DE10116788A1 - Non-pathogenic bacterial Poringen recipient strain of a mycolic acid-containing bacterium, process for its production and its use - Google Patents

Abstract

The invention relates to a non-pathogenic and/or rapidly growing bacterial porin gene recipient strain of a bacterium containing mycolic acid, said strain being transgenic for at least one inactivated porin gene, due to at least one corresponding porin gene from a pathogenic and/or slow-growing bacterium containing mycolic acid. The invention also relates to a method for producing one such inventive bacterial recipient strain and the use thereof for identifying substances having antibacterial action against pathogenic bacteria containing mycolic acid.

Description

The present invention relates to a non-pathogenic and / or rapidly growing bak Poringen recipient strain of a mycolic acid-containing bacterium, which for min least an inactivated porin gene by at least one corresponding porin gene from one pathogenic and / or slowly growing mycolic acid-containing bacterium is transgenic. Furthermore, the invention relates to a method for producing such an inventive moderate bacterial recipient strain and its use for the identification of anti bacterial substances against pathogenic mycolic acid-containing bacteria.

The development of new antibiotics against Mycobacterium tuberculosis is special urgently, since approx. 54 million people are newly infected every year and the number of infections with multi-resistant strains is increasing. Already about 2 die every year Millions of people with tuberculosis (Global Tuberculosis Control, WHO report 2001, http: / / www.who.int/health-topics/tb.htm)).

The genome sequence of M. tuberculosis and the availability of experimental techniques like the analysis of the "transcriptome" with DNA chips and the "proteome" by 2D Gel electrophoresis allows the identification of a variety of new "targets" for tuberculosis se (TB) therapeutics. The main disadvantage of in vitro high throughput methods for development However, the development of therapeutic agents is that they often do not reach their destination. This is particularly the case for M. tuberculosis, because its cell wall is unique due to its like structure for hydrophilic compounds is about 500 times less permeable than that of E. coli. (Jarlier, V. and Nikaido, H. (1990) Permeability barrier to hydrophilic solutes in My cobacterium chelonei. J Bacteriol 172: 1418-1423).

The cell wall of mycobacteria has a unique structure. Long chain fatty acids, the so-called mycolic acids form the inner layer of a lipid double membrane, the one represents a high permeability barrier for hydrophobic substances. Although mycobacteria are used for Belonging to the group of Gram-positive bacteria, they therefore have an outer membrane that functionally similar to that of gram-negative bacteria. Hydrophilic substances arrive through channel-forming proteins, the porins, in the mycolic acid layer into the mycobacterial  Periplasm. The inefficiency of this path is the cause of the general resistance of My cobacteria against many antibiotics. This makes the porins the key proteins for ent Development of new antibiotics with improved transport properties.

So far, porins have been found in both the fast growing Mycobacterium chelonae and M. smegmatis as well as in the slow growing M. bovis and M. tuberculosis sen. Despite intensive work in this area, only two genes that are known for encode mycobacterial proteins that show channel activity in vitro. The mspA gene ko is for an oligomeric porin from M. smegmatis, which consists of 20 kDa subunits and is extremely stable to chemical and thermal denaturation. MspA has none Homology to previously known proteins. This concludes a pro's N-terminal sequence mixture with low channel activity (Mukhopadhyay et al. (1997) Characterization of a porin from Mycobacterium smegmatis. J Bacteriol 179: 6205-6207). Because MspA also has to span a membrane of approx. 10 nm thickness, it seems the prototype of a new one Family of channel proteins.

The ompATb gene codes for a monomeric 35 kDa protein from M. tuberculosis, which is based on Due to the sequence similarity to OmpA from E. coli was found and a small channel shows activity in lipid bilayer experiments. Since the crystal structure shows that the membrane permanent part of OmpA does not form pores is unclear like the pore activity of E. coli OmpA and OmpATb comes about. It is not excluded that OmpA proteins forms high concentrations of intermolecular channels. M. tuberculosis expresses at least two porins different from OmpATb, as by analysis of channel activity of cell wall proteins has been detected. So far no data have been published that the Show function of the identified porins in mycobacteria in vivo.

The most attractive potential target proteins for antibiotics are in the cell wall of M. tuberculosis, which is very similar to other modifications of mycolic acids that of M. smegmatis. That is why M. smegmatis is already used in screening procedures today used for TB therapeutics. It is neglected that according to current knowledge the porins of M. smegmatis and M. tuberculosis appear to be very different.

It is therefore an object of the present invention to find a suitable non-pathogenic and / or rapidly growing strain of a mycolic acid-containing bacterium  that is suitable for finding the location quickly, safely and efficiently new effective therapeutic agents against pathogenic and / or slow growing mycolic acid to allow containing bacteria. Another object of the invention is to provide a method for Production of such a non-pathogenic strain of a mycolic acid-containing bacterium for To make available. Another object of the present invention is a method to find new effective therapeutic agents against pathogenic mycolic acid-containing bacteria to provide.

A first object of the invention is awakened by a non-pathogenic and / or quickly send bacterial recipient strain of a mycolic acid-containing bacterium, which for at least least an inactivated porin gene by at least one corresponding porin gene from one pathogenic and / or slow growing mycolic acid-containing bacterium is transgenic, ge solves.

Non-pathogenic strains of mycolic acid-containing bacteria are already being discovered today use of new potential therapeutic agents, but these may be due to the un Different porins have only limited permeability to the cell wall in the pathogenic strains simulate elongated. The construction of a non-pathogenic and / or rapidly growing Mycolic acid-containing bacterium that no longer has its own porins, but rather porins pathogenic mycobacteria, especially Mycobacterium tuberculosis, expresses a significant relief and improvement for the development of new Thera peutics. Such a strain, which is transgenic for one or more porin genes, is not yet known known.

The non-pathogenic and / or rapidly growing bacterial recipient according to the invention a mycolic acid-containing bacterium is responsible for at least one inactivated porin gene by at least one corresponding porin gene from a pathogenic and / or slow growing mycolic acid-containing bacterium transgenic. This means that at least one chromosomal porin gene is functionally inactivated. The inactivated gene is in his Function by at least one on a genetic element in the recipient strain guided poringen replaced from pathogenic mycobacteria. The ratio of inactivated Poringenes to "foreign poringenes" can be 1: 1, but a "foreign Poringen "functionally replace several inactivated Poringenes, but more can "Foreign poringenes" are present as inactivated poringenes in the recipient strain.  

Furthermore, the non-pathogenic and / or rapidly growing bacterial recipient strain characterized in that the at least one corresponding porin gene from one Bacterium of the same or a different genus of mycolic acid-containing bacteria.

According to the invention, non-pathogenic and / or rapidly growing bacterial Re recipient strains of the genera Mycobacterium, Corynebacterium, Nocardia, Rho dococcus, Gordona, Tsukarmurella, Dietzia or other Gram-positive bacteria that My contain colic acid, are used (see also: Tansill et al. (eds.) "Bergey's manual of systematic bacteriology "1984, Williams & Wilkins, USA).

