AU646886B2 - Corynebacteria integron, method of transformation of corynebacteria by said integron, and corynebacteria obtained - Google Patents

Description

OPI DATE 02/03/92 DEMNDE AOJP DATE 09/04/92 PCT NUM I D 8'4423 91 BER PCT/FR91/fl0656 RE DE BREVETS (PCT) i6ro de publication internationale: WVO 92/02627 (51) Classification internationale des brevets 5 C12N 15/77, 15/90, 1/21 (C12N 1/21, C12R 1/15) Al 011) Num (43) Date de publication internationale: 20 f~vrier 19923 (20.02.921) (21) Num~ro de la dennande internationale: PCT/FR91/00656 (22) Date de dip6t international: 8 ao~nt 1991 (08.08.91) Donn~es relatives i )a priorit6: 90/10126 8 aoi~t 1990 (08.08.90) FR (71) Diposant (pour tous les Etats d~signbs sauf US): CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) [FR/FR]; 15, quai Aratole-France, F-75007 Paris (FR).

(72) Inv~nteurs; et Inventeurs/D~posants (US seulement) GUYONVARCH, Armel, Andr6. Yves [FR/FR: 21, avenue Flouquet, F- 94240 L'Hay-Ies-Roses REYES ALVARADO, Oscar, Julio [FR/FR], 132. rue du ChAteau, F-75014 Paris LABARRE, Jean, Christian, Jocelyn [FR/FR], 8, residence des Quinconces. F-91 190 Gif-sur-Yvette (FR).

BONAMY. C&line. Anne-Marie [FR/FR]; 565, all~e du Bois Fleuri, F-78830 Bullion LEBLON, Gerard.

Louis, Andr6 5, al1~e des Bathes, F-9 1940 Les Vhis (FR).

(74) Mandataire: WARCOIN, acques; Cabinet Regimbeau, 26, avenue K.16ber, F-751 16 Paris (FR).

(81) Etats d~sign~s: AT (brevet europ~en), AU, BE (brevet europ~en), BR, CA, CH (brevet europ~en), DE (brevet europ~en), DK (brevet europ~en), ES (brevet europ~en), FI, FR (brevet europ~en), GB (brevet europ~en), GR (brevet europ~en), HU, IT (brevet europ~en), JP, KR, LU (brevet europ~en), NL (brevet europ~en), SE (brevet europ~en), SU, US.

Publi~e Avec rapport de recherchie internauionalc.

Avtant r 'expirauion du MaJi pr~vu pour la rnodtfication des revendications, sera repub~ie si de telles mnodifications sont re ,Ues.

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(54) Title: CORYNEBACTERIA INTEGRON. IMETHOD OF TRANSFORMATION OF CORYNEBACTERIA BY SAID1 INTEGRON. AND CORYNEBACTERIA OBTAINED (54)Titre: INTEGRON DE CORYNEBACTERIE, PROCEDE DE TRANSFORMATION D'UNE CORYNEBACTERIE PAR LEDIT INTEGRON ET CORYNEBACTERIE OBTEN1'E SltnQ Not IGeta Ce clonaot 5m41 56auence d S14uhtan d& clonagt d (57) Abstract 5 -co t _il A corynebacteria integron is characterized in that it comprises a gene ensuring efficient selection in the said corynebacteria and a homologous sequence of the genome of the said corx'nebacteria, said sequences having been adapted to said bacteria. Application is round particularly in the production of proteins by the culture of corynebacteria transformed by said integron when it comprises a sequence coding for a protein of interest.

(57) Abr~g6 La pr~sente invention concerne un int~gron de coryn~bact~re caract~is6 en ce qu'il comporte: un g~ne assurant une s~1ection eflicace dans ladite coryn~bact~rie, une sequence hornologue du genome de ladite corynebact~ric, lesdites sequences avant k6 adapt~es ti ladite bact~rie. Application notaniment An Ia production de prot~ines pat culture de corvinbact~ries transformies par ledit int~gron lorsqu'iI comporte une sequence codant. pour une prot~ine d'int~r~t.

Voir iu verso 1 The present invention relates to the integration of predefined DNA sequences in the genome of corynebacteria.

i This integration may have two main objectives. It may be desired, on the one hand, to produce a particular protein by a strain of corynebacterium, either because this protein is not normally expressed by the strain concerned, or in order to make the strain overproduce a homologous protein. But, on the other hand, it may be desired to block the expression of a gene by interrupting it, which will lead to the annulment of the corresponding enzymatic activity and to the accumulation of the reaction substrate in the cell.

