbiological properties are well known (attack points)
Research on a new principle of activity with the help of has been established in parallel. However, target selection by these methods is based on empirical methods, which have greatly complicated the discovery of new points of attack.

[0003] Recently, knowledge of the complete gene sequence of prokaryotes and eukaryotes, in conjunction with bioinformatics and molecular biology methods, offers the possibility of a systematic and rational selection of antibacterial points in attacks. Was opened.

[0004] Thus, analysis of various bacterial genomes has revealed a number of genes of unknown function.

[0005] Unknown protein from Escherchia coli, a Gram-negative model organism
(FR Blattner et al., Science 277: 1453-1462, 1997) have names that usually begin with Y, for example, YQGF. However, some proteins of unknown function have names off the system because the genes encoding them are located near functionally characterized genes, such as KDTB (TDT . Clemen
tz, CRH Raetz, J. Biol. Chem. 266: 9687-9696, 1991; T. Clementz, J. B.
acteriol. 174: 7750-7756, 1992).

[0006] In the context of the discovery of anti-infective agents, genes of unknown function and proteins derived from them, if this can be proven to be essential for the survival of microorganisms, are potential new attacks (F. Argoni et al., Nature Biotec
hnology, 16: 851, 1998; DT Moir et al., Antimicrob. Agents Chemother. 4
3: 439-446, 1999; United States Patent (US) 5,821,076; BJ Akerly et al., Proc.
Natl. Acad. Sc. USA 95: 8927-8932, 1998).

[0007] Knockout techniques can be used to analyze the essentiality of such gene sequences for an organism. Knockout strategies are based on the replacement of invariant (wild-type) gene sequence information in the bacterial chromosome with a gene sequence that has been altered by insertion or deletion. Thus, such gene sequences identify coding gene products that are essential for this cell from their ability to survive only in the presence of an invariant complementary copy in the cell (the plasmid or chromosomal sequence in the chromosome). Based on two artificial copies).

[0008] Knockout experiments are performed as homologous DNA sequence recombination events. Vectors that can be controlled to lose the ability to replicate plasmids are used for this, such as in the case of temperature-sensitive replicons (pKO3, pMAK700 or pSC101), or are not able to replicate in the desired target cells (E. coli). Vector, for example, a gram-positive plasmid (pC194) in a gram-negative bacterium (E. coli).
, PT181). The integrating vector, eg, pMAK700, contains not only sequences for temperature-sensitive replication, but also additional markers, eg, antibiotic resistance (eg, chloramphenicol) that can be used for selection of plasmid-containing cells.

The present invention points out these distributions within phylogenetically distinct cytospheres, and more particularly selects genes with no or at most very distant homology in eukaryotes. And genes with previously unknown functions that demonstrate the essentiality of these genes for the ability of microorganisms to survive.

[0010] Another requirement for the gene to be discovered is through appropriate choice of search strategy.
Based on these expected physicochemical properties, the only goal is to select only those proteins from which the derived proteins can be expected to be soluble in water and which can be suitably used in cell-free assays.

[0011] From this analysis, it is possible to select gene sequences that would not be selected by attack points to find antibiotically active substances in a conventional manner.

For this purpose, the gene product of the gene is expressed and produced within the scope of the present invention. The novel proteins obtained in this way are used in assays to discover agonists and antagonists of these proteins. In addition, over- or under-expressed mutants for the corresponding genes can also be used for the discovery of agonists and antagonists. Used in assays.

The present invention relates to YQGF, YHBC, YGGJ, YGBP, YCHB, YGBB,
YJEE, an essential gene encoding a protein from the group of KDTB and genes from other microorganisms encoding the corresponding orthologous gene products, and their controllable switch-off, can prove to lead to the arrest of growth of the host organism The above genes, and the above genes from E. coli, and the corresponding genes from E. coli, for which it can be demonstrated that 95-100% encode the corresponding gene products and that their controllable switch-off leads to the arrest of growth of the host organism. Others encode orthologous gene products with amino acids that are 42-100% identical or conservatively converted compared to the corresponding gene product and that their controllable switch-off can lead to the arrest of growth of the host organism. As a component of the above genes from organisms, and their nucleic acid sequences Gene comprising all or a portion of at least one gene from the group of the genes, and to the gene product of the gene.

[0014] The scope of the invention is a vector comprising one or more of the above genes, and a transformed microorganism comprising one or more of the above genes, and one of the above genes comprising Use of the engineered recombinant microorganism in which controllable expression is possible to find substances that bind to the product, and the gene product of the gene in an assay to find substances that bind to the gene product Use of the gene product in assays where the assay is based on the principle of affinity selection and use of the gene product of YQGF in assays where the assay uses flavin mononucleotide or flavin adenine dinucleotide as a substrate, and protein System or assay to indicate redox status Uses the gene product of YQGF in an assay when measuring the hydrolytic cleavage of a phosphodiester bond or other ester bond, and the use of the gene product of YJEE in an assay when the assay uses ATP / GTP as a substrate. Use and use of the gene product of KDTB in an assay when the assay used uses a nucleotide derivative as a substrate and measures its phosphorylation and / or dehydrogenation, and the assay uses a nucleoside diphosphate derivative as a substrate And in the assay when measuring its conversion
Use of the gene product of GBP, or the assay uses CDP-sorbitol or other polyol substrate and measures its hydrophosphorylation or measures nucleotide transfer to sorbitol 1- or other phosphate polyol. Use of the gene product of YGBP in cases where the main step of the assay is hydrolytic cleavage of the substrate or reduction of the substrate using NAD (P) H. Use of the gene product of YCHB in assays where the steps are either phosphorylation or dehydrogenation, and use of the gene product of YHBC in assays that measure binding to DNA, and the major steps of the assay are phosphorylation. Of YGGJ in assays where it is oxidation The use of gene products, and also includes the use or the use of a portion of the gene product of the gene product for discovering or preparing antibodies or other proteins that bind to the gene product. The scope of the present invention is the use of substances that bind the gene product for preparing medicaments, and the use of substances that bind the gene product for preparing medicaments having antimicrobial activity, and the use of these gene products. It also includes the use of the gene product to discover substances that bind, and the preparation and use of antisense constructs in the opposite orientation to the genes described above. Methods for purification of the gene products using antibodies are also within the scope of the invention.