Preferred is a non-pathogenic and / or rapidly growing bacterial strain, which consists of is selected from the group consisting of Mycobacterium smegmatis, Mycobacterium chelo nae (subsp. chelonae or abscessus), Mycobacterium fortuitum (subsp. fortiutum or pere grinum) Mycobacterium chitae, Mycobacterium senegalese, Mycobacterium agri, Mycobacte rium phlei, Mycobacterium thermoresistibile, Mycobacterium aichiense, Mycobacterium au rum, Mycobacterium chubuense, Mycobacterium duvalii, Mycobacterium flavescens, Mycob acterium gadium, Mycobacterium gilvum, Mycobacterium komossense, Mycobacterium neo aurum, Mycobacterium obuense, Mycobacterium parafortuitum, Mycobacterium rhodesiae, Mycobacterium sphagni, Mycobacterium tokaiense, or Mycobacterium vaccae, (see also: Tansill et al. (eds.) "Bergey's manual of systematic bacteriology" 1984, Williams & Wilkins, USA, section 16). Mycobacterium smegmatis is particularly preferred.

Another aspect of the present invention relates to a non-pa according to the invention thogenic bacterial strain in which the inactivated porin gene mspA or a homolog of it is. MspA or its homolog from other mycolic acid-containing bacteria provides the main component for the hydrophilic passage through the cell wall of mycolic acid containing bacteria, where MspA is described in Mycobacteria.

A non-pathogenic and / or rapidly growing bacteri according to the invention is preferred eller strain that is transgenic for all porin genes. Invention is particularly preferred moderate non-pathogenic and / or rapidly growing bacterial strain which is suitable for the porin Genes mspA, mspB, mspC and mspD, their homologues or previously unidentified porin Genes is transgenic.  

The donor strain for the porin gene (s) of the non-pathogenic and / or fast growing bacterial strain can be of a variety of pathogenic and / or slowly growing strains of mycolic acid-containing bacteria, for example from Mycobacterium africanum, Mycobacterium avium, Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium farcinogenes, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, My cobacterium leprae, Mycobacterium lepraemurium, Mycobacterium malmoense, Mycobacte rium marinum, Mycobacterium microti, Mycobacterium nonchromogenicum, Mycobacterium paratuberculosis, Mycobacterium scrofulaceum, Mycobacterium shimiodei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium triviale, Mycobacte rium tuberculosis, Mycobacterium ulcerans or Mycobacterium xenopi.

Most preferred according to the invention is an M. smegmatis strain which consists only of porins M. tuberculosis expressed. Such a M. smegmatis strain, which the permeability of the cell by M. tuberculosis for hydrophilic compounds by the expression of its Po imitated as closely as possible, should be very suitable to improve antibiotics Find transportation properties.

The strategy according to the invention thus addresses the weak point of previous concepts, whose goal is to identify new "targets" of M. tuberculosis, but the real one Problem in the treatment of tuberculosis, namely the poor transportation of most Disregard connections through the mycolic acid layer.

Thus, another object of the present invention is achieved by a method of manufacture treatment of a non-pathogenic and / or rapidly growing bacterial recipient strain, which is transgenic for at least one of its porin genes, the method resolving the Steps a) Providing a non-pathogenic strain of a mycolic acid-containing bacteri killed; b) Introduction of a genetic element that an expression cassette with a radio tional Porin gene together with a first resistance marker to be selected positively wearing; c) Introduction of a second genetic element that is one of recognition sequences a sequence-specific recombinase flanked deletion cassette of the to be deleted Porin gene together with a second resistance marker to be selected positively Selection of the genetic element and a third positive selection marker for selection  the deletion cassette and one or more selections to be selected negatively marker bears; d) selection for the selection markers to be selected positively; e) selection on the selection markers to be selected negatively; f) Identification of clones that the have replaced the porin gene to be deleted with the deletion cassette; g) Introducing a ge netic element, which is a suitable sequence-specific recombinase and a positive Selection marker for the selection of the genetic element as well as a negative selection rends selection marker; h) Selection for the selection to be selected positively marker; i) selection for the selection marker to be selected negatively; and given if repetition of steps a) to i) for deletion and replacement of a further porin Gene includes.

The inventive method can be further characterized in that the functio nale Porin gene from a bacterium of the same or a different genus mycolic acid containing bacteria and that the non-pathogenic and / or rapidly growing strain a mycolic acid-containing bacterium is selected from the genera Mycobacterium, Corynebacterium, Nocardia, Rhodococcus, Gordona, Tsukarmurella, Dietzia or others Gram-positive bacteria that contain mycolic acid.

Functional porin genes are characterized by the expression of the corresponding proteins. The synthesis of porins can begin by transcription from the own promoter. This is e.g. B. in the vector pMN101 (see Fig. 2) the case. The transcription can also be done by fusing the porin genes with any other promoters such as z. B. for the fusion of the mspA gene from M. smegmatis with the promoter p _smyc in pMN014 (see Examples 1 and 3). Such methods are known to the expert and are such. B. for Mycobacteria in (Harth, G., Lee, BY and Horwitz, MA (1997) High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect Immun 65: 2321-8 .; Triccas, JA, Patrick, T., Britton, WJ and Giequel, B. (1998) An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol Lett 167 : 151-6.). Functional porin genes can, however, also be characterized in that they code for fusion proteins in which the channel structure of the porins is preserved.

A method according to the invention is preferred in which plasmids, transposons and / or phages are used as genetic elements. A method according to the invention is further preferred, in which the selection markers used are selected from the group consisting of aph (from Tn 5 or Tn903; kanamycin resistance), hph (hygromycin resistance), aacC1 (gentamycin resistance), aac (3 ) -IVa (apramycin resistance), cat (chloram phenicol resistance), Sul ^r (sulfonamide resistance), Str ^r (streptomycin resistance), mercury salt resistance markers, rpsL, sacB and pAL5000ts, (see also Paget and Davies "Apramycin resistance as a selective marker for gene transfer in Mycobacteria" J. Bacteriol 178 (21); page 6357-6360; 1996) The method according to the invention can also be characterized in that the recombinases used are selected from the group consisting of FLP, Shufflon, CRE, FimB, XER, D protein, bacteriophage integrases (e.g. λ, HK22, P2, ϕ21, P22, HP1, SSV1, L5), plasmid integrases (e.g. pSAM2), transposon integrases (e.g. . Tn915, Tn1545, Tn916), AADA and AACA (see also Neidhardt et al. (Eds .) "Escherichia Coli and Salmonella Typhimurium" ASM Press 1996, p. 2367).

Most preferred according to the invention is a non-pathogenic and / or rapidly growing bacterial porin recipient strain which is produced by means of a method according to the invention is, the plasmids used are pMN101, pMN250 and pMN234.

The strategy according to the invention also has the advantage that it is the screening method to identify new therapeutic agents against pathogenic and / or slow-growing Mycol acidic bacteria considerably simplified. This happens in the case of mycobacteria especially because M. smegmatis is not pathogenic and grows about ten times faster than M. tuberculosis.