Corynebacteria, which include Brevibacterium in addition to Corynebacterium, are bacteria whose manipulation has so far proved to be quite delicate: firstly, because there are few or no methods permitting their transformation, and major restriction barriers exist as a result of which transformations using DNA from other bacter.al sources are most often ineffective.

Recently, it has been possible to demonstrate the possibility of transforming corynebacteria by electroporation methods. However, while the methods of transformation by autoreplicative vectors are useful, it is preferable in most cases and on an industrial scale to have available bacteria which have been transformed by integration inside the chromosome, that is to say strains which are stable with time with respect to the number of copies of the integrated element as well as with respect to its localisation.

The object of the present invention is to propose systems for integration inside the genome of corynebacteria.

The present invention relates to integrons of corynebacteria characterised in that they comprise: a gene ensuring an effective selection, hereafter called: "selection gene", in the said 40 corynebacterium, 2 a homologous sequence of the genome of the said corynebacterium, the said sequences having been adapted to the said bacterium.

"Integron" is understood as meaning a nonreplicative vector having the property of being integrated into the genome of a corynebacterium, it being possible for this integron to be in linear or circular form.

However, the integron is generally obtained from an autoreplicative plasmid which permits synthesis in a different host, for example Escherichia coli. But before the integration stage, all traces of DNA of noncorynebacterial origin will preferably be removed, except for the selection gene, and in particular all the sequences involved in the replication.

The genes for selection which are effective in the said corynebacteria are: either genes for resistance to a particular substance, an antibiotic in particular, or genes conferring a clearly identifiable phenotype, for example coloration and/or complementation.

In the present case, selection for resistance to an antibiotic is more particularly useful. Thus, there can be used: the AphIII genes conferring resistance to kanamycin, designated KmR, the Cat genes conferring resistance to chloramphehicol, designated CmR.

But other genes may be used, in particular genes for resistance to erythromycin.

"Homologous sequence" is understood as meaning sequences which correspond to those present in the transformed corynebacterium or which present a level of homology greater than 80%, they may be sequences from the same species or not, these sequences may furthermore be synthetic.

The sequences should be adapted or not, that is 3 to say they should take into account problems of restriction barriers existing in corynebacteria, using methods, some of which are described hereafter.

Preferably, this integron is obtained from a plasmid which comprises a replicative region in addition to the integron, the integron being flanked with restriction sites permitting its excision, and preferably with inverted repeat sequences corresponding to a restriction site not present in the integron, for example NotI, BstXI or SacI Thus, by digestion using the enzyme which recognises the said restriction site, the integron may be obtained directly and may be made circular, if necessary, before being used for the transformation of the corynebacterium, since the ends obtained are complementary.

Within the system according to the present invention, the integron is therefore preferably part of a plasmid vector which is capable of being replicated using origins of replication, or replicon, placed in the replicative region. Depending on the nature of the replicon present in this replicative cassette, the composite plasmid may be replicated either exclusively in corynebacteria if it consists of only one endogenous replicon, or in corynebacteria as well as in a foreign host, for example Escherichia coli, when two different replicons are combined with the plasmid, each permitting replication in its own host.

In this case, construction of the plasmid may be carried out inside systems different from corynebacteria, which may sometimes be useful given the difficulty of construction inside Corynebacterium and Brevibacterium.

By virtue of the presence of homologous sequences present at the same time in the integron and in the genome, the integron will be inserted inside the chromosome through recombination. Thus, inside the primary transformant, the gene initially cloned in the multiple cloning site of the integron is duplicated inside the bacterial chromosome (see diagram Figure 2a).

Such a duplication may have a technological

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4 usefulness in that the doubling of the gene may cause an increase in the activity encoded by this gene, in particular the corresponding enzymatic activity.

As the structure of the primary integrant corresponds to a direct tandem duplication of the homologous sequences surrounding the selection gene, it is possible to provide an amplification of this structure by selection of the growth of the primary integrant on a medium permitting detection of strains overexpressing the selection gene. Thus, when the selection gene is the gene for resistance to an antibiotic, the most resistant strains may be selected on media with an increasing antibiotic content, which strains should be suitable for an overexpression of the genes corresponding to the homologous sequences, but also to any gene or DNA sequence which would have been inserted inside the integron.

Naturally, the integron will preferably comprise, in addition to the homologous sequence(s), a sequence encoding a useful sequence, in particular a useful peptide or protein which may be homologous, that is to say be obtained from a corynebacterium, or heterologous, that is to say be obtaine,: from other bacterial species, but may also be of eucaryotic or synthetic origin.

These sequences will preferably comprise elements ensuring their expression in corynebacteria or they will be inserted inside a frame so they can be expressed by the expression elements of the host bacterium.