[0015] Substances that bind to the above gene products and are discovered using the methods described in detail above, and drugs prepared from these substances, are useful in the treatment of infectious diseases caused by pathogens in humans and animals. Can be used for

This is preferably the case of the family Bacillaceae, the family Bacteroides.
idaceae), Chlamydiales, Chlamydiales, Enterobacteriaceae, Micrococcaceae, Micrococcaceae, Mycobacteriaceae, Neisseriaceae, Pasteurellaceae, Pseudomonaceae, Pseudomonadaceae Rickettsiaceae (Rickettsiaceae), Spirulinaceae (Spirill
aceae), spirochaetaceae (Spirochaetaceae), Streptococcus family (Streptoco
ccaceae) and diseases caused by bacterial pathogens selected from the family Vibrionaceae.

[0017] The treatment is particularly preferably carried out with Porphyromonas gingivalis (Prophyromonas
gingivalis), Chlamydia trachomatis, Escherichia coli, Yersinia pestis, Staphylococcus aureus, M. tuberculosis (M
ycobacterium tuberculosis), Neisseria gonnorhoeae, Neisseria meningitidis (Neisseria
eria mengitidis), pertussis (Bordetella pertussis), Haemophilus influenzae (Haemo
philus influenza), Pseudomonas aeruginosa, typhus ricketsia
(Rickettsia prowazekii), Campylobacter jej
uni), Helicobacter pylori, Borrelia burgdorf
eri), Treponema pallidum, Enterococcus faecal
is), caries pathogen (Streptococcus mutans), pneumococcus (Streptococcus pneumonia)
e), a disease caused by one or more bacterial pathogens selected from the genera of Streptococcus pyogenes, Vibrio chloerae, and Bacillus subtilis.

The substances which bind to the above gene products and are found using the methods described in detail above, and the medicaments prepared from these substances, are useful for the treatment of diseases in humans and animals, in particular Infections affecting the blood, cardiovascular system, central nervous system and its appendages, bone, bone marrow, muscle and fascia, teeth, joints and its appendages and lumens, visceral, these lumens, intima and Adventitia and its appendages, gastrointestinal tract and its lumen, intima and adventitia and its appendages, skin, skin appendages and soft tissues and its appendages, genitourinary system and its appendages, local and / or systemic inflammation And / or bacteremia and sepsis due to systemic disease, trauma, polytrauma, and / or surgical intervention, other diseases such as blood disease, virus disease, wasting It can be used for the treatment of opportunistic infections and sepsis and / or antineoplastic diseases and / or immunosuppression that are also associated with sexual diseases.

By way of example, Porphyromonas spp. In the area of the throat, nose, ears, head and neck, in the wound area in the mouth after surgical and / or dental treatment, and / or after human and animal bites. Diseases that occur after infection with gingivalis, genitourinary organs including the urinary tract, E. coli in the areas of the bladder, kidneys, renal pelvis, and sepsis after urinary tract sepsis and surgical intervention and other trauma, airway and nasopharyngeal cavity, lung Staphylococcus aureus as a pathogen of inflammatory diseases of the bones, skin, skin appendages and soft tissues, heart and heart valves and toxic diseases caused by this pathogen, such as hemolysis of blood, epidermal blisters of the skin, enteritis of the digestive tract, toxic shock syndrome Mycobacterium tuberculosis, gonococci and pathology of upcoming gonorrhea as tuberculosis, tuberculosis and / or tuberculosis-like diseases of the lungs, skin, and other organs and organ systems Conditions, meningococci as pathogens of acute purulent meningitis, B. pertussis as pathogens of pertussis, N. meningitidis as pathogens of meningitis, B. pertussis, meningitis, epiglottis Inflammation, osteomyelitis, pneumonia, sepsis, laryngotracheitis, pharyngitis, sinusitis, otitis media, septic arthritis, acute endocarditis, influenzae as a pathogen of cellulitis, Pseudomonas aeruginosa and upcoming wounds, urine Reproductive tract, endocarditis, inflammation of the trachea and gastrointestinal tract, typhus ricketsia and upcoming typhoid, trachoma chlamydia and upcoming conjunctivitis of the eyes, granulosa trachomatis, pestis and upcoming plague, Campylobacter jejuni and upcoming Chronic inflammatory diseases of the gastrointestinal tract such as enteritis, colitis, proctitis, and peptic ulcers, H. pylori and the gastrointestinal tract that arises Inflammatory diseases such as gastritis and duodenal and septic ulcers, Lyme disease Borrelia and Lyme disease and erythema migrans, Treponema pallidum and the pathological condition of syphilis that will occur, the enterococcus and the urinary tract that will arise, the urogenital system, Endocardial diseases and wound infections, caries pathogens and upcoming endocarditis and / or heart valve inflammation, pneumococci and upcoming upper and lower tracheal diseases such as pneumonia, otitis media, sinusitis, and meninges flame,
Peritonitis, and diseases of the endocardium and pericardium and caries, streptococcus pyogenes and the inflammatory diseases of the upper trachea to come, such as streptococcal throatitis, scarlet fever, otitis media, and cutaneous pyoderma and erysipelas and sepsis, Vibrio cholerae and their upcoming pathological conditions, Bacillus subtilis and upcoming opportunistic infections, secondary infections associated with multiple dispersions and other bacterial infections, such as otitis, mastoiditis, urinary tract infections, bacteremia It is also possible to treat pathological conditions of the disease, meningitis, endocarditis. Bioinformatic analysis of various bacterial genomes The amino acid sequence of the 4289 protein of Escherichia coli K12MGG1655 having Genebank / EMBL entry U00096 was compared with the Bacillus subtilis of a Gram-positive model organism (Genebank / EMBL accession number AL009126).
) And the gram-negative pathogen Haemophilus influenzae (Genebank / EMBL accession number L42023) and all other public databases (Genpept, Swiss-Pr)
ot) was compared to the sequence of the complete protein set.