Another object of the present invention is thus achieved by a method for identi Treatment of antibacterial substances against pathogenic Mycolic acid-containing Bak solved in series, the method comprising the following steps: a) bringing into contact min at least one potentially antibacterial substance with at least one invention according to non-pathogenic and / or rapidly growing bacterial strain, and b) measuring the survival rate of the at least one non-pathogenic and / or rapidly growing bak serial strain. According to the invention, this method cannot do with all of the invention pathogenic and / or rapidly growing bacterial strains are carried out Contain mycolic acid. A method is preferred in which the non-pathogenic bacterial  The strain is selected from the genera Mycobacterium, Corynebacterium, Nocardia, Rho dococcus, Gordona, Tsukarmurella, Dietzia or other Gram-positive bacteria that My contain colic acid.

Survival rate can be measured by conventional methods such as counting Colonies occur on culture media plates or measure the density of a cell culture. Insbeson their preferred are methods in which, as step b), the expression is additionally measured a marker gene, for example the luciferase gene and / or the GFP gene. The Use of the latter genes and the construction of the necessary genetic Constructs are readily familiar to the person skilled in the art and can easily be introduced into the bacteria be introduced using conventional methods.

Alternatively, instead of (or in parallel) measuring the survival rate of the bacteria according to another embodiment of the identification method according to the invention tion of antibacterial substances as step b) measuring the porin Permeability properties of the at least one potentially antibacterial sub punching. This can be done, for example, on the living cell in vivo leads, the transport of radioactive, fluorescent, heavy metal or on their suitable way labeled potentially antibacterial substances by the Porine is measured. Another option is to use the carpenter Rosselet tests (Nikaido H, "Role of permability barriers in resistance to beta-lactam antibio tics "Pharmacol Ther 27: 197-231, (1985) and analog tests for antibacterial sub punch except for β-lactams.

According to the invention, a method is preferred which is characterized in that as poten tially antibacterial substances modified known tuberculosis therapeutics and / or other known antibiotics are examined. The modifications can either introduced randomly or specifically into the known substances. It is that For example, it is known to those skilled in the art that modifications of side groups of the beta Lactam scaffold on changed chemical and physical properties of beta Lactams can lead. However, the method according to the invention can also be used for this collections of already known tuberculosis therapeutics and / or others antibiotics for their suitability for the treatment of pathogenic mycolic acid-containing  To investigate bacteria, since the method according to the invention is also automated High throughput method allowed.

Furthermore, a method according to the invention for identifying antibiotics is preferred, which is characterized in that substances from natural product banks or molecular banks to be examined for their antimycobacterial effectiveness. Such banks are the subject known and sometimes even commercially available. Another aspect of the invention relates to an antibacterial substance which by means of a method according to the invention rens was obtained by screening the above banks.

Another aspect of the present invention relates to the use of an inventive non-pathogenic and / or rapidly growing bacterial strain for identification of antibiotics against pathogenic and / or slow-growing mycolic acid-containing bacteria s, especially Mycobacteria. Modified known Tu are preferably used Over-the-counter therapeutics and / or other known antibiotics to identify the anti examined biotics. However, the use according to the invention can also be characterized thereby is characterized in that substances from natural product banks and / or molecular banks for identification Antibiotic can be used.

Another aspect of the present invention relates to an in vitro test element for identification Decoration of antibiotics against pathogenic mycolic acid-containing bacteria, especially My cobacteria, the at least one non-pathogenic bacterial strain according to the invention includes. Such test elements can be found in the strains according to the invention, for example in Mi microplates for high throughput screening using suitable robo tertechnik or other analysis techniques include.

Another aspect of the invention relates to a kit that has at least one according to the invention non-pathogenic bacterial strain together with appropriate reagents and equipment gene includes. Such kits can be suitable, for example, all for implementation of a method according to the invention for identifying potentially antibacterial to contain the necessary components of the substances. Another option is a Kit for carrying out the Zimmermann-Rosselet test (see below).  

The expression "transgenic" strains in the sense of the present invention means bacteria in which one or more of our own porin genes have been deleted or modified which and / or which express one or more porin genes from other bacteria. above in addition, these transgenic strains can also be characterized in that they are their own Porin genes in a different genetic environment than that originally in the exi chromosome own.

The invention will now be described below by way of examples with reference to the accompanying Figures will be explained further.

Seq ID Nos. 1 to 4 show oligonucleotide sequences of Oligonu used in Example 1 kleotiden.

Fig. 1 shows maps of the plasmids of the two-plasmid system for M. smegmatis. pMycVec1 contains the pAL5000 origin of replication for mycobacteria, the pBR322 origin of replication for E. coli and a kanamycin resistance cassette. pMycVec2 contains the pJAZ38 origin of replication for mycobacteria, the COLE1 origin of replication for E. coli and a hygromycin resistance cassette. Both plasmids have an identical polylinker, which enables easy recloning of expression cassettes. pMy cVec1 can be replaced by all plasmids with the PAL5000 replicon, e.g. B. pMS2 to be replaced.

Figure 2 shows a map of plasmid pMN101 which carries the "auxiliary sporin" mspA. pMN101 is a derivative of pMycVec2 and contains the pJAZ38 origin of replication for mycobacteria, the COLE1 origin of replication for E. coli, the sacB cassette for counter-selection in mycobacteria and a hygromycin resistance cassette (hyg). The restriction sites shown are singular. T4g32 and rrnBT2 are two transcription terminators.

Fig. 3 shows a map of the sequence-specific removable gentamycin knockout cassette in plasmid pMN250 which is flanked by FRT sequences and can be cut again by the FLP recombinase. For homologous recombination, DNA fragments of any length in the upstream and downstream region of the gene to be deleted from the chromosome can be amplified by PCR and inserted into PMN250 by PmeI / SpeI or PacI / SwaI. The FRT sequences can be exchanged for the target sequences of other recombinases via SpeI / HpaI or SacI / PacI. The FRT-aacC1 cassette itself can be cut out with NruI. The entire cassette including the homologous sequences can be cut out via PmeI / SwaI. SpeI can also be replaced by SexAI.

Fig. 4 shows a map of Deletionssvektors pMN250 for mycobacteria. pMN250 is a derivative of ptrpA-rpsL + [Sander, 1995 # 3315] and contains only one COLE1 origin of replication for E. coli and one ampicillin resistance cassette. The rpsL gene serves as a counter-selectable marker. The restriction interfaces shown are singular. T4g32 and rrnBT2 are two transcription terminators.

Fig. 5 shows a map of the expression vector for the pMN234 FLPE recombinase. pMN234 is a derivative of pMS2 and contains the pA15000 origin of replication for My cobacteria, the COLE1 origin of replication for E. coli and a kanamycin resistance cassette. The rpsL gene serves as a counter-selectable marker. The restriction interfaces shown are singular. T4g32 and rrnBT2 are transcription terminators.

Fig. 6 shows the cloning strategy for the basic vectors of the recombination process (see also Example 1).

The restriction interfaces shown are singular. The polylinker contains the through the Arrows labeled gene 32 transcription terminators from bacteriophage T4 and from the E. coli rrnB gene. The bases of the transcription terminators, which are the stem of the termi nators are shown in small letters. The bold letters indicate the Stop codons. The aph and hyg genes confer resistance to kanamycin and hy, respectively gromycin.