In the case of Corynebacterium, it may be useful for example to obtain an overexpression of some enzymes, in particular gltA or gdhA.

As previously stated, it is possible to use this system to ensure interruption of a gene by using an integron according to the invention. Through interruption or through substitution, as described in Figure 2b, the corresponding gene is inactivated, this leads to an overproduction of the substrate of the enzyme encoded by the corresponding gene.

In general, the integron will be designed in the 5 form of an integration cassette, that is to say that in addition to the selection gene and the homologous sequence, which in some cases may be identical, a sequence comprising several cloning sites will be provided which will permit insertion of DNA sequences and/or of genes as desired.

In all cases, for the purpose of genetic confinement, attempts will be made to provide an integron lacking replicative DNA from another species.

It is also possible to provide more complex integration systems, in particular integration systems which comprise in addition the sequences of a transposable element. The sequences of transposable element may be all the sequences which ensure transposition with the exception of the proteins encoded by the corynebacterium, but they may also be transposable elements which lack the sequences encoding transposases.

Among the transposable elements, the Mu phage should be mentioned, in particular in the form of a miniMu phage. In the case of transposable elements such as the miniMu phage, as will be seen hereafter, markers were used which may be part of the phage or of different origin, for example coloured markers.

The invention also relates to integrons using the transposable elements obtained from various corynebacteria, in particular from Brevibacterium, more specifically ISaBI such as described in Figure 9.

Example 10 presents the characterisation of the element ISaB1 and Example 11 gives a general method which makes it possible to select and identify this type of transposable element.

The present invention also relates to integrons comprising a sequence obtained from transposed elements, in particular elements encoding transposases and/or repressors of transposition.

The integrons according to the invention may comprise all or part of the relevant sequences, in particular the one corresponding to ISaBi.

This type of integration structure comprising a f* 6 fragrrnt of miniMu phage, a homologous DNA sequence and a selection gene may make it possible to obtain, as in the case using the structures previously described, either an overexpression of a particular gene, or the disruption of a gene when necessary.

The latter structures, although different from the preceding structures, will nevertheless be called, for simplicity, "integron".

The present invention also relates to corynebacterium strains obtained by integrative transformation using the integrons previously described, in particular when the integron has been introduced by electrotransformation.

Among the corynebacteria strains which may be used, there should be mentioned more particularly: B. lactofermentum, B. flavum, C. glutamicum, C. melassecola, because of their industrial usefulness.

In the case where the cloning is carried out in a different host from the corynebacterium which is final host of the integron, the transfer of the integron may take advantage of the possible replicative properties of the construct inside the corynebacterium in order to adapt the integron to the corynebacterium. Thus, the procedure will start by introducing the plasmid comprising a replicative region, in addition to the integron, inside the very strain in which the integration will be carried out, this integron thus adapted will then be recovered by extraction of the plasmid and then digested with the enzyme or enzymes which release the integron, the purified fragment then being autoligated or not and the ligation product being used for the integrative transformation; in this case, the restriction barriers no longer constitute a problem.

In some cases it may be useful to go through an intermediate corynebacterium, in particular in the case S.where the DNA is of E. coli origin, it may be useful to

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7 adapt the integron to Brevibacterium lactofermentum before adapting it to Corynebacterium melassecola.

Finally, the invention relates to the use of Corynebacteria according to the invention in industrial processes, in particular for the preparation of proteins or metabolites employing the integron according to the invention.

The examples hereafter will make it possible to better illustrate the advantages of the present invention.

In the attached figures: Figure 1 is a schematic representation of the preparation of an integron starting with a replicative plasmid, Figure 2 is a schematic representation of the insertion of an integron, by single recombination (a) by double recombination S Figure 3 is a schematic representation of the structure of pCGL519, Figure 4 is a schematic representation of the percentage of resistance to kanamycin as a function of time for two transformed strains, Figure 5 is a schematic representation of the structure of miniMu phages, MudII1681, MudII1681-Cat, Figure 6 is a schematic representation of the plasmids pCGL107 and pCGL107::Mud+, S Figure 7 is a schematic representation of the integration of the desired integrons containing miniMu or not, Figure 8 represents the restriction map of the insertion element of Brevibacterium lactofermentum CGL 2005 (B115) cloned from an insert in the 3' terminal end of the lac operon, Figure 9 represents the ISaBl sequence, t"0- 9 8 Figure 10 represents the restriction map of ISaB1, Figure 11 represents the restriction map of the pCGL330 plasmid, Figure 12 represents the restriction map of the pCGL331 plasmid.