The comparative analysis was performed using the FASTA program (FASTA version 2; Pearson and
Lipman 1988).

The proteins of Haemophilus influenzae and Bacillus subtilis are remarkable [expected value E (N) <1
0 ^-4 ] All E. coli proteins with sequence homology were selected.

Sequences exhibiting expected homology to yeast proteins (HW Mewes et al., Nature supp. Vol. 387: 9, 1997) with expected values E (N) <10 ^-4 , and others Of known functions or proteins or all proteins with homology were excluded.

Proteins from E. coli in which the homologous protein sequence is present in the E. coli species itself [expected value E (N) <10 ^-4 ] were also excluded.

Furthermore, a sequence having one or more predicted transmembrane regions [program “Peptidestruct
ure ", Wisconsin Sequence Analysis Package (Vers. 9, Genetics Computer Gr
oup, Madison WI, USA] were excluded. Results of biological information analysis of various bacterial genomes From the possible target sequences obtained by this method, 27 protein sequences were selected. KDTB, SMPB, YBAB, YBAD, YBAK, YBAX, YBEA, Y
BEY, YCHB, YFGB, YGAG, YGBB, YGBP, YGGJ, YG
GV, YHBC, YHBJ, YHBY, YHIN, YIBK, YIDA, YIG
Z, YJEE, YJEQ, YQGF, YRAL, YRFI “Knockout” construction and essentiality testing using temperature sensitive plasmids Materials and Methods Bacterial strains and plasmids

[0025]

[Table 1]

[0026]

[Table 2]

Nutrient medium used LB (Luria-Bertani) medium 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl in ₁ l of H ₂ O. M9 minimal medium 6 g of Na ₂ HPO ₄ , ₃ g of KH ₂ PO ₄ , 0.5 g of NaCl in ₁ l of H ₂ O
, 1 g of NH ₄ Cl.

[0028]

[Table 3]

Molecular Biology Techniques Basic Molecular Biology Techniques Basic molecular biology techniques such as plasmid isolation, cleavage with restriction endonucleases, denaturation of DNA (dephosphorylation, filling reaction, ligation), Preparation and transformation are performed using conventional protocols known to those skilled in the art, for example, J. Am. Sambrook et al., Molecular Cloning (J. Sambrook et al., Molecular Cloning) and Ausbel et al., "Recent Protocols in Molecular Biology" (Ausbel et al., Curr.
ent Protocols in Molecular Biology). Polymerase Chain Reaction (PCR) All PCR primers used had a calculated melting temperature between 58 ° C and 62 ° C.

The following DNA polymerases were used for cloning purposes: Pwo and T
aq DNA polymerase (Boeringer, Mannheim, Germany), Akku-Taq
DNA polymerase (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) and Pfu DNA polymerase (Stratagene, Heidelberg, Germany).

[0031] The gene and its adjacent region were ligated in a reaction volume of 25-100 μl in
Amplified from 100 ng of chromosomal DNA from OF. The primer concentration was 1 μM in each case and the dNTP concentration was 250 μM in each case. A 25-30 reaction cycle was performed at 94 ° C. for 30 to 60 seconds, 48 to 55 ° C. for 30 to 60 seconds, and 72 ° C. for 1 to 3 minutes, and finally a step of 72 ° C. for 5 minutes was performed. Protocols: a Guide to Method and Application, Ed.M. Inn
is et al., Academic Press (1990)]. PCR construction The amplified 5'- and 3'-proximal gene regions were fused by mixing 1 μl of the two previously described PCR mixtures as templates for a second PCR, in each case using primers located distally. did. This PCR was performed under the above conditions. Diagnostic colony PCR Diagnostic colony PCR is performed using Taq DNA polymerase (Boeringer) and T
This was performed using a mixture of hermosequenase (Amersham).

In each case one colony was resuspended in 50 μl of H ₂ O. This 5μ
1 was used for PCR, which was performed in a volume of 50 μl under the conditions described above. Overexpression of essential proteins in E. coli Various vector systems for overexpression of essential proteins in E. coli: pBAD /
His (Invitrogen) and pQE vector (Quiagen) were used.

The pBAD / His plasmid (pUC vector derivative) constructed for controlled dose-dependent expression and purification of the recombinant protein in E. coli was used.

The pBAD / His vector system contains a sequence encoding six histidines containing a linker for fusion to the N- or C-terminus of the desired recombinant protein. Maximal expression of the soluble recombinant protein was achieved by specific activation of the araBAD promoter using arabinose. Protein was purified by affinity chromatography on nickel-NTA agarose.

For purification of the fusion protein, the cells containing the pBAD construct were
It is necessary to culture to an optical density of 0.6 (OD _600nm ). Specific protein production is then started with arabinose [0.002-0.2% w / v] and incubation is continued for 2-3 hours. After completion of the incubation, the cells are harvested by centrifugation and the cell pellet is frozen. Cell disruption and purification procedures are described in the handbook "The QIAexpressionist, A handbook for high-level expression and
purification of 6xHis-tagged proteins, Quiagen, Hilden, Germany 1997. The purification steps were examined using polyacrylamide gels (and subsequent staining with Coomassie blue).

The pQE vector system contains a sequence encoding six histidines, including a linker for fusion to the N- or C-terminus of the desired recombinant protein. Maximal expression of the soluble recombinant protein is due to specific activation of the phage T5 promoter system and IPTG
This was achieved by induction with The protein was purified by affinity chromatography on nickel-NTA agarose.

Cell culture and purification of fusion proteins from cells containing the pQE construct is described in the handbook “The QIAexpressionist, A handbook for high-level expression and
purification of 6xHis-tagged proteins, Quiagen, Hilden, Germany 1997 ". Cloning strategy Knockout plasmid The essentiality of the open reading frame of the selected protein is pSC10
The test was performed using a knockout plasmid having a single temperature-sensitive origin of replication.
This means that these plasmids are capable of autonomous replication in E. coli at 30 ° C, but not at 42-44 ° C. D that is homologous to the E. coli chromosome
Once the NA region has been cloned into this plasmid, homologous recombination at 42-44 ° C allows homologous recombination of these plasmids into the locus to be studied in the E. coli chromosome. Subsequent excision of the plasmid at 30 ° C. can lead to the replacement of the wild-type sequence by the sequence originally cloned into the plasmid.