Fig. 7 shows the change in permeability of the outer membrane for cephaloridine (A) and the uptake of glucose (B) at a M. smegmatis mspA deletion mutant. A. The hydrolysis of cephaloridine by β-lactamases was measured spectrophotometrically for the wild type (gray bars), the mspA deletion mutant (white bars) and the complemented mspA deletion mutant (striped white bars). The first bar shows the rate of cephaloridine hydrolysis by cell lysates and indicates the total β-lactamase activity. The second bar shows the speed of cephaloridine hydrolysis through whole cells and is a measure of the permeability of the cell wall.

B. Absorption of glucose by M. smegmatis (black dot) and an mspA deletion mutant (white dots). The glucose concentration was 3.3 mM, the temperature was 37 ° C.

Figure 8 shows the results of growth tests of M. smegmatis (black symbols) and an M. smegmatis-mspA deletion mutant (white symbols). The cells were grown in minimal medium with 1 mM glucose (spots) and 1 mM glucosamine.

FIG. 9 shows sensitivity tests of M. smegmatis (dark gray bars), an mspA deletion mutant (light gray bars) and an mspA deletion mutant (white bars) complemented with an mspA expression vector (pMN014). The percentage of surviving cells (cfu) is plotted with increasing amounts of ampicillin (A) and cephaloridine (B).

Four M. smegmatis porin genes have been identified so far: mspA, mspB, mspC and MSPD. To find a suitable bacterial mutant and a screening system for antibiotics To develop M. tuberculosis, all porin genes from M. smegmatis should be deleted and inserted into the this mutant the genes for the porins from M. tuberculosis are expressed.

This was exemplified by a ΔmspA mutant as a first step in this direction (Stahl, et al. "MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis "Mol. Microbiol. (2001) (in press)). Since the presence of Is essential for the survival of mycobacteria, the consecutive knock- Out of the porin genes a two plasmid system. An auxiliary sporin, e.g. B. MspA, expressed in order to be able to delete the chromosomally encoded poringenes. Um now the desired porin without being able to examine the presence of other porins must be ses are introduced into the cell on a second plasmid and the expression of the auxiliary spo rins can be prevented.

Previously published vectors for use in a two-plasmid system are not suitable for complex cloning. The plasmids pMycVec1 and pMy cVec2 (see FIG. 1) were therefore constructed which carry two different, compatible origins of replication for mycobacteria and, apart from the polylinker, have no common sequences in order to minimize the probability of recombination between the two plasmids.

Then two expression cassettes with different constitutive promoters and gfp + and ebfp were constructed and by measuring the energy transfer from EBFP to GFP + ge showed that not only both vectors replicate stably over many generations in M. smegmatis, but also genes from both plasmids are expressed , Details of the polylinker are shown in Fig. 1.

In a next step, an expression cassette for mspA with a very strong constitutive promoter was constructed and cloned into pMS2. The functionality of this expression cassette was shown in the plasmid pMN014, by transforming it into the Δ mspA mutant whose permeability defects could be complemented. An mspA expression cassette with a weaker promoter was already constructed (pMN012) and has now been cloned into pMycVec2 to give plasmid pMN104. Through the subsequent insertion of the sacB cassette, which is also already present, the plasmid-independent expression of the auxiliary sporine MspA can be ended at any time by adding sucrose to the medium. This task can be accomplished with any mspA expression vector that carries a sacB cassette. We have shown with the plasmid pMN101 (see FIG. 2) that the chromosomal DNA fragment from M smegmatis carries the mspA gene and its own promoter.

The analysis of the function and properties of porins in vivo is only possible in bacteria that only express the porin of choice. Although this has already been recognized for E. coli Despite numerous publications, there are allegedly "porin-free" E. coli strains me, no E. coli strain in which all porin genes are physically derived from the chro mosom were removed. However, this is imperative since insertions of resistance casas set, transposons or reporter genes can lead to revertants that are due to the growth advantage quickly overgrows the parent strain with fewer porins. There but porins are essential for the survival of bacteria with an outer membrane Porin genes can only be deleted from the chromosome if a plasmid is transient auxiliary spore is expressed. This strategy has not yet been used in E. coli and is described below.

The above-mentioned strategy is necessary above all for the investigation of the porins of pathogenic My colic acid-containing bacteria, such as in particular M. tubereulosis, since these cannot otherwise be purified in sufficient quantities. It was therefore decided to remove all porin genes from M. smegmatis, the model organism for mycobacteria, from the chromosome as an example. However, this project was by no means trivial; several difficulties had to be overcome when implementing this strategy in M. smegmatis:

• 1. In addition to the porin genes described so far, there could be other porin genes in M. smegmatis, which are not very similar to mspA. But these should The course of the successive deletion of the previously known porin genes can be discovered when in parallel approaches the expression of the auxiliary sporine does not take place. In these experiments So far, silent porin genes could then be activated by mutations, like that in E. coli was observed for nmpC and lc.
• 2. The expression of the auxiliary porin must be transient. The mspA gene was therefore cloned with its own promoter into the low-copy vector pMycVec2 of our two-plasmid system and the plasmid pMN101 was obtained. The vector pMN101 (see FIG. 2) additionally carries a sacB cassette which allows selection for cells which no longer have this plas mid and only carry the pMycVec1 derivative with the poringenenes from M. tuberculosis. The sacB cassette has already proven to be very effective in counter-selection in recombination attempts in M. smegmatis. Additional regulation of gene expression is not necessary with this strategy.
• 3. Due to the low recombination frequency in, the positive selection for recombination events by the introduction of resistance cassettes is imperative. So far, only a few resistance cassettes have been described as functional in My cobacteria, namely the aph, the hyg and the aacC1 cassettes, which give mycobacteria resistance to camycin, hygromycin and gentamycin. Since the first two resistance casings have already been used for the two plasmids, only the gentamycin cassette remains as a positive selection marker. In order to be able to use the gentamycin cassette for consecutive recombination experiments, it has to be cut out of the chromosome again after each knock-out. For this purpose, sequence-specific recombination noses are to be used which can carry out precise DNA deletions in vivo if the sequence to be deleted is flanked by the recognition sequences for a sequence-specific recombination. Such cassettes are e.g. B. has already been used in E. coli and mammalian cells. Such experiments have not yet been described for mycobacteria. For these experiments, we constructed a gentamycin knockout cassette in the vector pMN236 (see FIG. 3) and the expression plasmid pMN234, which was functional in M. smegmatis, for expressing a modified form of the FLP recombinase (see FIG. 5).

For homologous recombination, approximately 1 kbp long DNA fragments of the upstream and downstream region of the gene to be deleted from the chromosome are amplified by PCR and inserted into PMN250 by PmeI / SpeI or PacI / SwaI (see FIG. 4). Alternatively, the SexAI interface can be used instead of the SpeI interface. This deletion plasmid was transformed into M. smegmatis SMR5, which already carries the auxiliary plasmid pMN101. By selection for gentamycin and counter-selection against the deletion plasmid by streptomycin, selection is made for recombinant strains which no longer have the respective porin gene. These strains are genetically and functionally verified. The gentamycin cassette is then cut out by transforming the Flp recombinase plasmid pMN234 and the recombinase plasmid is removed again by counter-selection with streptomycin. Theoretically, this process could be repeated any number of times until all porin genes were deleted without a resistance cassette remaining in the chromosome. However, since one of the two recognition sequences remains in the genome during recombination, undesirable side reactions could occur from the second recombination. These can be achieved by using other sequence-specific recombinases such as e.g. B. Cre from the bacteriophage P1, Int (Tn916) or FimB (E. coli) can be avoided.