EXAMPLE 1 Construction of a chromosomal DNA library of Corynebacterium melassecola ATCC17965 and cloning of the qltA gene The chromosomal DNA of the C. melassecola strain ATCC 17965 was prepared following the modified method of Ausubel et al. (1987). A controlled digestion by the MboI restriction endonuclease (Boehringer) was carried out on pg of this DNA following the procedure described in Maniatis et al. (1982). The DNA fragments were separated according to their size on a sucrose gradient as described by Ausubel et al. (1987). The fragments of size between 6 and 15 kb were selected for constructing the library.

The pUN121 cloning plasmid (Nilsson et al., 1983) was prepared by the method of Birnboim and Doly, (1979) from the freely available E. coli strain GM2199. The plasmid was linearised by the BclI restriction endonuclease (Boehringer).

The library was constructed by ligation with the T4 DNA ligase (Boehringer) under the conditions described by Ausubel et al. (1987), from 1 pg of pUN121 plasmid linearised by BclI and 2 pg of the 6 to 15 kb DNA fragments described above. The ligation mixture was introduced into the E. coli strain DH5alpha by electroporation following the procedure described by Dower et al. (1988). The E. coli clones bearing the recombinant plasmids were directly selected by their ability to grow on LB medium containing 10 pg/ml of tetracycline. The plasmids of all the tetracycline-resistant clones were prepared by the method of Birnboim and Doly (1979). These plasmids taken as a whole correspond to the DNA library.

The E. coli strain W620 deficient in citrate synthase activity was transformed with the C. melassecola /1,7 9 ATCC17965 DNA library. An E. coli W620 transformant clone capable of growth on minimum selection medium containing tetracycline was selected. This clone bears a pCGL508 recombinant plasmid. Various subclonings have made it possible to shorten the C. melassecola DNA fragment bearing the complete gltA gene to a 3.5 kb DNA fragment delimited by two HindIII restriction sites.

EXAMPLE 2 pCGL519 inteqron The preparation diagram of the integron is represented in Figure 1.

The aphIII gene was chosen as selection gene; it confers resistance up to 600 pg/ml of kanamycin when one copy is integrated in the chromosome. 25 Ag/ml are normally used. The 3.5 kb HindIII fragment bearing the gltA structural gene encoding the C. melassecola citrate synthase obtained in Example 1 was chosen at the start as homologous DNA fragment of the corynebacterial genome and inserted in one of the unique sites of the multiple cloning site (the HindIII site). The restriction sites delimiting the integron which are intended to be the most widely used in the integration strategy are the NotI sites which correspond to an 8-nucleotide sequence, and BstXI sites. The enzyme BstXI recognises the sequence

(CCAN

5 NTGG); consequently, it cuts as frequently as a 6nucleotide enzyme and it can be expected that several fragments will be produced. But the fragments released are recombined depending on the nature of the internal sequences of the different BstXI sites, which should finally lead to the reconstitution of the starting fragment alone.

The pCGL519 plasmid (Figure 3) is an example of a versatile plasmid capable of producing an integron. The integration of its integrative cassette in C. melassecola has been tested using a plasmid initially obtained from E. coli. pCGL519 consists of two replicative and integrative fragments delimited by the NotI sites. The first fragment corresponding to the integron which comprises a multiple cloning site, the aphIII selection 10 gene and a homologous HindIII fragment of the chromosome bearing the gltA gene which encodes the citrate synthase.

The second fragment comprises the replicative region of the pBLI plasmid (3 kb SspI-HpaI fragment) which is replicated in corynebacteria, the replicative region of the pACY184 plasmid which is replicated in E. coli, ori pl5A, and the origin of replication of the transcomplementable M13 phage. The pCGL519 plasmid was constructed in E. coli by insertion of a 3.5 kb HindIII fragment containing the gltA gene inside a pCGL243 vector.

As represented in Figure 1, after cloning, pCGL519 is used to transform B. lactofermentum. At the time of the transfer of pCGL519 into Brevibacterium lactofermentum CGL2002, the NotI fragment containing the origins of replication was substituted because the replicative region of pBLI was inactivated in E. coli.

This shows a further advantage of the cassette structure.

EXAMPLE 3 Integration The transfer of pCGL519 into Corynebacterium melassecola ATCC17965 which is very restrictive towards E. coli was achieved using the same plasmid extracted from B. lactofermentum. The system proposed makes it possible to isolate the pCGL519 plasmid from the C. melassecola strain ATCC17965 which is completely restrictive towards E. coli but only p'artially towards B. lactofermentum. Thus, an integrative cassette possessing the alteration of the recipient strain was prepared.

