WO2000061792A1 - Novel essential bacterial genes and their proteins - Google Patents

Abstract

The invention relates to nucleic acids and to protein sequences derived from said nucleic acids which possess functions that are essential to the survival of Escherichia coli. The invention also relates to vectors and host cells that have been transformed with these vectors for expressing essential proteins of the type described above and to proteins produced with these vectors. Finally, the invention also relates to the use of the proteins produced with these vectors in screening methods for identifying antibacterial substances and to the antibacterial substances found with these proteins.

Description

New essential bacterial genes and their proteins

In the past, new and innovative antibiotics were primarily developed by modifying and optimizing known classes of substances such as ß-Lactams (penicillin-cephalosporin I-V) and testing their effect on microorganisms in so-called MIC tests researched and developed.

In parallel, the search for new active principles with the help of biochemically, microbiologically well-characterized targets (points of attack) has been established in recent years. However, the selection of targets based on these methods was based on empirical procedures, which made finding new targets very complex.

Knowledge of complete genome sequences of pro- and

Eukaryotes in connection with methods of bioinformatics and molecular biology the possibility for the systematic and rational selection of antibacterial targets.

A large number of genes of unknown function were discovered by analyzing different bacterial genomes.

Unknown proteins from the gram-negative model organism Escherichia coli (FR Blattner et al, Science 277: 1453-1462, 1997) generally have names that begin with "Y": z. B. YQGF. Some proteins of unknown function have names that deviate from this system because the genes coding for them are in the vicinity of functionally characterized genes: e.g. B. KDTB (T. Clementz and CRH Raetz. J. Biol. Chem. 266: 9687-9696, 1991; T. Clementz. J. Bacteriol. 174: 7750-7756, 1992). Against the background of the discovery of anti-infectives, genes of unknown function and proteins derived from them represent new potential targets if it can be shown that they are essential for the survival of microorganisms (F. Argoni et al., Nature Biotechnology, 16: 851, 1998 ; DT Moir et al., Antimicrob. Agents Chemother. 43: 439-446, 1999; US 5821076; BJ Akerly et al,

Proc. Natl. Acad. Si. USA 95: 8927-8932, 1998).

Knockout technologies can be used to analyze the essentiality of such gene sequences for an organism. Knockout strategies are based on the exchange of intact (wild-type) gene sequence information in the chromosome of a

Bacterium against a gene sequence destroyed by insertion or deletion. From the ability to survive only with an intact complementary copy in the cell (on a plasmid or a second artificial copy in the chromosome), such a gene sequence thus qualifies the coding gene product as essential for this cell.

Knockout experiments are carried out as recombination events of homologous DNA sequences. For this, vectors are used which can controllably lose the ability to replicate the plasmid, as in the case of temperature-sensitive replicons (pKO3, pMAK 700 or pSCIOl) or vectors which cannot replicate in the desired target cell (E. coli): e.g. Gram-positive plasmids (pC194, pT181) in Gram-negative bacteria (E. coli). In addition to sequences for the temperature-sensitive replicon, integration vectors such as pMAK700 also carry markers such as antibiotic resistance (e.g. chloramphenicol), which can be used for selection on plasmid-carrying cells.

The invention relates to genes with hitherto unknown function, to show their distribution in the phylogenetically diverse bacterial realm and to select in particular those genes which have no or at most only a very distant homology in eukaryotes, and for these genes their essentiality for

To prove the viability of microorganisms. A further requirement for the genes to be found is to select, by suitable selection of the search strategy, only those whose derived proteins can be expected to be water-soluble due to their physicochemical properties to be expected, so that these can advantageously be used in cell-free assays.

From this analysis, gene sequences can be selected that would not have been selected in the conventional way as a target for antibiotic drug binding.

For this purpose, the gene products of said genes are expressed and purified within the scope of the invention. The new proteins thus obtained are used in assays for the detection of agonists and antagonists of these proteins. In addition, over- or under-expression mutants for the corresponding genes are also used in assays for the detection of agonists and antagonists.

The present invention relates to essential genes which encode the proteins from the group YQGF, YHBC, YGGJ, YGBP, YCHB, YGBB, YJEE, KDTB, and genes from other microorganisms which encode the corresponding orthologic gene products, and genes as indicated above for which can be shown that their controllable elimination leads to growth inhibition of the host organism, as well as genes from Escherichia coli as above, which encode gene products which are 95-100% identical and for which it can be shown that their controllable elimination to growth inhibition of the host organism, as well as genes as stated above from other microorganisms, which encode orthologic gene products which have 42-100% identical or conserved exchanged amino acids to the corresponding gene product from E. coli, and for which it can be shown that their controllable deactivation for growth inhibition of the host organism, as well as genes that are part of their nucleins acid sequence completely or partially contain at least one gene from the group of the genes specified above, and the

Gene products of the above genes. The scope of the invention further includes vectors which contain one or more of the abovementioned genes, and transformed microorganisms which contain one or more of the abovementioned genes, and the use of constructed, recombinant microorganisms in which one of the abovementioned genes can be regulatedly expressed to find substances that bind to said gene products and the use of the gene products of the above-mentioned genes in assays to find substances that bind to said gene products and to use said gene products in assays, said assays being based on the principle of affinity selection , as well as the use of said YQGF gene products in assays, the assay using a flavin mononucleotide or a flavin adenine dinucleotide as a substrate, and an indicator system which indicates the redox state of the protein, or the use of said YQGF gene products in assays, wherein in the assay the hydrolytic gap a phosphodiester bond or another ester bond is measured, and the use of said gene products from YJEE in assays, the assay using an ATP / GTP as substrate, and the use of said gene products of KDTB in assays, where the assay is a nucleoside derivative as substrate is used and its phosphorylation and / or dehydrogenation is measured, as well as the use of said gene products of YGBP in assays, the assay using a nucleoside diphosphate derivative as substrate and its conversion being measured, or the use of said gene products of YGBP, where the assay uses a CDP sorbitol or another polyol substrate and its pyrophosphorylyseis measured or the nucleotide transfer to sorbitol 1-phosphate or another polyol phosphate is measured, and the use of said gene products of YGBB in assays, the key step of Assays is the hydrolytic cleavage of the substrate, or the reduction of the substrate with NAD (P) H, and the use of said YCHB gene products in assays. wherein the key step of the assay is either phosphorylation or dehydrogenation, and the use of said gene products of YHBC in assays, wherein the