The contiguous region of the gene under study in the region of 400-800 bp in each case was cloned into pMAK705 or pKO3. Instead of the gene to be studied, a cassette resistant to either tetracycline or kanamycin was integrated into the respective plasmid. For the cloning strategy, two routes were followed. Integration of the resistance cassette into the gene The relevant gene containing the adjacent region (400-800 bp) was inserted into the chromosomal E. coli W311.
Amplified by PCR from 0F DNA and cloning vector (pCR-
Blunt). Due to restriction digestion and subsequent ligation, the majority of the coding region of the gene of interest was replaced by an antibiotic action cassette or the gene was interrupted by this cassette (tetracycline or kanamycin resistance cassette). Then, using the sequence that was initially closest to the gene,
The cassette was cloned into the knockout plasmid pMAK705 or pKO3. Cloning of in-frame deletions with integration of resistance cassettes 500-800 bp long DN flanking the 5 'and 3' ends of specific genes
The A region was amplified in two separate PCR reactions from chromosomal E. coli W3110F DNA. The primers required for this are 21 bp, with the ends of the PCR product covering the 5 'and 3' ends of the gene additionally having one or two restriction cleavage sites.
Selected to have tags. Thus, the two PCR products contain a 21 bp extension complementary in length to each other. These were used to construct the two products in a second PCR step with the help of the outer primers.

Via a restriction cleavage site incorporated at the 5 ′ end of the outer primer,
Cloning of the fusion PCR product into pKO3 was possible. The fusion PCR product contains a contiguous region (400-800 bp each) and exactly 18 nucleotides at the 5 'end and 36 nucleotides at the 3' end of the gene to be studied, while the 5 'and 3' ends of the gene are interrupted by a 21 bp tag. Was done. So-called gene in-frame deletions were generated in this way. The kanamycin resistance cassette was incorporated into a 21 bp tag via a restriction cleavage site. Temperature shift experiments (integration and excision) For integration of the pMAK705 and pKO3 constructs, E. coli JM101 and E. coli MC1061 were transformed with the plasmids according to standard protocols, respectively, and at 43-44 ° C. with chloramphenicol. For LB agar plates.

To allow the plasmid to be excised, the 43/44 ° C. clone (co-integration) was then transferred to 30 ° C. LB. The route used for this differed according to the vector system (pMAK705 or pKO3). Temperature shift using pMAK705 The 43/44 ° C clone was transferred into 100 ml of LB-chloramphenicol-liquid medium and incubated at 30 ° C for 16 hours. The plasmid was isolated from the last 1 ml of the cultivated medium after twice in fresh medium, in each case 16 hours of renewed incubation at 30 ° C. The prepared plasmid was tested by restriction digestion for the presence of a wild-type copy of the gene in the plasmid. Cultures in which positive plasmids were found were isolated on LB chloramphenicol plates at 30 ° C.

A number of colonies were picked and cultured for plasmid preparation. The prepared plasmid was tested by restriction digestion for the presence of a wild-type copy of the gene in the plasmid. The presence of the antibiotic resistance cassette in the appropriate chromosomal locus was confirmed by PCR on positive clones. Chromosomal knockouts of the tested genes were produced in this way in E. coli with simultaneous supplementation with a functional gene copy on plasmid pMAK705. Temperature Shift Using pKO3 Co-incorporation of a pKO3 derivative in E. coli MC1061 was performed using the described method.
[AJ Link et al., J. Bacteriol. 179: 6228-6237 (1997)]
The KO3 plasmid was excised simultaneously. This is Luria-Bertani (LB)
Involve three 43/44 ° C. colonies diluted in medium and LB-5
Plated out on a% (w / v) sucrose-kanamycin plate and incubated overnight. 50-100 clones resulting from each integrated clone were transferred again to LB-sucrose-kanamycin and KB-chloramphenicol plates (replica plates). Chloramphenicol sensitive but kanamycin resistant colonies were analyzed by PCR using primers that eventually brought the gene into proximity. Based on the size of the PCR product, it was possible to prove whether a particular clone had a kanamycin resistance cassette instead of the wild type gene. In this way, chromosomal knockouts for the gene studied were produced in E. coli without functional complementation. Demonstration of essentiality The essentiality of the gene was proven in various ways. Demonstration of Essentiality by Means of Temperature Shift Using the pMAK705 Vector System An aliquot sample was taken from 5 ml of overnight LB-chloramphenicol medium of each supplemented knockout strain obtained at the end of the pMAK705 experiment. These aliquot samples were adjusted to an even OD _{600 nm of} 0.1. These dilutions were plated out on LB plates with or without chloramphenicol in each case and incubated at 30 ° C. and 43/44 ° C. overnight.

The clones formed a significantly lower number of colonies on LB-chloramphenicol plates at 43/44 ° C. compared to the number of colonies on LB +/− chloramphenicol at 30 ° C. This indicates that most of the clones were pMA at 43/44 ° C.
This is because the K705 construct is lost and thus cannot exhibit resistance to chloramphenicol. The number of colonies is reduced by at least a factor of 10 ² . Under these conditions, the clone is considered non-viable. As the number of colonies on LB without using chloramphenicol in standard 43/44 ° C. to chromosomes knockout in essential genes, LB on, 30 ° C. of at least 10 ² times greater than (chloramphenicol or without) less and Clones that formed no more than 10 times more colonies than on LB plates when chloramphenicol was used at 43/44 ° C.
According to our criteria, there is a chromosomal knockout within the essential gene. Clones cannot survive if they lose the plasmid at 43/44 ° C. on LB without chloramphenicol (measured by “number of viable colonies”).