Bacterial strains and growing conditions

Mycobacterium smegmatis SMR5 is a streptomycin-resistant mutant of M. smegmatis mc ² 155 (Sander, P., Meier, A. and Böttger, EC (1995) rpsL +: a dominant selectable marker for gene replacement in mycobacteria. Mol Microbiol 16: 991 -1000) and was grown in Middle brook 7H9 medium (Difco) supplemented with 0.2% glycerin and 0.05% Tween 80 at 37 ° C. Escherichia coli DH5α was used for all cloning experiments and was routinely grown in LB medium at 37 ° C. If necessary, antibiotics were added in the following concentrations: ampicillin (100 µg / mL for E. coli), kanamycin (30 µg / mL for E. coli, 10 µg / mL for M. smegmatis), hygromycin (200 µg / mL for E. coli, 50 µg / mL for M. smegmatis), gentamycin (15 µg / mL) and streptomycin (400 µg / mL).

example 1 Construction of plasmids (see also FIG. 6)

The plasmid pMycVec1 was digested with FseI, the protruding 3 'ends removed with Klenow polymerase and then cut with KpnI. This vector fragment was ligated to the rpsL gene, which was excised from the plasmid ptrpA-1-rpsL ⁺ (Sander et al., Supra) with SmaI and KpnI. The plasmid thus obtained was called pMN210. The pAL5000 origin of replication pMN210 was replaced by its temperature-sensitive variant from pCG63 (Guilhot, C., Gicquel, B. and Martin, C. (1992) Temperature-sensitive mutants of My cobacterium plasmid pAL5000. FEMS Microbiol Lett 77: 181- 186). For this, pMN210 was cut with ApaI and the temperature-sensitive origin of replication was cut out of pCG63 with EcoRV / KpnI. The ends of both DNA fragments were blunt-ended with the Klenow polymerase and ligated. The resulting plasmid pALEX1 contained two counter-selectable markers (rpsL gene and a temperature-sensitive origin of replication) and was used as a vector for recombination in mycobacteria. In a first step for deleting the mspA gene, a 2.8 kbp genomic DNA fragment with the mspA gene with SpeI and EcoRV was cut out of pPOR6, treated with Klenow polymerase and cloned into the filled SalI site of pBluescriptII KS + (Strata gene). The plasmid thus obtained was called pBlue-mspA. Then a 536 bp fragment with FseI and StyI was excised from pBlue-mspA, which carries the suspected promoter, Shine-Dalgarno, translation start and a part of the mspA gene coding for 54 amino acids. The ends of the DNA of the vector fragment were blunt-ended with T4 DNA polymerase and ligated with a 1180 bp gentamicin resistance cassette, which was obtained from pMV361 (Stover, CK, de la Cruz, VF, Fuerst, TR, Burlein, JE, Benson, LA, Bennett, LT, Bansal, GP, Young, JF, Lee, MH, Hatfull, GF, Snapper, SB, Barletta, RG, Jacobs, WR and Bloom, BR (1991) New use of BCG for recombinant vaccines. Nature 351: 456-60) with the oligonucleotides GenR-fwd (AGAGAGAATTCGATCGAAATCCAGATCCTTGACC; Seq. ID No. 1) and GenR-rev (TCTCTGAATTCGTTGTGACAATTTACCGAACAACTCC; Seq. ID No. 2) The plasmid thus obtained was called pMN224. The genomic fragment containing the ΔmspA :: aacC1 cassette was excised from pMN224 with ApaI and SacI, treated with T4 DNA polymerase and cloned into the unique PmeI site of pALEX1 to obtain the mspA deletion vector pMN226inv.

In order to obtain a mycobacterial expression vector for mspA, the mspA gene of pPOR6 with the oligonucleotides MP-fwd (GATTAGTTAATTAAGGGAGAACATGAAGGCAATCAGTCG; Seq. ID a No. 3) and MP- rev (AAATCGAACGGCGGTCCAGGAATCAG; Seq. ID No. 4). The PCR Fragment was cloned into pMS2 via PacI and SwaI. The plasmid thus obtained was  called pMN006. A strong promoter of M. tuberculosis was derived from pUV 5 with PmeI and PacI cut out and cloned the same interfaces from pMN006 to the mspA Ex to obtain the pressure vector pMN014. All plasmid constructs were sequenced checked.

Example 2 Construction of a mspA deletion mutant from M. smegmatis

M. smegmatis SMR5 was transformed with the plasmid pMN226inv. Gentamicin is the marker for positive selection on the ΔmspA :: aacC1 cassette. The growth of the cells which still contain the plasmid pMN226inv should be inhibited by streptomycin and at the temperature of 39 ° C. which is not permissive for the replication of pMN226inv. After selection on 7H10 plates containing gentamicin and streptomycin at 39 ° C for four days, six clones were obtained. These clones were isolated on 7H10 plates with and without kanyincin to confirm their gene ^R Str ^R Kan ^S phenotype and to check whether these clones had actually lost the plasmid pMN226inv. This procedure was repeated twice for each clone. Three of these clones were grown in 7H9 medium with Genta micin and streptomycin up to an OD ₆₀₀ of 1. Then their chromosomal DNA was isolated. NruI cuts the wild-type chromosomal DNA twice within the cloned mspA fragment, releasing a 936 bp fragment. This fragment was detected in a DNA hybridization experiment. One of the three clones examined no longer had this fragment. Instead, this strain had an additional NruI fragment of approximately 4400 bp. This result showed that the mspA gene was deleted from this strain. This strain was called M. smegmatis MN01.

Example 3 Quantification of the permeability of the outer membrane of M. smegmatis for cephaloridine (Zimmermann-Rosselet test)

The Zimmermann-Rosselet test is one of the very few theoretically correct methods to measure diffusion rates through bacterial cell walls (Nikaido, H. (1985) Role of permeability barriers in resistance to beta-lactam antibiotics. Pharmacol Ther 27: 197-231.) And was therefore selected to determine the permeability of the cell walls M. smegmatis and the ΔmspA mutant. In this test the permeabili the cell wall for the zwitterionic β-lactam antibiotic cephaloridine through its Hydrolysis by periplasmic β-lactamases measured in intact cells (Zimmermann, W. and Rosselet, A. (1977) Function of the outer membrane of Escherichia coli as a permea bility barrier to beta-lactam antibiotics. Antimicrob Agents Chemother 12: 368-372; Jarlier, V.  and Nikaido, H. (1990) Permeability barrier to hydrophilic solutes in Mycobacierium che lonei. J Bacteriol 172: 1418-1423). The hydrolysis of cephaloridine becomes spectrophotome measured trically.