Binding to the DNA is measured, and the use of said gene products of YGGJ in assays, the key step of the assay being phosphorylation, and the use of said gene products or the use of parts of said gene products to find or produce antibodies or other proteins that bind to said gene products. The scope of the invention further includes the use of substances which bind said gene products

Manufacture of drugs, and the use of substances that bind said gene products for the production of antimicrobial drugs, and the use of said gene products for finding substances that bind to these gene products, and the production and use of antisense constructs, directed against the above genes. Procedures also belong to

Purification of said gene products using antibodies within the scope of the invention.

The substances found by the methods listed above, which bind to the gene products mentioned above, and the medicaments produced from these substances can be used for the treatment of infections caused by pathogens in humans and animals.

This preferably includes diseases caused by bacterial pathogens selected from the families of the Bacillaceae, Bacteroidaceae, Chlamydiales, Enterobacteriaceae, Micrococcaceae, Mycobakteriaceae, Neisseriaceae, Pasteurellaceae, Pseudomonadaceae. Rickettsiaceae, Spirillaceae, Spirochaetaceae, Streptococcaceae and Vibrionaceae.

The treatment of diseases which are selected by one or more bacterial pathogens from the genera Porphyromonas gingivalis, Chlamydia trachomatis, Escherichia coli, Yersinia pestis, Staphylococcus aureus, Mycobacterium tuberculosis, Neisseria gonnorhoeaert, Neisseria meningitellaidisidellaidisidellaiditis is very particularly preferred. Haemophilus influenzae, Pseudomonas aeruginosa, Rickettsia prowazekii, Campylobacter jejuni, Helicobacter pylori,

Borrelia burgdorferi, Treponema pallidum, Enterococcus faecalis, Streptoccus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Vibrio cholerae, Bacillus subtilis.

The substances found by the methods listed above that bind to the gene products mentioned above and those prepared from these substances

Medicines can be used in humans and animals for the treatment of diseases and in particular for the treatment of infections affecting the blood; the cardiovascular system; the central nervous system and its appendages; the bones, the bone marrow; the muscles and fascia, the teeth; the joints and their appendages and cavities; the internal organs, their cavities, inner and outer skins and appendages; the gastrointestinal tract and its cavities, inner and outer skins and appendages; the skin, skin appendages and soft parts and their appendages; the genitourinary system and its annexes; localized and / or generalized inflammation and / or general bacteremia and sepsis, as may occur in connection with general illness, trauma, polytrauma, and / or after surgery; Opportunistic infections and sepsis also relate to other diseases such as blood disorders, viral diseases, consuming diseases and / or antineoplastic and / or immunosuppressive therapy.

For example, diseases can be treated that after infection with Porphyromonas gingivalis in the area of the neck, nose, ear, head and neck, in the wound area of the oral cavity after surgical and / or dental treatment, and / or after bites by humans and animals; Escherichia coli in the area of the

Genitourinary system including urinary tract, bladder, kidney, pelvis, urosepsis and sepsis after surgery and other trauma; Staphylococcus aureus as a causative agent of inflammatory diseases of the respiratory tract and nasopharynx, the lungs, bones, skin, appendages and soft tissues, the heart and heart valves, as well as toxic diseases caused by the pathogens, such as haemolysis of the blood, the Epidermolysis of the skin, enterocolitis of the gastrointestinal tract, toxic shock syndrome; Mycobacterium tuberculosis as tuberculosis and / or tuberculoid diseases of the lungs, skin, and other organs and organ systems; Neisseria gonnorhoeae and the resulting clinical picture of gonorrhea; Neisseria meningitidis as a causative agent in acute purulent meningitis; Bordetella pertussis as a cause of whooping cough; Haemophilus influenzae as a causative agent in meningitis, epiglottitis, osteomyelitis, pneumonia, septicemia, laryngotracheitis, pharyngitis, sinusitis, otitis media, septic arthritis, acute endocarditis, cellulitis; Pseudomonas aeruginosa and the resulting inflammation of wounds, the genitourinary tract, the inner lining of the heart, the respiratory tract, the gastrointestinal tract; Rickettsia prowazekii and resulting typhus; Chlamydia trachomatis and resulting conjunctivitis granulosa trachomatosa of the eye; Yersinia pestis and the resulting plague disease; Campylobacter jejuni and resulting inflammatory diseases of the gastrointestinal tract such as enteritis, colins, proctitis and ulcer disease; Helicobacter pylori and resulting inflammatory chronic diseases of the gastrointestinal tract such as gastritis and duodenal ulcer and ulcer disease; Borrelia burgdorferi and the resulting Lyme disease and Erythema-Migrans disease; Treponema pallidum and the resulting clinical picture of syphilis; Enterococcus faecalis and the resulting disease of the urinary tract, urogenital system, the

Cardiac membranes and wound infection; Streptoccus mutans and resulting endocarditis and / or inflammation of the heart valves; Streptococcus pneumoniae and resulting inflammatory diseases of the upper and lower respiratory tract such as pneumonia, otitis media, sinusitis, as well as meningitis, peritonitis, and inflammation of the cardiac membranes and the pericardium, as well as caries; Streptococcus pyogenes and resulting inflammatory diseases of the upper respiratory tract such as streptococcal pharyngitis, scarlet fever, otitis media, and pyoderma and erysipelas of the skin and sepsis; Vibrio cholerae and the resulting clinical picture of cholera; Bacillus subtilis and the resulting clinical picture of oppurtunistic infections, disseminated spread and secondary infections in the context of other bacterial infections such as otitis, mastoiditis, urinary tract infection. Bacteremia, meningitis, endocarditis occur.