In contrast, a similar number (within a factor of ± 10) of colonies on LB at 30 ° C. (with or without chloramphenicol) on LB without chloramphenicol at 43/44 ° C. All clones that form have chromosomal knockouts in non-essential genes. Deletion of a plasmid with a complementary functional copy from E. coli cells on LB without chloramphenicol at 43/44 ° C. does not lead to reduced viability of the clone (see “Number of viable colonies”). "). Demonstration of Essentiality by Generating Knockout Limited to the Presence of Complementary Gene Copy (pKO3 System) Clones generated using the pKO3 system and sensitive to chloramphenicol and containing a kanamycin resistance cassette instead of the wild-type gene Had a chromosomal knockout in a non-essential gene. Plasmid code complementarity was no longer present.

If all colonies remained chloramphenicol resistant, intrachromosomal knockouts could not be generated simultaneously with loss of plasmid.

To further examine the essentiality of these genes, we attempted to generate knockouts of the corresponding genes in chromosomes with functional complementation at the same time.

An E. coli strain MG1 that differs from MC1061 by having a complete araBAD locus
655 was used for this purpose. First, the araBAD gene of E. coli MG1655 was replaced with a functional copy of the particular gene to be studied. Plasmid p
AL761 was used for this purpose. After transformation of the pAL761 derivative and subsequent integration and excision steps, replacement of the araBAD gene with a functional copy of the gene is impossible to grow on M9 minimal medium with 0.2% (w / v) arabinose PCR was performed on all clones.

Subsequently, positive clones were transformed with the pKO3 derivative of the appropriate gene. Subsequent simultaneous integration and excision were performed as described above, except that the integration and excision steps were performed in the presence of 0.2% (w / v) arabinose.

PCR was used to diagnose the phenotype of a deleted chromosomal locus using a kanamycin cassette between chloramphenicol-sensitive colonies. If the resulting clones show a dependence on arabinose for growth (ie, no single colony formation on LB agar plates without arabinose as opposed to growth on LB agar plates with arabinose) Are considered essential. Demonstration of essentiality by means of in-frame deletion (pKO3 system) In some cases, it is not possible to generate a gene interruption with the appropriate interruption or deletion copy using an antibiotic resistance cassette even in the presence of complementation Met. The reason may be a polar effect caused by the cassette leading to the inhibited expression of essential genes located downstream from the gene under study. For this reason, the in-frame deletion plasmid of the corresponding gene described above was used without kanamycin cassette in the following experiments.

An in-frame deletion clone was formed in E. coli MC1061. Incorporation, excision and detachment of pKO3, and subsequent replica plating were performed as described above, with modifications, no longer using kanamycin selection. Observed statistically, 50% of the chloramphenicol-sensitive colonies must carry the gene deletion. If none of the 50 clones tested by PCR contained the deletion, another test was performed to confirm the essentiality of the gene being tested.

For this purpose, gene deletions within the chromosome were generated simultaneously with complementation as described above, indicating a dependence on arabinose for growth. Demonstration of Essentiality for Complementary Knockout Showing No Arabinose-Dependent Growth An attempt was made to replace a functional copy of the gene in the araBAD locus with the kanamycin cassette using a complementary knockout strain that did not show arabinose-dependent growth. For this purpose, the corresponding strain was transformed with pAL767, a pKO3 derivative with the kanamycin resistance cassette and the araBAD flanking region.

After integration, excision and removal of pAL767, the resulting clone (clone 2
PAL767 was still shown to be integrated within the chromosome when 100% of the (100 tested) were resistant to both kanamycin and chloramphenicol. Therefore, it is not possible to delete an invariant copy of the gene from the araBAD locus. This finding underscores the essentiality of related genes. In any kanamycin-resistant and chloramphenicol-resistant clones, the gene is non-essential if PCR allows successful replacement of the functional gene copy by the kanamycin resistance cassette. Results of proof of essentiality

[0052]

[Table 4]

[0053] Thus, of the 27 genes tested, 8 were identified as essential. The protein sequences derived from these genes were used to perform a homology search comparing to the nucleotide sequence of the fully and incompletely decoded bacterial genome using the TBLASTN program.

Using this method, all significantly homologous protein sequences can be distinguished from those that happen to be coincident through the selection of the threshold, ie the expected value P (N). Using stringent thresholds (expected value P (N) <10 ^-4 ), it was possible to find gene products with significant sequence homology in many disease-associated bacteria.

Homologs of the bacterial species with the greatest similarity selected from those found in this way will have the maximum number of identical and / or conservatively altered amino acids compared to the corresponding E. coli protein. (Table 5). Such proteins are
They are called orthologous proteins (gene products) because the skilled person knows that they perform the same function as E. coli.

The orthologous proteins listed in Table 5 have at least 21% identical or 42% identical or conservatively altered amino acids, ie, 21% identical / 42% similarity. Table 5: Distribution of orthologous proteins of the identified essential gene products of E. coli in pathogenic bacteria. TBLASTN results using E. coli protein sequences can be obtained from fully or incompletely decoded bacterial genomes, for example, as deposited in a publicly accessible genome database of the National Center for Biotechnology Information (NCBI). Was found to be essential compared to the nucleotide sequence of The criterion for significant homology is an expected value P (N) <10 ^-4 .
Orthologous identity and similarity by comparison with the corresponding E. coli proteins were described as% identity /% similarity. Completely sequenced genomes were marked with (*). Lack of homology in incompletely decoded genomes is marked with (?).

[0057]

[Table 5]

Based on the sequence motifs within the amino acid sequences of the eight essential gene products and / or their spatial structure predicted from the amino acid sequences, the following functions can be predicted.

YJEE probably stands for ATP- or GTP-binding protein. This protein will have ATPase / GTPase or ATP / GTP synthase activity.

[0060] KDTB is probably a saccharide- and / or nucleoside and / or nucleoside derivative binding protein with phosphorylation or dehydrogenation activity.

YQGF probably represents a flavin derivative-binding protein. It can be a flavomononucleotide (FMN) or a flavin adenine dinucleotide (FA
D) and will transport electrons to other substrates / proteins. YQG
F represents a hydrolysis, possibly breaking a phosphodiester bond or other ester bond.