M. smegmatis SMR5 and MN01 are grown to an OD ₆₀₀ of 1, harvested by centrifugation for 5 min at 3000 g at room temperature, with PBS (4.3 mM Na ₂ HPO ₄ , 1.4 mM KH ₂ PO ₄ , pH 7.5, 137 mM NaCl, 2.7 mM KCl) and resuspended in 2.5 mM PIPES (pH 6.5) at a concentration of 80 mg cells (wet weight) / mL. 100 .mu.L of this cell suspension are mixed with 400 .mu.L of a 2.5 mM PIPES (pH 6.5) buffer which contains 1 mM cephalo ridin (Sigma). 300 µL of this mixture are quickly transferred into a cuvette with a light path of 1 mm and the absorption at 260 nm and 241 nm is measured for 40 min at 25 ° C. In order to determine the activity of the periplasmic β-lactamases of M. smegmatis, 160 mg cells (wet weight) were resuspended in 1 mL 2.5 mM PIPES (pH 6.5), disrupted by ultrasound and the hydrolysis of cephaloridine by half Volume of the supernatant determined. The hydrolysis rate was 25.4 ± 4 nmol min ^-1 .mg ^-1 cells (dry weight) and corresponds to the maximum activity of the β-lactamases from M. smegmatis. The mean dry weights were 0.57 mg and 0.78 mg for wild type M. smegmatis and the ΔmspA mutant, respectively. Special precautions were taken to avoid mechanical stress cell lysis that would release periplasmic β-lactamases into the supernatant. This was controlled by measuring the hydrolysis of cephaloridine through the supernatant of 80 mg (wet weight) of whole cells. The permeability coefficients P were calculated according to the published method (Zimmermann and Rosse let, supra) using a K _M value of 146 µM for the β-lactamases from M. smegmatis (Trias and Benz, supra) and a surface weight Ratio of 132 cm ² / mg (dry weight) for mycobacteria (Jarlier and Nikaido, supra).

0.8 mM cephaloridine were hydrolyzed by the cell lysate of M. smegmatis SMR5 at a rate of 25.4 ± 4 nmol.min ^-1 .mg ^-1 . Cephaloridine hydrolysis by intact cells was approximately five times slower at 4.4 ± 0.3 nmol.min ^-1 .mg ^-1 . This shows that the diffusion through the outer membrane is rate limiting ( Fig. 7A). Therefore, the cephaloridine hydrolysis rate is a direct measure of the permeability of the cell wall of M. smegmatis. The β-lactamase activity of the supernatant from M. smegmatis SMR5 cells was 0.3 ± 0.09 nmol.min ^-1 .mg ^-1 . This value corresponds to approximately 1% of the total β-lactamase activity and lies in the error range of the measurements of cephaloridine hydrolysis by whole cells. This allows the calculation of the permeability coefficient of the cell wall of M. smegmatis to (7.2 ± 1.4) .10 ^-7 cm / s in good agreement with the previously published value of (10 ± 1.2) .10 ^{- 7} cm / s (Trias, J. and Benz, R. (1994) Permeability of the cell wall of Mycobacterium smegmatis. Mol Microbiol 14: 283-290). The activity of the β-lactamases of the ΔmspA mutant M. smegmatis MN01 was identical to that of the parent strain ( FIG. 7A). The evaluation of cephaloridine hydrolysis by whole cells of M. smegmatis MN01 showed a permeability coefficient of (8.4 ± 2.2) .10 ^-8 cm / s. From this it can be concluded that the deletion of the mspA gene reduces the permeability of the cell wall to cephaloridine by a factor of 9. This result indicated an important role of MspA for the diffusion of hydrophilic molecules into the cell of M. smegmatis.

In order to demonstrate whether the permeability defect of M. smegmatis MN01 was actually caused by the deletion of the mspA gene, a fusion of the mspA gene with the constitutive promoter p _smyc from M. smegmatis was made. The transformation of M. smegmatis MN01 with the vector pMN014, which contained the p _smyc- mspA fusion, did not change the β-lactamase activity, but increased the permeability for cephaloridine back to the wild-type level ( FIG. 7A). From this it could be concluded that the MspA porin is responsible for approx. 90% of the permeability of M. smegmatis to cephaloridine.

Example 4 Change in glucose uptake by smegmatis-mspA deletion mutant

In order to investigate whether mspA is important for the uptake of sugars by M. smegmatis, the uptake rate of ¹⁴ C-labeled glucose by whole cells of M. smegmatis was measured. For this, the strains M. smegmatis SMR5, MN01 and MN01, which was complemented with an episomal copy of mspA (pMN014), were grown to an OD ₆₀₀ of 1.5, harvested by centrifugation at 3000 g and 4 ° C. for ten minutes, twice washed with 2 mM PIPES (pH 6.5), 0.05 mM MgCl ₂ and resuspended in 32 mL of the same buffer. [ ¹⁴ C] -glucose (specific activity 311 mCi / mmol, Amersham) was added to the cell suspension and mixed with glucose in such a way that glucose concentrations of 50 μM to 3.3 mM were obtained. The mixture was incubated at 25 ° C and 37 ° C, respectively. 1 mL samples were taken at times from 15 seconds to 16 minutes. The cells were filtered through a filter with a pore size of 0.45 μm (Sartorius), washed with 0.1 M LiCl and the radioactivity of the filter was measured in a scintillation counter. One mL of the cell suspension was dried and weighed to determine the cell size used in the test. The mean dry weight per test was 0.84 ± 0.02 mg. Glucose uptake was expressed in nmol per mg (dry weight) of cells. The recording speed was determined by fitting a straight line to at least the first three data points from 15 to 45 seconds. The correlation coefficients were greater than 0.97 for all fits. This shows that the data did not deviate significantly from a straight line. Analysis of the uptake speeds determined in this way at different glucose concentrations using the Michaelis-Menten equation gave K _M and V _max values for the total transport of glucose into the cell. The data analysis was confirmed by the Lineweaver-Burk application for wild type M. smegmatis. An estimate of the minimum value of the permeability coefficient was obtained from the following equation,

which was derived from Jarlier and Nikaido (supra) by assuming that the substrate diffuses passively through the cell wall and then is actively transported through the cytoplasmic membrane at a higher rate. Then the substrate concentration in the periplasm can be neglected compared to the initial substrate concentration in the buffer. We used a value of 132 cm ² / mg (dry weight) as an estimate for the surface to weight ratio for smegmatis (Jarlier and Nikaldo, supra).

The uptake kinetics for 3.3 mM glucose showed a rapid saturation after approx. 5 min ( FIG. 7B). The uptake rate of the ΔmspA mutant for glucose was 2.4 times slower than that of the wild type. Complementation of the ΔmspA mutant with the mspA expression vector pMN014 increased the uptake rate for glucose at the wild-type level (data not shown). This confirms that the deletion of mspA causes a permeability defect in M. smegmatis.

A series of uptake experiments with glucose in concentrations of 50 µM to 2 mM were carried out in order to determine the apparent V _max and K _m values. The effect of the mspA deletion on the uptake of glucose was in agreement with results in Gram-negative bacteria at lower concentrations (Ferenci, T. (1996) Ad aptation to life at micromolar nutrient levels: the regulation of Escherichia coli Glucose transport by endoinduction and cAMP.FEMS Microbiol Rev 18: 301-17). For example, the uptake of glucose in the ΔmspA mutant was 6.7 times slower at 50 µM than in the wild type (data not shown). Data analysis using the Michaelis-Menten equation showed v _max and K _m values of 11.2 nmol.mg ^-1 .min ^-1 and 2.6 nM for the wild type, and 1.3 nmol.mg ^{- 1} .min ^-1 and 1.1 nM for the ΔmspA mutant. Assuming that glucose first diffuses passively through the cell wall and then is actively transported through the cytoplasmic membrane (Jarlier and Nikaido, supra), estimates for the minimum permeability coefficient P of 2.6.10 ^-7 cm / s for the wild type and 7 , 2.10 ^-8 cm / s for the ΔmspA mutant. This shows that the deletion of mspA reduces the permeability of M. smegmatis to glucose by a factor of 4 to a value which is 200,000 times less than the value determined for E. coli (Bavoil, P. , Nikaido, H. and von Meyen burg, K. (1977) Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein. Mol Gen Genet 158: 23-33). The permeability of M. smegmatis ΔmspA mutant for glucose is the lowest value ever measured for bacteria.