Bioinformatic analysis of various bacterial genomes: The amino acid sequences of the 4289 proteins from Escherichia coli K12 MG1655 with the Genbank / EMBL entry U00096 were compared with the sequences of the complete protein sets from the Gram-positive model organism Bacillus subtilis (Genbank / EMBL accession no. AL009126) and from the Gram -negative pathogen Haemophilus influenzae (Genbank / EMBL accession no. L42023) and compared with all other amino acid sequences in the public database (Genpept, Swiss-Prot).

Comparative analyzes were carried out with the FASTA program (FASTA vers. 2; Pearson and Lipman 1988).

All E. coli proteins with significant [expected value E (N) <10 ^"4 ] sequence homology to proteins from H. influenzae and B. subtilis were selected.

Sequences which have a homology of an expected value E (N) <10 ^{"" 1} to yeast proteins (HW Mewes et al., Nature. Suppl. Vol. 387: 9, 1997), as well as all

Proteins with known function or with homologies to known proteins from other organisms were excluded.

The proteins of E. coli for which homologous protein sequences occur within the species of E. coli were also excluded [expected value E (N)

<10 ^"4 ].

In addition, sequences with more than one predicted transmembrane domain [program: "Peptidestructure", Wisconsin Sequence Analysis Package (Vers. 9, Genetics Computer Group, Madison WI, USA)] were excluded. Results of the bioinformatic analysis of different bacterial genomes:

From the sequences of potential targets thus obtained, for example, 27

Protein sequences selected:

KDTB, SMPB, YBAB, YBAD, YBAK, YBAX, YBEA, YBEY, YCHB, YFGB,

YGAG, YGBB, YGBP, YGGJ, YGGV, YHBC, YHBJ, YHBY, YHIN, YIBK, YIDA, YIGZ, YJEE, YJEQ, YQGF, YRAL, YRFI.

"Knockout" construction and essentiality tests using temperature-sensitive plasmids

material and methods

1. Bacterial strains and plasmids

Table 1: E. coli strains used

Table 2: Plasmids used

Culture media used

LB (Luria Bertani) medium

10 g tryptone, 5 g yeast extract. 10 g NaCl on 11 H ₂ O

M9 minimal medium

6 g Na ₂ HPO ₄ , 3 g KH ₂ PO ₄ , 0.5 g NaCl, 1 g NH ₄ C1 on 11 H ₂ O

Table 3: Antibiotic concentrations in the media used

Molecular biological techniques

Basic molecular biological techniques Basic molecular biological techniques such as plasmid isolation. Cutting with restriction endonucleases, modification of DNA (dephosphorylation, replenishment reactions, ligation), production of competent E. coli cells and transformation have been carried out using standard regulations known to the person skilled in the art, such as those described in e.g. in J. Sambrook et al., Molecular Cloning, and Ausubel et al., Current Protocols in Molecular Biology.

Polymerase chain reaction (PCR)

All PCR primers used had calculated melting temperatures of 58 ° C to

62 ° C The following DNA polymerases were used for cloning purposes: Pwo, Taq (Boehringer, Mannheim, Germany), Akku Taq (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) and Pfu DNA polymerase (Stratagene, Heidelberg, Germany) .

Genes or their flanking regions were amplified from 100 ng chromosomal DNA from E. coli W3110 F in a reaction volume of 25 to 100 μl. The primer concentrations were 1 μM and dNTP concentrations were 250 μM. Twenty-five to thirty reaction cycles of 30-60s at 94 ° C, 30-60s at 48-55 ° C and 1-3 min at 72 ° C were followed by a final 5-minute

Step at 72 ° C [ref: PCR Protocols: a Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990)].

Assembly PCRs Amplified 5 'and 3' flanking gene regions were fused by

1 μl of the two previous PCR approaches were mixed as template for a second PCR with the distally located primers. This PCR was carried out under the conditions described above.

Diagnostic colony - PCRs

Diagnostic Colony - PCRs were performed with a mixture of Taq DNA polymerase (Boehringer) and ThermoSequenase (Amersham).

One colony at a time was resuspended in 50 μl H ₂ 0. 5 μl of this was used for the PCR, which was carried out in a 50 μl volume under the conditions described above.

Overexpression of essential proteins in E. coli

Various vector systems have been used to overexpress essential proteins in E. coli: pBAD / His (Invitrogen), pQE vectors (Quiagen). There were pBAD His plasmids (pUC vector derivatives) that were regulated for. Dose dependent expression and purification for recombinant proteins constructed in E.coli were used:

The vector system pBAD / His contains the sequence which codes for six histidines, including a linker for fusion with the N or C terminus of the desired recombinant protein. The maximum expression of the soluble, recombinant protein is achieved by targeted activation of the araBAD promoter with arabinose. The purification of the proteins was carried out with the aid of affinity chromatography on nickel-NT A-agarose.

To purify the fusion proteins, the cells containing the pBAD construct must first be grown to an optical density (OD _600nm ) of 0.3 to 0.6. The targeted protein production is then started with arabinose [0.002-0.2% (w / v)] and incubated for a further 2-3 hours. After completing the

Incubation, the cells are harvested by centrifugation, the cell pellet is frozen. Cell disruption and purification procedure were carried out in accordance with "The QIAexpressionist, A handbook for high-level expression and purification of 6xHis tagged proteins, Quiagen, Hilden, Germany 1997". The cleaning steps were carried out with polyacrylamide gels (and then stained with

Coomassie blue) checked.

The vector system pQE contains the sequence which codes for six histidines, including a linker for fusion with the N or C terminus of the desired recombinant protein. The maximum expression of the soluble, recombinant

Protein is achieved by targeted activation of the phage T5 promoter system and induction with IPTG. The purification of the proteins was carried out with the aid of affinity chromatography on nickel-NT A-agarose.

Cell culture and purification of the fusion proteins from cells containing pQE constructs were carried out according to the protocol from “The QIAexpressionist, A handbook for high-level expression and purification of 6xHis-tagged proteins, Quiagen, Hilden, Germany 1997 ".