[0062] YHBC is a putative DNA-binding protein with regulatory / regulatory functions. Y
GGJ is a phosphotransferase. YGBP is probably a nucleoside diphosphate derivative phosphorylase or pyrophosphorylase. YGBP probably cleaves CDP-sorbitol or other polyol nucleoside diphosphates with pyrophosphate or cleaves nucleotide CMP with sorbitol 1
-Will transfer to phosphoric acid or other phosphoric acid polyols. YCHB is a putative kinase or oxidoreductase. YGBB is a putative hydrolase or NAD (P) H-dependent reductase.

[0063] For Example Example 1 Construction integrative plasmid of integration vector carrying the gene sequences for yjeE for YJEE pMAK705 (5.5kb), it is possible to incorporate E. coli JM101 into the chromosome by homologous recombination . In addition to the temperature-sensitive origin of replication from pSC101, this plasmid contains the chloramphenicol resistance gene for selection of the plasmid in the host organism.

The coding region for yjeE and the upstream 420 base pairs (bp) and 413 bp downstream of the coding region for yjeE were amplified using PCR (Perkin Elmer 480 Cycler). E. coli W
100 ng of chromosomal DNA from the 3110F was ligated with Pfu DNA polymerase in a 100 μl reaction volume at 94 ° C. for 45 seconds, 48 ° C. for 45 seconds and 72 ° C.
Amplification was performed with 25 cycles of 3 minutes at, and a final incubation of 5 minutes at 72 ° C. The amplification primer used was primer A (5'-CCTG G
CT GGC AAT CAA TCC CGA TA-3 ') and primer B (5'-AGG CGG TGG CGG CAC ATCGGC GTT
-3 '). The amplification product (1482 bp) was converted into Qiaex (Qiagen, Hi
lden, Germany) and then subcloned into the vector pCR-Blunt using the "pCR-Blunt Cloning Kit" (Invitrogen Corporation, Carlsbad, CA). The tetracycline resistance cassette was cloned into the BpmI restriction cleavage site using pCR-blunt / yjee. The tetracycline resistance cassette was isolated from plasmid pBR322 as a 1400 bp EcoRI / AvaI fragment, and blunt ends were made using Klenow polymerase.

The plasmid pCR-blunt / yjeE :: tet produced in this way is
Digestion was performed using the nI / XbaI restriction enzyme. The vector pMAK705, in which a DNA band of size 2.8 kb was isolated and pretreated with KpnI / XbaI.
(Host organism E. coli JM101).

The resulting construct (pMAK705yjee) was used for integration experiments. Preparation and analysis of yjeE knockout Cells carrying the plasmid pMAK705yje were cultured overnight in LB medium at 30 ° C. and plated out the following day on LB test plates containing chloramphenicol and 16 ° C. at 43 ° C. Incubated for hours. Escherichia coli JM
Integration of the plasmid into the 101 chromosome was identified by selection of chloramphenicol-resistant colonies capable of growing at 43 ° C. After identification of integration (43 ° C.)
Cells were incubated in 100 ml of LB medium at 30 ° C. for 16 hours. Cells were transferred twice to fresh medium and incubated for an additional 16 hours at 30 ° C.

After this series of incubations, rapid plasmid preparation was performed using Qiapr (Qiapr
ep) (Qiagen, Hilden, Germany) using 1 ml of each of the last incubation mixtures. The prepared plasmid was checked for the presence of a wild-type copy of the gene in the plasmid by restriction digestion. Cells from a mixture containing positive clones (yjeE gene from wild-type chromosome on plasmid) were isolated at 30 ° C. on LB plates containing chloramphenicol.

The plasmid DNA of the isolated clone was tested again for the invariant yjeE gene from the chromosome and positive clones were identified. The integration of the yjeE copy, inactivated by insertion of the tetracycline cassette in the chromosome, was tested by PCR.

The clones tested in this way were then cloned into the target yjeE for E. coli survival.
Was available due to its essentiality. Cells were incubated at 30 ° C and 43 ° C on LB test plates with and without chloramphenicol.

[0070]

[Table 6]

As can be seen from the table, the cells can grow at 30 ° C. with or without chloramphenicol. At 43 ° C., growth declines rapidly, ie, with and without chloramphenicol at a factor of 10 ⁴ . This clearly indicates that the presence of the plasmid with the yjeE complementary sequence is necessary for the growth of E. coli cells at 43 ° C. without chloramphenicol, and that yjeE is essential for the survival of these cells. Is shown.

Example: Demonstration of essentiality for ygbP In-frame deletion with and without integration of the resistance cassette Cloning of the DNA region flanking the 5 'and 3' ends of the ygbP gene
From 100 ng of 3110F DNA, two separate PCs in a reaction volume of 25 μl
Amplified in R reaction. The primer concentration was 1 μM in each case and the dNTP concentration was 250 μM in both cases. 52 seconds at 94 ° C. for 30 seconds
A 25 reaction cycle of 30 seconds at 72 ° C and 1-2 minutes at 72 ° C, and a final 5 minute step was performed at 72 ° C. Primer YGBP1A (5′-cgcggatcc
CCCACATGGTCACTGCCTGG-3 ') and YGBP1B (5'-
cccatccactaactgcagctCAAATGAGTGGTTTGC
The PCR product prepared using CATGTT-3 ′) was the same as the 1 ′ at the 5 ′ end of ygbP.
8 bp and 776 bp of the chromosomal region upstream from ygbP. Primer YGBP2A (5′-agctgcagttttaggtgatggggTAC
CTCACCCCGAACCCATCC-3 ') and YGBP2B (5'-cgc
The PCR product prepared using ggatccACCTGGCAGCCTTCCAGTTG-3 ′) comprises 36 bp at the 3 ′ end of ygbP and 807 bp of the chromosomal region downstream from ygbP.

The internal primers YGBP1B and YGBP2A additionally contained a complementary 21-member tag at their 5 ′ end (lowercase in the above sequence). These were used to construct the two PCR products in a second PCR step using the externally located primers YGBP1A and YGBP2B. For this purpose,
1 μl of each of the two above-mentioned PCR mixtures was mixed with YGBP1A and YGBP2B.
Was mixed as a template for a second PCR. This PCR was performed under the above conditions. PCR products of the expected size were prepared using QIAEX (QIAGEN, Hilden, Ger.
Many) were purified by agarose gel electrophoresis.