Because of the diffusion of such different molecules as glucose and cephaloridine the cell wall of M. smegmatis can be affected by the deletion of the mspA gene concluded that MspA is a general diffusion path for hydrophilic molecules in M. smegmatis represents. This assumption is supported by the finding that the admission me of fructose by the ΔmspA mutant was significantly slower than by the wild type. The results of the two transport tests are therefore in good agreement with the lower channel share of detergent extracts and the lower porin content of the ΔmspA mutant. The physiological function of MspA in M. smegmatis is thus similar to that for the Porine OmpF and OmpC has been described by E. coli (Nikaido, H. and Nakae, T. (1979) The outer membrane of gram-negative bacteria. Adv Microb Physiol 20: 163-250). The deed thing that the cell wall of the ΔmspA mutant about 90% or 75% of its permeability for Ce lost phaloridine and glucose, shows that MspA is the major porin of M. smegmatis and was insufficiently replaced by the smaller number of other porins.

Example 5 growth experiments

It has been suggested that the low permeability of the mycobacterial cell wall to nutrients causes their slow growth (Ratledge, C. (1982) Nutrition, growth and me tabolism. In The biology of the mycobacteria, pp. 186-212. Edited by C Ratledge & J. Stan ford. London, United Kingdom: Academic Press, Ltd). This hypothesis was supported by theoretical considerations for M. chelonae (Jarlier and Nikaido, supra). In order to investigate whether the deletion of mspA influences the growth rate of M. smegmatis, the wild type and the ΔmspA mutant were used in minimal medium with glucose as the only carbon source in concentrations of 10 μM to 10 mM. In these experiments, M. smegmatis SMR5 and MN01 were used for 36 to 40 hours in 5 mL precultures in MMT minimal medium (60 g / L Na ₂ HPO ₄ , 30 g / L KH ₂ PO ₄ , pH 7.4, 5 g / L NaCl, 10 g / L NH ₄ Cl, 2 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.05% Tween 80) with 10 mM glucose.

The cells were washed twice with MMT minimal medium and supplemented in 100 mL MMT with 0.01, 0.1, 1 and 10 mM glucose, 1 mM succinate and 1 mM glucosamine as the only carbon source. The growth rates were determined in three independent experiments by OD ₆₀₀ measurements. The growth of both strains in the presence of glucose up to a concentration of 100 µM was very slow, stopped at an OD ₆₀₀ of approx. 0.1 and did not differ significantly. Higher glucose concentrations supported the growth of both the wild type smegmatis and the ΔmspA mutant up to an OD ₆₀₀ of approximately 0.3 (see FIG. 8). The growth rates of both strains were similar despite the slower glucose uptake of the ΔmspA mutant (see Example 4). In the presence of 1 mM glucosamine (see FIG. 8) or 1 mM succinate, a significant delay in the growth of the ΔmspA mutant compared to the wild type was observed.

These results showed that even the low permeability of the ΔmspA mutant restricted ge allow sufficient rapid glucose transport that slow the growth rate of M. smegmatis not affected. This speaks against the low permeability of the cell wall Mycobacteria as a determinant of slow growth of wild-type M. smegmatis. the This result is due to the existence of at least three other porin genes M. smegmatis not surprising. So z. B. in E. coli a strong growth defect only in Mutants were observed that had lost both main porins, OmpF and OmpC (Bavoil et al. supra). In addition, sugar-specific porins such as LamB in E. coli exist that are even important for the growth of E. coli among limiting sugars concentrations in the presence of OmpF and OmpC are (Death, A. and Ferenci, T. (1993) The importance of the binding-protein-dependent Mgl system to the transport of glucose in Escherichia coli growing on low sugar concentrations. Res Microbiol 144: 529-37). Yet indicates the slightly reduced growth rate of the ΔmspA mutant in the presence of  Glucosamine indicated that the deletion of mspA increased the permeability of the cell wall of My cobacteria reduced to a level that is just limiting growth. More Experi elements are necessary to check whether this effect is due to the slower transport of glucosamine is caused by the mycolic acid layer of M. smegmatis or by Different speeds of absorption via the cytoplasmic membrane compared to Glu cose.

Example 6 Antibiotic sensitivity tests

Measurements of the rate of cephaloridine diffusion through the mycolic acid layer show ten that the ΔmspA mutant a 10 times lower permeability of the outer Has membrane, which complements by inserting an episomal copy of mspA (Stahl et al. "MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis "Mol. Microbiol. (2001) (in press). This resulted in the Fra ge, whether the changed permeability of the mycolic acid layer also with a lower Sen sensitivity to hydrophilic antibiotics. To investigate this question an antibiotic sensitivity test was carried out.

Plates consisting of 19 g / l Middlebrook 7H10 agar (Difco) 5 ml / l 100% glycerol were used for the antibiotic sensitivity tests. These plates contained 8, 16, 32, 64, 128, 256 and 512 µg / ml ampicillin or 1, 2, 4, 8, 16, 32, 64 µg / ml cephaloridine. 100 μl of a preculture of the M. smegmatis strain to be tested with a cell density of OD ₆₀₀ / ml = 1.2 in the dilution levels 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^{-6 were} plated onto these plates. To determine the total cell titer, the same dilution levels were applied to 7H10 plates without antibiotics. The plates were incubated at 37 ° C. for 72 h. The colony numbers of the plates were then determined and the values converted to cell number / ml. From this, the mean for each antibiotic concentration and each strain was formed and set in a percentage relationship to the total live titer.

The sensitivity of the three different M. smegmatis strains wt, ΔmspA and komple mented mutant against the antibiotics ampicillin, cephaloridine, streptomycin, Ri fampicin, kanamycin, chloramphenicol and erythromycin and the tuberculosis Therapeutics measured isonicotinic hydrazide and ethambutol.  

It can be seen that the ΔmspA mutant is significantly less sensitive to ampicillin than the wt strain and the complemented mutant ( FIG. 9A). These two strains showed visible colonial growth only at concentrations ≦ 16 µg / ml, while the colony number of the ΔmspA mutant at this concentration is still 55% of the live titer. Only at concentrations ≧ 64 µg / ml did the relative number of colonies of the ΔmspA mutant drop to 5%.

All strains showed significant growth at cephaloridine concentrations ≦ 2 µg / ml ( FIG. 9B). However, twice as many colonies grew from the ΔmspA mutant as from the wild type and from the complemented mutant. At a concentration of 4 µg / ml, this difference becomes even greater because the wt 4% growth and the ΔmspA mutant is still 40%.