Cloning strategy

Knockout plasmids:

In order to test the essentiality of the open reading frames of the selected genes, "knockout" plasmids were used which have the temperature-sensitive origin of replication of pSCIOl. This means that these plasmids can reproduce independently in E. coli at 30 ° C., but not at 42-44 ° C. If DNA regions which are homologous to the E. coli chromosome are cloned into such plasmids, these plasmids can at 42-44 ° C using homologous recombination in the gene locus of the E. coli chromosome to be examined. The subsequent excision of the plasmid at 30 ° C can lead to the exchange of the wild type sequence for the sequence originally cloned in the plasmid.

The flanking areas of the examined genes in the range of 400-800 bp each were cloned into pMAK705 and pKO3. Instead of the gene examined, either a tetracycline or a kanamycin resistance cassette was integrated into the respective plasmids. Two paths were taken for the cloning strategy:

Integration of the resistance cassette into the gene:

The gene of interest, including its flanking regions (400-800 bp), was amplified by means of PCR from chromosomal E. coli W3110-F DNA and converted into one

Cloning vector cloned (pCR blunt). With the aid of restriction digests and subsequent ligations, the major part of the coding region of the gene of interest was replaced by an antibiotic cassette or the gene was interrupted by such a cassette (tetracycline or kanamycin resistance cassette). Then the cassette with the original flanking the gene

Sequences cloned into the "Knockou 'plasmid pMAK705 or pKO3. Cloning of "in-frame" deletions with integration of a resistance cassette: DNA regions with lengths of 500 to 800 bp, which surround the 5 'and 3' end of the respective gene, were separated in two separate PCR reactions from chromosomal 1 E. coli W31 10-F DNA amplified. The primers required for this were selected such that the ends of the PCR products, which covered the 5 'and 3' ends of the gene, additionally had a 21 bp "tag" with one or two restriction sites. Accordingly, the two PCR products contained two 21 bp long, complementary extensions. These were used to assemble both products in a second PCR step using the external primers.

The fusion PCR product could be cloned into pKO3 via restriction sites built into the 5 'ends of the external primers. The fusion PCR product contained the flanking areas (400-800 bp each) as well as exactly 18 nucleotides of the 5 'end and 36 nucleotides of the 3' end of the examined

Gene, the 5 'and 3' ends of the gene being interrupted by a 21 bp "tag". In this way, a so-called "in-frame" deletion of the gene was generated. A canamycin resistance cassette was integrated into the 21 bp "tag" via a restriction interface.

Temperature - "Shift" experiments (integration and excision)

To integrate pMAK705 and pKO3 constructs, E. coli JM101 and E. coli MC 1061 were transformed with the plasmids according to standard protocols and incubated at 43-44 ° C. on LB agar plates with chloramphenicol.

To enable excision of the plasmids, the 43/44 ° C clones (cointegrates) were then inoculated to 30 ° C LB. Depending on the vector system (pMAK705 or pKO3), different approaches were taken: Temperature shifts with pMAK705:

43/44 ° C clones were inoculated in 100 ml LB - chloramphenicol liquid medium and incubated at 30 ° C for 16 h. After two passages in fresh medium with renewed incubation at 30 ° for 16 h each, the plasmids were isolated from 1 ml of the last grown culture. The prepared plasmids were examined for the presence of the wild-type copy of the gene in the plasmid by means of restriction digestion. The culture in which positive plasmids were found were isolated on LB - chloramphenicol plates at 30 ° C.

A number of colonies were picked and grown for plasmid preparation. The prepared plasmids were examined by restriction digestion for the presence of the wild type copy of the gene in the plasmid. Positive clones were verified by PCR for the presence of the antibiotic resistance cassette in the corresponding chromosomal locus. In this way, a chromosomal knockout for the gene under investigation was generated in E. coli with simultaneous complementation by a functional gene copy on the plasmid pMAK705.

Temperature shifts with pKO3: The cointegrates of the pKO3 derivatives in E. coli MC 1061 were excised while simultaneously eliminating the pKO3 plasmid from the cell according to the method described [AJ Link et al., J. Bacteriol. 179: 6228-6237 (1997)]. Three 43/44 ° C colonies were diluted in Luria-Bertani (LB) medium and plated on LB-5% (w / v) sucrose - kanamycin plates and incubated overnight. Subsequently, 50 to 100 of the resulting clones were inoculated again on LB - sucrose - kanamycin and LB - chloramphenicol plates per integration clone ("replica - plating"). Colonies sensitive to chloramphenicol but kanamycin resistant were finally analyzed by PCR using primers flanking the gene. Based on the size of the PCR product, it could be shown whether the respective clone was replaced by a kanamycin resistance cassette of the wild type gene. In this way, a chromosomal knockout for the gene under investigation was generated in E. coli without functional complementation.

Evidence of essentiality: The essentiality of the genes has been demonstrated in different ways:

Proof of essentiality by means of temperature shifts with the pMAK705 vector system: Aliquots were taken from 5 ml of the overnight LB-chloramphenicol cultures of the complemented knockout strains obtained at the end of the pMAK705 experiments. These were based on a uniform OD _6OÖ

0.1 set. Dilutions thereof were plated out on LB plates with or without chloramphenicol and incubated at 30 ° C. and 43/44 ° C. overnight.

The clones form a significantly reduced number of colonies on LB chloramphenicol plates at 43/44 ° C compared to the number of colonies on LB +/- chloramphenicol at 30 ° C, since the majority of the clones at 43/44 ° C loses the pMAK705 construct and can therefore no longer develop resistance to chloramphenicol. The number of colonies is reduced by at least a factor of 10. Under these conditions, the clones are not considered to be viable.

Criterion for chromosomal knockout in an essential gene: clones which, after colony counting on LB without chloramphenicol at 43/44 ° C, formed at least 10 ² fewer colonies than at 30 ° C on LB (with or without chloramphenicol) and at most 10 times more Colonies formed as on LB plates with chloramphenicol at 43/44 ° C, according to our criteria had a chromosomal knockout in an essential gene. The clones were not viable if they lost the plasmid at 43/44 ° C on LB without chloramphenicol (measured in "number of viable colonies").