The fusion PCR product was ligated into BamHI-cut and dephosphorylated pKO3 with Ba integrated at the 5 ′ end of the externally located primers YGBP1A and YGBP2B.
Cloning was possible via the mHI cleavage site (lowercase letters in the above sequence). Plasmid pAL759 containing an in-frame deletion of ygbP was generated in this manner. The kanamycin resistance cassette obtained by digesting PstI from pUC4K was
pAL7 via the PstI cleavage site in the bp tag (5'-ctgcag-3 ')
Cloned in 59. The resulting construct is called pAL759a. Temperature shift using pAL759a E. coli MC1061 was transformed with plasmid pAL759a and incubated at 44 ° C. on LB agar plates with chloramphenicol.

The three 44 ° C. clones (co-integrated) thus obtained were diluted in LB medium, and LB-5
Plated out on a% (w / v) sucrose-kanamycin plate and incubated overnight. One hundred sucrose-kanamycin clones obtained for each integrated clone were again transferred onto LB-sucrose kanamycin and LB-chloramphenicol plates (replica plating) and incubated overnight at 30 ° C. One of kanamycin resistant clones
00% were also chloramphenicol resistant. Therefore, it was not possible to successfully replace ygbP with the kanamycin cassette. Temperature shift using pAL759 E. coli MC1061 was transformed with plasmid pAL759 and incubated at 44 ° C. on LB agar plates with chloramphenicol.

Three of the resulting 44 ° C. clones (simultaneous integration) were diluted in LB medium and LB-5
% (W / v) sucrose-plated out and incubated overnight.

One hundred sucrose clones obtained for each integrated clone were again transferred onto LP-sucrose and LB-chloramphenicol plates (replica plating) and incubated at 30 ° C. overnight. In each case, 50 chloramphenicol-sensitive colonies were used as primers for Y
It was finally analyzed by PCR using GBP1A and YGBP2B. PCR
Based on the size of the product, it could be shown that none of the clones tested had a ygbP deletion (PCR product with ygbP deletion: 1637b
p). In all colonies, amplification of the wild-type locus of ygbP was possible (
2294 bp). Therefore, it was not possible to successfully carry out the replacement of ygbP by deletion. Generation of ygbP Deletion in the Presence of Chromosomal Supplementation In the next step, we attempted to generate a deletion of ygbP in the chromosome with a co-functional complement. E. coli strain MG1 which differs from MC1061 in having the complete araBAD locus
655 was used for this purpose. First, the araBAD gene of E. coli MG1655 was replaced with a functional copy of ygbP. Plasmid pAL763 was used for this purpose. Plasmid pAL763 contains its ribosome binding site (18 bp upstream from the 5 'end) and 8 bp downstream from the 3' end of ygbP;
From the chromosomal E. coli W3110F DNA, the primer YGBPR (5'-atcg
ctagcATCAGCCCGGGAATTAACATG-3 ′) and YGB
PT (5'-atcctcgagAATTCGCATTATGTTATTCTCC
TG-3 ') was used to amplify and produce the complete YGBP gene. The primers contain restriction cleavage sites for XhoI (YGBPT) and NheI (YGBPR) at their 5 ′ ends. The PCR product is transferred to the vector pAL761 via the cleavage site.
Cloned into After transformation of E. coli MG1655 with pAL763 and subsequent integration and excision steps (see above), replacement of the araBAD gene by a functional copy of ygbP is 0.2% by PCR on the clone.
(W / v) demonstrated by the inability to grow on M9 minimal medium with arabinose. Diagnostic PCR was performed using primers YGBPR (see above) and ARA
2B (atcgcggccgcAAAGCCGTGCTCGCGC). Primer ARA2B bound in the 3 'region 865 bp downstream from the araD gene, and along with primer YGBPR, a 1655 bp product was produced using a positive clone. The positive clones were then replaced with the in-frame deletion plasmid pA.
Transformation was performed using L759. Subsequent simultaneous integration and excision was performed as described above, except that the integration and excision steps were performed in the presence of 0.2% (w / v) arabinose. The genotype of the deleted chromosomal locus was diagnosed by PCR between chloramphenicol-sensitive colonies. araBA
YgbP-deleted clones containing an arabinose-regulatable ygbP copy in the D locus showed arabinose dependence on growth. It was not possible for single colonies to form on LB agar plates without arabinose, and single colonies were obtained on LB agar plates with arabinose. Therefore, y
The gbP gene is essential.

Example 3 Preparation and Purification of Recombinant YJEE Protein Expression and purification of YJEE protein was performed using the pBAD / Hisa vector system (Inv
Itrogen) was used. The sequence of yjeE was converted from 100 ng of chromosomal DNA from E. coli W3110F using pfu DNA polymerase to 100 reaction mixture.
Amplification was performed in 25 μl of 25 cycles of 94 ° C. for 1 minute, 52 ° C. for 1 minute, and 72 ° C. for 3 minutes in μl. The primer used was YJEE-START (5'-TGA AG
A TCT AAT CGA GTA ATT CCG CTC CCT GA
T-3 ') and YJEE-STOP (5'-TCA GAA TTC TTA
ACC GGC TAA ACG CGC CAG CAA CAA TC-
3 '). The 5 'end of YJEE-START contains the sequence for the restriction enzyme BglII. The 5 'end of YJEE-STOP contains the sequence for EcoRI. The PCR mixture was fractionated in an agarose gel and the 450 bp band was excised. Bands were then isolated using Qiaex (Qiagen, Hilden, Germany) and incubated with restriction enzymes BglII and EcoRI at 37 ° C. for 3 hours.

The DNA fragment treated in this manner was cloned into the vector pBAD / HisB treated with BglII / EcoRI [host organism: E. coli strain TO
P10 (Invitrogen)]. Successful cloning was demonstrated by plasmid isolation of the resulting transformants and restriction with EcoRI. Plasmid pBAD /
A clone using HisB-YjeE30 was used for expression and purification.