These results exemplify that the deletion of mspA leads to an increased resi of smegmatis leads to hydrophilic antibiotics. These experiments prove that MspA is important for the entry of antibiotics into the cell. From this it can also be seen on an important role of the porins of M. tuberculosis for the transport of antibiotics conclude.  

SEQUENCE LISTING

Claims (27)

1. Non-pathogenic bacterial recipient strain of a mycolic acid-containing bacteri um that corresponds to at least one inactivated porin gene by at least one Porin gene from a pathogenic and / or slowly growing mycolic acid containing bacterium is transgenic. 2. Non-pathogenic bacterial recipient strain according to claim 1, characterized records that the at least one corresponding Porin gene from a bacterium of the same or another genus of mycolic acid-containing bacteria. 3. Non-pathogenic bacterial recipient strain according to claim 1 or 2 of the genus gene Mycobacterium, Corynebacterium, Nocardia, Rhodococcus, Gordona, Tsukarmu rella, Dietzia or other Gram-positive bacteria that contain mycolic acid. 4. Non-pathogenic bacterial strain according to claim 3, selected from the group consisting of Mycobacterium smegmatis, Mycobacterium chelonae and Mycobacteri around flavescens or another non-pathogenic and / or fast growing mycob stem is. 5. Non-pathogenic bacterial strain according to claim 3 or 4, in which the porin gene mspA or a homologue of it. 6. Non-pathogenic bacterial strain according to one of claims 1 to 4, which is for all Porin genes are transgenic. 7. Non-pathogenic bacterial strain according to one of claims 1 to 4, which for the Porin genes mspA, mspB, mspC and mspD or their homologues is transgenic. 8. Non-pathogenic bacterial strain according to one of the preceding claims, in which the donor strain for the Porin gene (s) Mycobacterium leprae, Mycobacterium bo vis BCG, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum,  Mycobacterium gastri, Mycobacterium terrae, Mycobacterium nonchromoge nicum, Mycobacterium triviale, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gastri, Mycobacterium malmoense, Mycobacterium gordonae, My cobacterium szulgai, Mycobacterium simiae, Mycobacterium scrofulaceum, Mycob acterium xenopi, Mycobacterium shimiodei, Mycobacterium ulcerans, Mycobacterium haemophilum, Mycobacterium farcinogenes, Mycobacterium lepraemurium, Mycob acterium paratuberculosis, Mycobacterium tuberculosis. or another pathogenic and / or slow growing strain of Mycobacteria. 9. A method for producing a non-pathogenic bacterial recipient strain that is transgenic for at least one of its porin genes, comprising:

• a) providing a non-pathogenic strain of a mycolic acid-containing bacterium;
• b) introduction of a genetic element which carries an expression cassette with a functional porin gene together with a first resistance marker to be selected positively;
• c) Introduction of a second genetic element, which is a deletion cassette flanked by recognition sequences of a sequence-specific recombinase of the porin gene to be deleted together with a second resistance marker to be selected positively for selection of the genetic element and a third positive selection marker for selection of the deletion cassette, and one or carries several selection markers to be selected negatively;
• d) selection for the selection markers to be selected positively;
• e) selection for the selection marker to be selected negatively;
• f) introduction of a genetic element which bears a suitable sequence-specific recombinase and a positive selection marker for the selection of the genetic element and also a selection marker which is to be selected negatively;
• g) selection for the selection markers to be selected positively;
• h) selection for the selection marker to be selected negatively; and if necessary
• i) repeating steps a) to h) for deletion and replacement of a further porin gene.

10. The method according to claim 9, characterized in that the functional porin gene from a bacterium of the same or another genus of mycolic acid-containing Bak terie originates. 11. The method according to claim 9 or 10, characterized in that the non-pathogenic A strain of a mycolic acid-containing bacterium is selected from the genera Mycobacterium, Corynebacterium, Nocardia, Rhodococcus, Gordona, Tsukarmurella, Dietzia or other Gram-positive bacteria that contain mycolic acid. 12. The method according to any one of claims 9 to 11, characterized in that as ge Netic elements plasmids, transposons and / or phages can be used. 13. The method according to any one of claims 9 to 12, characterized in that the selection markers used are selected from the group consisting of Km ^r (from Tn 5 or Tn903), chloramphenicol resistance markers, rpsL, sacB, Am ^r (e.g. aacC1 or aac (3) -IVa), Sul ^r , mercury salt resistance marker, aph, Hm ^r and Str ^r . 14. The method according to any one of claims 9 to 13, characterized in that the ver Recombinases used are selected from the group consisting of FLP, Shuf flon, CRE, FimB, Int, XER, D protein, bacteriophage integrases (e.g. λ, HK22, P2, ϕ21, P22, HP1, SSV1, L5), plasmid integrases (e.g. pSAM2), transposon integrases (e.g. Tn915, Tn1545), AADA and AACA. 15. Non-pathogenic bacterial recipient strain, produced by a process according to one of claims 9 to 14, wherein the plasmids used pMN101, pMN250 and pMN234 are. 16. A method for identifying antibacterial substances against pathogenic mycolic acid-containing bacteria, comprising

• a) bringing at least one potentially antibacterial substance into contact with at least one non-pathogenic bacterial strain according to one of claims 1 to 8 and 15, and
• b) measuring the survival rate of the at least one non-pathogenic bacterial strain.

17. A method for identifying antibacterial substances according to claim 16, characterized in that the non-pathogenic bacterial strain is selected from the genera Mycobacterium, Corynebacterium, Nocardia, Rhodococcus, Gor dona, Tsukarmurella, Dietzia or other Gram-positive bacteria, the mycolic acid contain. 18. A method for identifying antibacterial substances according to claim 16 or 17, characterized in that as step b) additionally measuring the Ex pression of a marker gene, for example the luciferase gene and / or the GFP gene includes. 19. A method for identifying antibacterial substances according to claim 16 or 17, characterized in that as step b) a measurement of the porin Permeability properties of the at least one potentially antibacterial Substance is carried out. 20. Method for identifying antibiotics according to one of claims 16 to 19, characterized in that as potentially antibacterial substances modifi graced known tuberculosis therapeutics and / or other known antibiotics un be searched. 21. A method for identifying antibiotics according to one of claims 16 to 19, characterized in that as potentially antibacterial substances Sub punching from natural product banks or molecular banks. 22. Antibacterial substance obtained by a method according to one of the Claims 16 to 21. 23. Use of a non-pathogenic bacterial strain according to one of claims 1 to 8 and 15 for the identification of antibiotics against pathogenic mycolic acid-containing Bacteria, especially Mycobacteria.   24. Use according to claim 23, characterized in that modified known Tuberculosis therapeutics and / or other known antibiotics for identification of antibiotics are examined. 25. Use according to claim 23, characterized in that substances from nature Substance banks are used to identify the antibiotics. 26. In vitro test element for the identification of antibiotics against pathogenic mycolic acid containing bacteria, especially Mycobacteria, comprising at least one nonpa thogenic bacterial strain according to one of claims 1 to 8 and 15. 27. Kit comprising at least one non-pathogenic bacterial strain according to one of the Claims 1 to 8 and 15, together with suitable reagents and equipment.