On the other hand, all the clones that formed as many (+/- factor 10) colonies on LB without chloramphenicol at 43/44 ° C as on LB (with and without chlorine amphenicol) at 30 ° C, a chromosomal knockout in a non-essential gene. Removal of the plasmid with the complementary functional copy from the E. coli cells on LB without chloramphenicol at 43/44 ° C. does not lead to a reduction in the viability of the clones (measured in “number of viable colonies”).

Proof of essentiality by means of knockout generation only in the presence of a complementary gene copy (pKO3 system):

The clones generated with the pKO3 vector system, which were chloramphenicol-sensitive and contained a kanamycin resistance cassette instead of the wild-type gene, had a chromosomal knockout in a non-essential gene. A plasmid-coded complementation no longer existed.

If all colonies remained chloramphenicol - resistant, no knockout could be generated in the chromosome with simultaneous loss of the plasmid.

To further check the essentiality of these genes, an attempt was made to knock out the corresponding genes in the chromosome with simultaneous functional complementation.

For this purpose, the E. coli strain MGI 655 was used, which in contrast to MC 1061 has a complete araBAD locus. First, the araBAD genes from E. coli MGI 655 were replaced by the functional copy of the gene to be examined in each case. The plasmid pAL761 was used for this. After transformation of the pAL761 derivatives and subsequent integration and excision steps, the exchange of the araBAD genes for the functional copy of a gene was detected by means of PCR on the clones that were not on M9 minimal medium with 0.2% (w / v) arabinose could grow.

Positive clones were subsequently identified with the pKO3 derivative of the corresponding

Gene transformed. The subsequent cointegration and excision was as above described performed with the exception that the integration and excision step was carried out in the presence of 0.2% (w / v) arabinose.

The genotype of the deleted chromosomal gene locus with kanamycin cassette was diagnosed by means of PCR among the chloramphenicol-sensitive colonies. If the clones obtained showed a dependence on the growth of arabinose (i.e. no single colony formation on LB agar plates without arabinose in contrast to the growth on LB agar plates with arabinose), the corresponding genes were considered essential.

Proof of essentiality with the help of "in-frame" deletions (pKO3 system):

In some cases, an exchange of the genes for corresponding interrupted or deleted copies with antibiotic resistance cassettes could not be generated even in the presence of complementation. The cause of this can be polar effects caused by the cassettes, which lead to the expression of essential genes located downstream of the examined gene being impaired. For this reason, the above-described "in-frame" deletion plasmids of the corresponding genes without the kanamycin cassette were used for the following experiments.

"In-frame" deletion clones were transformed into E. coli MC 1061. Integration,

Excision and elimination of pKO3 and the subsequent "replica plating" were carried out as described above with the modification that no more selection was made for kanamycin. The chloramphenicol-sensitive colonies should statistically have 50% of the gene deletion. If among 50 colonies tested by PCR, no clone carried the deletion, further experiments followed to confirm the essentiality of the gene under investigation.

For this purpose, a deletion of the gene in the chromosome with simultaneous complementation as described above was generated and the growth dependence of arabinose was shown. Proof of essentiality in complemented knockouts without arabinose-dependent growth:

Complementary knockout strains that did not show arabinose-dependent growth were attempted to test the functional copy of the gene in araBAD -

Replace locus with a Kanamycin cassette. For this purpose, the corresponding strains were transformed with pAL767, a pKO3 derivative with a kanamycin resistance cassette and regions flanking araBAD.

After integration, excision and elimination of the pAL767, 100% of the resulting clones (200 clones considered) were both kanamycin and chloramphenicol-resistant, it was shown that pAL767 was still integrated in the chromosome. It was therefore not possible to remove the intact copy of the gene from the araBAD locus. This finding underlines the essentiality of the gene in question. If the successful cloning of the functional gene copy against the kanamycin resistance cassette could be shown in the possibly existing clanamycin - resistant and chloramphenicol - sensitive clones via PCR, the gene was not essential.

Results of the proof of essentiality:

Table 4: Genes whose essentiality according to the above Methods were assessed:

Accordingly, eight essential genes were identified from the 27 genes examined. With the protein sequences derived from these genes, a homology search against nucleotide sequences of completely and incompletely decoded bacterial genomes was carried out with the aid of the program TBLASTN.

With this method, by choosing a threshold value, the expected value P (N), all significant homologous protein sequences can be distinguished from randomly matching sequences. Using a stringent threshold value (expected value P (N) <10), gene products with a significant sequence homology could be found in a large number of disease-relevant bacteria.

From the homologues of a bacterial species found in this way, the most similar ones were selected, which have the maximum number of identical and / or conserved exchanged amino acids compared to the corresponding E. coli protein (Table 5). Such proteins are referred to as orthologous proteins (gene products) because the person skilled in the art knows that they have the same function as in Escherichia coli.

The orthologic proteins listed in Table 5 have at least 21% identical or 42% identical or conserved exchanged amino acids, ie 21% identity / 42% similarity. Table 5:

Distribution of the orthologic proteins of the identified essential E. coli gene products in pathogenic bacteria. Results of a TBLASTN search with the protein sequences found to be essential from E. coli against nucleotide sequences of fully or incompletely decoded bacterial genomes, such as those stored in the publicly accessible genome database of the National Center for Biotechnology Information (NCBI): criterion for significant homology is an expected value P (N) <10 ^"4. The identity and similarity of the orthologists in comparison to the corresponding E. coli protein are given as

% Identity /% similarity. Fully sequenced genomes are marked with (*). Missing homology in incompletely decoded genomes is marked with a (?).

The following functions can be predicted on the basis of sequence motifs in the amino acid sequence of the eight essential gene products and / or their spatial structure to be expected from the amino acid sequence: YJEE is likely to be an ATP or GTP binding protein. The protein may have ATPase / GTPase or ATP / GTP synthase activity.