The cultivation and purification of YjeE from E. coli TOP10 using plasmid pBAD / HisB-YjeE30 was performed according to a protocol for purification of histidine-tagged protein on Ni-NTA agarose (The QIAexpressioni).
st, A Handbook for high-level expression and purification of 6xHis-tagge
d proteins, Quiagen, Hilden, Germany, 1997). Induction of YjeE expression was initiated at an optical density of culture OD ₆₀₀ = 0.5 with arabinose 0.02% (w / v). Various concentrations of imidazole (50, 100, 150 and 200
mM) was used to elute the bound protein. Maximum protein yield is 1
Obtained at 50 mM. Purification was checked by polyacrylamide gel fractionation and subsequent Coomassie staining. 10 mg of protein could be isolated from 1 L of medium (LB medium) with a purity of 90% or more.

Example 4 Screening a Library of Low Molecular Weight Compounds with Targets of Unknown Function Based on Affinity Selection and Mass Spectrometry Target proteins whose function is unknown but essential for bacterial survival (eg, Y
To find inhibitors against JEE), it is possible to use screening methods that test substance libraries for their affinity for proteins. One possible method for screening is affinity selection from a mixture of substances and subsequent detection of the ligand in mass spectrometry. It is necessary to use this defined substance mixture in which the individual substances can be identified by mass detection. Therefore, substance mixtures prepared by combinatorial synthesis are particularly suitable for this screening method.

Liquid chromatography electrospray mass spectrometry was used as the screening device, replacing the HPLC column with ultrafiltration. The ultrafiltration chamber consists of a filtration device in which the filter is replaced by an ultrafiltration membrane (excluded molecular weight 10 kDa). Mass spectra were obtained by mass spectrometry (Hewett-Packard, Palo Alto, C
Prepare from A using "5989 MS Engine Quadrupole Mass Spectrometer"). This was operated as described (von Bremen et al., Pulsed Ultrafiltrati
on Mass Spectrometry.A New Method for Screening Combinatorial: Anal.Ch
em. 69 [11], 2159-2164, 1997).

An equimolar mixture of 96 compounds (about 200 μM each) in 10 μl of DMSO was added to 90 μl of 2 μM protein (eg, YJEE) present in 20 mM ammonium acetate buffer. The protein / substance mixture was incubated for at least 15 minutes at room temperature. The injection of the mixture into the ultrafiltration chamber takes 50 l
This was done after a wash using a water flow of 8 minutes per minute. The mobile phase was then changed to methanol / water (50:50 v / v) to degrade possible ligand-protein complexes.
) And filter out the ligand. Ligands are detected by electrospray mass spectrometry. Substances identified from the 96 mixtures as possible ligands based on their mass peaks were again used as sole substances in affinity tests to demonstrate their affinity for the target protein (eg, YJEE).

Example 5 The substances taken up in the affinity selection are subjected to further tests, for example the MIC test.

For this purpose, the MIC value is determined by the microdilution method in BH medium.
Individual test substances were dissolved in the nutrient medium. A series of test substance concentrates are prepared in microtiter plates by serial dilution. An overnight culture of the pathogen is used for inoculation after a 1: 250 dilution in nutrient medium. Each inoculation solution 10
0 ml was added to 100 ml of nutrient medium containing the diluted active substance.

The microtiter plate is incubated at 37 ° C. and read after about 20 hours or 3-5 days. The MIC (mg / ml) indicates the lowest concentration of the active that is clearly not growing.

Another method is to determine the minimum inhibitory concentration (MIC) in a liquid dilution test. Overnight cultures of the test organism (S. aureus 133) in allogeneic sensitization test broth were diluted 1: 1000 in fetal calf serum (FCS) or allogeneic sensitization test broth and the test substance With the dilutions (1: 2 dilution steps).

The culture is incubated at 37 ° C. for 18-24 hours. The lowest concentration of an individual substance at which there is no visible bacterial growth is defined as the MIC.

[0089]

[Table 7]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/19 1/19 1/21 1/21 G01N 33/15 Z G01N 33/15 33 / 50 Z 33/50 C12N 15/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL , PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, (72) invention PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW Freiberg, Christoph Deutsche D-42115 Butzpertal Nijurase-Julase 85 (72) Inventor Spartman, Frank 02138 Cambridge Bridge Apartment No. 403, Massachusetts Avenue 1008 (72) Invention Beerland, Bernd Germany Day-42349 Butzpertal Wunderkirchen 38 (72) Inventor Labisinski, Harald Germany Day-42113 Butzpertal Katelberger Schulbeek 80 F-term (reference) 2G045 AA40 DA36 FB06 4B024 AA01 BA80 CA02 DA06 EA10 4B065 AA26Y AB01 BD25 CA44 4C084 AA07 AA17 NA14 ZB072 ZB332 ZB352

Claims (10)

[Claims] 1. YQGF, YHBC, YGGJ, YGBP, YCHB, YG
Use of these proteins in assays to discover substances that bind to proteins from the group of BB, YJEE, KDTB and their orthologous gene products. 2. Use according to claim 1, wherein the protein corresponds to the listed proteins by 95-100%. 3. A vector comprising one or more genes encoding the proteins of claims 1 and 2. 4. A transformed microorganism comprising one or more genes encoding the proteins of claims 1 and 2. 5. A compound that binds to the protein according to claim 1 or 2. 6. A compound which binds to the proteins of claims 1 and 2 and modulates their activity. 7. A medicament comprising one or more compounds according to claim 5 or 6. 8. Use of a compound according to claim 5 or 6 for the preparation of a medicament. 9. YQGF, YHBC, YGGJ, YGBP, YCHB, YG
A method for discovering a compound that modulates a protein from the group of BB, YJEE, KDTB and their orthologous gene products, comprising contacting the compound with the protein and testing their binding. 10. Having antibacterial activity and YQGF, YHBC, YGGJ,
In a method for discovering compounds that modulate proteins from the group of YGBP, YCHB, YGBB, YJEE, KDTB and their orthologous gene products, the compounds are contacted with proteins, their binding is examined, and these are tested. Then, a MIC test is performed.