KDTB is likely to be a sugar and / or nucleoside and or nucleoside derivative binding protein with phosphorylation or dehydrogenase activity.

YQGF may be a flavin derivative binding protein. It can bind flavomononucleotide (FMN) or flavin adenine dinucleotide (FAD) and transfer electrons to another substrate / protein. YQGF may represent hydrolysis that cleaves phosphodiester bonds or other ester bonds.

YHBC is a putative DNA-binding protein with a modulatory / regulatory function. YGGJ is a possible phosphotransferase. YGBP is probably a nucleoside diphophate derivative phosphorylase or pyrophosphorylase. YGBP probably cleaves CDP sorbitol or another polyol nucleoside diphosphate with the aid of pyrophosphate or transfers the nucleotide CMP to sorbitol 1-phosphate or another polyol phosphate. YCHB is a putative kinase or putative oxidoreductase. YGBB is a putative hydrolase or NAD (P) H-dependent reductase.

Examples

Example I to YJEE

Construction of the integration vector with the gene sequence for yjeE

The integration plasmid pMAK705 (5.5 kb) can be integrated into the chromosome of E. coli JM101 via homologous recombination. In addition to a temperature-sensitive origin of replication from pSCIOl, the plasmid contains a chloramphenicol resistance gene for the selection of the plasmid in the host organism.

The coding sequence for yjeE and 420 base pairs (bp) upstream and 413bp downstream of the coding sequence for yjeE were amplified by PCR (Cycler Perkin Elmer 480). 100 chromosomal DNA from E.coli W3110 F were in 25 cycles with 45 sec. at 94 ° C, 45 sec. at 48 ° C and 3 min. at 72 ° C and a subsequent incubation at 72 ° C for 5 minutes with Pfu DNA polymerase in

Reaction volume amplified by 100 μl. Primer A (5'-CCT GCT GGC AAT CAA TCC CGA TA-3 ') and primer B (5'-AGG CGG TGG CGG CAC ATCGGC GTT-3') were used as amplification primers. After purification with Qiaex (Qiagen, Hilden, Germany), the amplification product (1482 bp) was converted into the vector pCR - blunt using the "pCR - blunt cloning kit" (Invitrogen

Corporation, Carlsbad, CA). A tetracycline resistance cassette was cloned into the restriction site Bpml of pCR - blunt / yjee. The tetracycline cassette was isolated from plasmid pBR322 as a 1400bp EcoRI / Aval fragment, and blunt ends were made with Klenow polymerase.

The resulting plasmid pCR - blunt / yjeE :: tet was digested with Kpnl / Xbal restriction enzymes. A 2.8 kb DNA band was isolated and cloned into the vector pMAK705 (host organism E. coli JM101) pretreated with Kpnl / Xbal. The resulting construct (pMAK705yjee) was used for the integration experiments.

Production and analysis of the yjeE knockout cells with the plasmid pMAK705yjee were grown overnight in LB medium at 30 ° C. and plated the next day on LB test plates with chloramphenicol and incubated at 43 ° C. for 16 h. The integration of the plasmid into the chromosome of E.coli JM101 was identified by selection of chloramphenicol-resistant colonies that could grow at 43 ° C. After identification of the integration (at 43 ° C), the cells were incubated in 100 ml LB medium at 30 ° C for 16 h. This

Cells were transferred twice into fresh medium and further incubated at 30 ° C. for 16 h each.

After this incubation series, plasmid rapid preparations of 1 ml each of the last incubation batches were carried out with Qiaprep (Qiagen, Hilden, Germany).

The prepared plasmids were examined by restriction digestion for the presence of the wild type copy of the gene in the plasmid. Cells from the batches containing positive clones (yjeE gene from the wild-type chromosome on the plasmid) were separated on LB plates with chloramphenicol at 30 ° C.

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

The clones checked in this way could now be used for the vitality of the target yjeE for the survival of the E.coli cell. The cells were incubated on LB test plates with and without chloramphenicol at 30 ° and 43 ° C. Table 6: Cell numbers of the incubation +/- chloramphenicol at 30 ° C / 43 ° C

As can be seen from the table, the cells can grow at 30 ° C. both with and without chloramphenicol. At 43 ° C the growth with chloramphenicol as well as without chloramphenicol is drastically reduced, ie by a factor of 10 ⁴ . This clearly shows the need for the presence of the plasmid with the complementing sequence of yjeE for the growth of the E. coli cell at 43 ° C. without chloramphenicol and thus the essentiality of yjeE for the survival of these cells.

Example: proof of essentiality for ygbP

Cloning of "in-frame" deletions with or without integration of a

Resistance cassette:

DNA regions surrounding the 5 'and 3' end of the ygbP gene were amplified in two separate PCR reactions from 100 ng chromosomal E. coli W31 10-F DNA in a reaction volume of 25 μl. The primer concentrations were 1 μM and dNTP concentrations were 250 μM. Twenty-five reaction cycles of 30s at 94 ° C, 30s at 52 ° C and 1-2 min at 72 ° C were carried out with a final 5 minute step at 72 ° C. The PCR product made with the primers YGBP1A (5'-cgcggatccCCACATGGTCACTGCCTGG-3 ') and YGBP1B (5'-cccatccactaaactgcagctCAAATGAGTGGTTGCCATGTT-3') comprises 18 bp of the 5 'bend-on end and the 6' bend-on end by ygbP. The PCR product that is primed with YGBP2A (5'- agctgcagtttagtggatgggTACCTCACCCGAACCATCC-3 ') and YGBP2B (5'- cgcggatccACCTGGCAGCCTTCCAGTTG-3 '), comprises 36 bp of the 3' end of ygbP and 807 bp of the chromosomal region downstream of ygbP.

The inner primers YGBPIB and YGBP2A additionally contained 21mer "tags" at their 5 'ends, which were complementary to one another (small letters of the sequences mentioned above). These were used to assemble both PCR products in a second PCR step using the external primers YGBP1A and YGBP2B. To this end, 1 μl of the two previous PCR batches were mixed as template for a second PCR with YGBP1A and YGBP2B. This PCR was carried out under the conditions described above. The PCR

Product of the expected size was purified using an agarose gel electrophoresis using QIAEX (QIAGEN, Hilden.Germany).

Via the BamHI interface which was installed at the 5 'ends of the external primers YGBP1A and YGBP2B (small letters of the above-mentioned

Sequences), the fusion PCR product could be cloned into the BamHI-cut, dephosphorylated pKO3. In this way, the plasmid pAL759 was generated which contained the "in-frame" deletion of ygbP. The kanamycin resistance cassette was cloned into pAL759 via the Pstl interface in the 21 bp "day" (5'-ctgcag-3 ') and was obtained from pUC4K by means of a Pstl digest. The resulting construct was named pAL759a.

Temperature shifts with pAL759a:

E. coli MC 1061 was transformed with the plasmid pAL759a and opened at 44 ° C

LB agar plates incubated with chloramphenicol.

Three of the 44 ° C. clones (cointegrates) obtained were diluted in LB medium, plated onto LB-5% (w / v) sucrose-kanamycin plates and incubated overnight. For each integration clone, 100 of the resulting sucrose - kanamycin - clones were changed again to LB - sucrose - kanamycin and LB - chloramphenicol Plates were inoculated ("replica - plating") and incubated at 30 ° C. overnight. 100% of the kanamycin-resistant clones were also chloramphenicol-resistant. An exchange of ygbP for a kanamycin cassette could therefore not be carried out successfully.

Temperature shifts with pAL759:

E. coli MC 1061 was transformed with the plasmid pAL759 and incubated at 44 ° C. on LB agar plates with chloramphenicol.

Three of the 44 ° C. clones (cointegrates) obtained were diluted in LB medium, plated on LB-5% (w / v) sucrose plates and incubated overnight.

For each integration clone, 100 of the resulting sucrose clones were inoculated again on LB sucrose and LB chloramphenicol plates ("replica -

Plating ") and incubated overnight at 30 ° C. 50 chloramphenicol-sensitive colonies were finally analyzed by PCR using YGBP1 A and YGBP2B as primers. Based on the size of the PCR product, it could be shown that none of the clones tested had the deletion of ygbP (PCR product for ygbP deletion: 1637 bp). The wild-type gene locus of ygbP could be amplified (2294 bp) in all colonies. An exchange of ygbP for a deletion could therefore not be carried out successfully .

Generation of a ygbP deletion in the presence of a chromosomal complement:

In the next step an attempt was made to create a deletion of ygbP in the chromosome with simultaneous functional complementation. For this the E. coli strain MG 1655 was used, which in contrast to MC 1061 has a complete araBAD locus. First, the E. coli MG 1655 araBAD genes were replaced with the functional copy of ygbP. The plasmid pAL763 was used for this. The plasmid pAL763 was generated from the chromosomal E. coli W3110-F-DNA with the primers YGBPR (5'-atcgctagcATCAGCCCGGGAATTAACATG-3 ') and YGBPT (5'-atcctcgagAATTCGCATTATGTATTCTCCTG-3P) including its full gene Binding site (18 bp upstream from the 5 'end) and including 8 bp downstream from the 3' end of ygbP was amplified. The primers contain the restriction sites for Xhol (YGBPT) and Nhel (YGBPR) at their 5 'termini. The PCR product was cloned into the vector pAL761 via the interfaces. After transformation of E. coli MG1655 with pAL763 and subsequent integration and excision steps (see above), the exchange of the araBAD genes for the functional copy of ygbP was detected by means of PCR on the clones which were not on M9 minimal medium with 0, 2% (w / v) arabinose could grow. The diagnostic PCR was carried out with the primers YGBPR (see above) and ARA2B (atcgcggccgcAAAGCCGTGCTCGCGC). The primer ARA2B binds in the 3 'region 865 bp downstream of the araD gene, so that together with the primer YGBPR a 1655 bp product was formed in positive clones. Positive clones were subsequently transformed with the "in-frame" deletion plasmid pAL759. The subsequent cointegration and excision was carried out as described above, except that the integration and excision step was carried out in the presence of 0.2% (w / v) arabinose. The genotype of the deleted chromosomal gene locus was diagnosed by means of PCR among the chloramphenicol-sensitive colonies. The ygbP deletion clones, which contained an arabinose-regulated ygbP copy in the araBAD locus, showed a growth dependence on arabinose. No single colonies could be formed on LB agar plates without arabinose, single colonies being obtained on LB agar plates with arabinose. The ygbP gene is therefore essential. Example 3

Production and purification of recombinant YJEE protein

The expression and purification of YJEE protein was carried out using the pBAD His vector system (Invitrogen). The sequence of yjeE was made from 100 chromosomal DNA from E. coli W3110F for 1 min at 94 ° C. for 1 min. at 52 ° C, 3 min at 72 ° C in 25 cycles with Pfu DNA polymerase in 100 μl reaction mixture. YJEE-START (5'-TGA AGA TCT AAT CGA GTA ATT CCG CTC CCT GAT-3 ') and YJEE-STOP (5'-TCA GAA TTC TTA ACC GGC TAA ACG CGC CAG CAA CAA TC-3') were used as primers. used. The 5 'end of YJEE

START contains the sequence for the restriction enzyme Bglll. The 5 'end of YJEE-STOP contains the sequence for EcoRI. The PCR mixture was separated in an agarose gel and a band was cut out at 450 bp. The band was then isolated with Qiaex (Qiagen, Hilden, Germany) and incubated with the restriction enzymes BglII and EcoRI for 3 hours at 37 ° C.

The DNA fragment treated in this way was cloned into the vector pBAD / HisB pretreated with BglII / EcoRI [host organism: E. coli strain TOP 10 (Invitrogen)]. Successful cloning was demonstrated by plasmid isolation of the transformants obtained and restriction with EcoRI. The clone with the plasmid pBAD / ΗisB-YjeE30 has been used for expression and purification.

The cultivation and purification of YjeE from E. coli TOP 10 with plasmid pBAD HisB-YjeE30 was carried out according to the protocol for the purification of histidine-labeled proteins on Ni-NTA agarose (The QIAexpressionist, A handbook for high-level expression and purification of 6xHis- tagged proteins, Quiagen, Hilden, Germany 1997). The induction of the YjeE expression was started with 0.02% arabinose (w / v) at an optical density of the culture of OD ₆ oo ⁼ 0.5. Various concentrations of imidazole (50, 100, 150 and 200 mM) were used to elute the bound protein. The highest yield was obtained at 150 raM

Get protein. With polyacrylamide gel separation and subsequent Coomassie Staining was checked for purification. 10 mg of protein with a degree of purity> 90% could be isolated from 1 l of culture medium (LB medium).

Example 4 Screening of banks of low molecular weight chemical compounds with

Targets of unknown function based on affinity selection and mass spectrometry

To find inhibitors for the target proteins with an unknown function, but essential for bacterial survival (such as YJEE), screening methods can be used that test substance banks with regard to their affinity for the protein. A screening option is the affinity selection from substance mixtures with subsequent detection of the ligands in the mass spectrometer. Defined substance mixtures must be used for this, from which individual substances can be identified with the aid of mass detection. Mixtures of substances that have been prepared from combinatorial syntheses are therefore particularly suitable for this screening method.

A liquid chromatography-electrospray mass spectrometer serves as a screening apparatus, the HPLC column being replaced by an ultrafiltration chamber. The

Ultrafiltration chamber consists of a filtration unit in which the filter disk is replaced by an ultrafiltration membrane (exclusion molecular weight 10 kDa). Mass spectra are generated using a mass spectrometer (from Hewlett-Packard; Palo Alto, CA; "5989B MS Engine Quadrupole Mass Spectrometer"). It is operated as described (van Breemen et al., Pulsed Ultrafiltration Mass Spectrometry. A New Method for Screening Combinatorial: Anal. Chem., 69 [11], 2159-2164, 1997).

An equimolar mixture of 96 compounds (each approx. 200 μM) in 10 μl DMSO is added to 90 μl 2 μM protein (e.g. YJEE), which is in a 20 mM

Ammonium acetate buffer is present. The protein substance mixture is at least Incubated for 15 min at room temperature. After the mixture has been injected into the ultrafiltration chamber, it is washed with water for 8 min at a flow rate of 50 l / min. The mobile phase is then changed to methanol / water (50:50 v / v) in order to dissociate possible ligand-protein complexes and to filter out the ligands. The ligands are detected using electrospray mass spectrometry. The substances from a 96 mixture identified as possible ligands on the basis of their mass peaks are used again as individual substances in the affinity test in order to verify their affinity for the target protein (eg YJEE).

Example 5

Substances that are noticeable in the affinity selection are subjected to further tests such as the MIC test.

For this purpose, the MIC values are determined using the microdilution method in BH medium. Each test substance is dissolved in the nutrient medium. Serial dilutions of the test substances are made in the microtiter plate. Overculture cultures of the pathogens are used for inoculation, which are previously diluted 1: 250 in the nutrient medium. 100 ml of the inoculated solution are added to 100 ml of the diluted nutrient solutions containing the active ingredient.

The microtiter plates are incubated at 37 ° C and read after about 20 hours or after 3 to 5 days. The MIC value (mg / ml) indicates the lowest active substance concentration at which no growth can be seen.

Another method is to determine the minimum inhibitory concentration (MIC) in the liquid dilution test. Overpower cultures of the test germs (S. aureus 133) in isosensitest broth are diluted 1: 1000 in fetal calf serum FKS) or isosensitest broth and incubated with dilutions of the test substances (dilution levels 1: 2). The cultures are incubated at 37 ° C for 18 to 24 hours. The lowest substance concentration at which no visible bacterial growth occurs is defined as the MIC.

Credentials:

Blattner et al. Science 277: 1453-1462

F. Argoni et al., Nature Biotechnology, 16: 851 (1998)

H.W. Mewes et al. Nature, suppl. Vol. 387, p 9 (1997)

Hamilton et al. J.Bacteriol. 171: 4617-4622 (1989) Link et al. J. Bacteriol. 179: 6228-6237 (1997)

US 5821076 Identification of Essential Survival Genes

B.J. Akerly et al. Proc. Natl. Acad. Si. USA 95: 8927-8932, 1998

T. Clementz and C. R. H. Raetz: J. Biol. Chem. 266: 9687-9696, 1991

T. Clementz, J. Bact. 1992 174: 7750-7756, 1992 D. T. Moir et al., Antimicrob. Agents chemother. 43: 439-446, 1999;

Claims

Claims:

1. Use of the proteins from the group YQGF, YHBC, YGGJ, YGBP, YCHB, YGBB, YJEE, KDTB and their orthologic gene products in an assay to find substances which bind to these proteins.

2. Use according to claim 1, the proteins being 95-100% identical to those listed.

3. Vectors containing one or more of the genes which are responsible for the proteins

Coding claims 1 and 2.

4. Transformed microorganisms containing one or more of the genes which code for the proteins according to claims 1 and 2.

5. Compounds that bind to the proteins according to claims 1 and 2.

6. Compounds that bind to the proteins according to claims 1 and 2 and modulate their activity.

7. Medicaments containing one or more compounds according to Claim 5 or 6.

8. Use of compounds according to claims 5 and 6 for the manufacture of medicaments.

9. Method for finding compounds containing proteins from the group YQGF, YHBC, YGGJ, YGBP, YCHB. Modulate YGBB, YJEE, KDTB and their orthologic gene products, characterized in that the compounds are brought together with the proteins and checked whether they bind.

0. A method for finding antibacterially active compounds which modulate proteins from the YQGF, YHBC, YGGJ, YGBP, YCHB, YGBB, YJEE, KDTB proteins and their orthologic gene products, characterized in that the compounds are combined with the proteins and tested, whether they bind and then undergo an MIC test.