After digesting the pCGL519 plasmid obtained from C. melassecola ATCC17965 with the NotI restriction endonuclease (Boehringer), the integron containing the gltA gene and the AphIII selection gene was isolated and purified from a low-melting-point agarose gel. The integron thus purified was then subjected to an autorecircularisation by ligation. The ligation mixture was then introduced into the C. melassecola strain ATCC17965 by electroporation (Bonamy et al., 1990). The

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11 C. melassecola clones resistant to 25 pg/ml of kanamycin were subjected to analysis.

500 transformants were obtained. 31 of the analysed did not possess the pCGL519 plasmid and 20 of them were analysed by Southern blot after XbaI digestion.

They all correspond to the same integration event which is carried out by homologous recombination in the gltA region. Depending on the nature of the ligation product (circular monomeric molecule or linear or circular polymeric molecule), the primary integrants may be int; ~.pretated as being obtained from a single or double "crossing-over". Pulsed field analysis after NotI digestion followed by a hybridisation with a probe corresponding to the 3.5 kb HindIII fragment containing gltA was performed. The integration of a single copy of the integron is confirmed by this analysis.

The model involves duplication of the wild copy of the gltA gene: the enzymatic activity was measured, it is multiplied by a factor of 1.82 which is consistent with the interpretation of the blots and shows that the copy integrated is not inactivated. The results are assembled in Table 1 with respect to the wild strain and the strain transformed by a replicative plasmid. The stability of the integrated structure was measured with respect to the percentage of kanamycin resistant cells after approximately thirty generations (Figure 4) and with respect to the enzymatic activity of citrate synthase which remain stable.

Amplification of the integrated structure was performed by selection of growth on a dish containing an excess of kanamycin, selection at 800 pg/ml, then 1,000 pg/ml and then 1,000 pg/ml of kanamycin and neomycin. A direct tandem amplification was obtained.

Despite the stability of the amplified structure and the kanamycin resistance, the high level of enzymatic activity initially obtained in the case of citrate synthase was not subsequently maintained. It is possible that a specific inactivation of the gltA gene occurred.

>1" .0/ 12 EXAMPLE 4 Construct based on miniMu The Mu derivatives chosen in this example are MudII 1681 and MudII 1681-Cat (KmR and CmR respectively) which have a size of 14.8 kb and 16.6 kb respectively (Figure The miniMu MudII 1681-Cat is a derivative of the transposon MudII 1681 (Castilho et al., 1984). It possesses the elements required for the transposition previously outlined (except HU) and also the heatsensitive repressor gene C (regulator of the expression of the transposases A and a gene for resistance to antibiotics (aphlI and cat respectively) and the lacA, lacY and lacZ' genes. The latter starts at the 8th codon and makes it possible to detect translational fusions of proteins when Mud is inserted in a reading frame.

These transposons have been transferred into Corynebacteria. After integration of the transposons inside the chromosome, a method, described in Example 8, made it possible to amplify the integrated copy. In combination with this amplification, the target gene of the integration event (the structural gene for glutamate dehydrogenase) has, in numerous cases, also been amplified with an increase in the corresponding enzymatic activity up to a factor of EXAMPLE Construction of.vectors for the transfer of miniMu The integrative vector (pCGL107 Figure Ci contains the gdhA gene (Gdh') interrupted by the kanamycin resistance marker KmR (aphIII), the pUN121 replicon (ori) (Nilsson et al., 1983) and the genes conferring the resistance to tetracycline (TetR) and to ampicillin (AmpR). Given that this vector is not replicative inside Brevibacterium lactofermentum, it is integrated by single "crossing over" into the homologous site of the gdhA gene.

MudII 1681-Cat was introduced inside the integrative vector pCGL107 by minimuduction from E. coli 0R1836 into the MC4100 strain. Among the various inserts obtained, one which gave a lac- phenotype in E. coli 13 (pCGL107::Mud+, Figure 6) was selected. A disabled Mud (pCGL107::Mud-, Figure in which the genes for the transposases A and B were deleted bj .indIII digestion, was prepared from this same plasmid.

Other transfer strategies were tested; MudII 1681 was introduced into Corynebacteria by placing the transposon inside two other types of vector: A suicide vector (nonreplicative, nonintegrative) pEVll::Mud, which is a derivative of pUC18 Jacking the lac genes, in which MudII 1681 has been introduced.

A shuttle vector (pCGL229) possessing the pBL1 replicon (HindIII-Hpal fragment), the pl5A replicon and the Tn9 cat gene.

MudII 1681 was introduced inside the shuttle vector by minimuduction in E. coli Rec+. Among the various inserts obtained, one which gave a Lac- phenotype (pCGL229::Mud+) was selected. A disabled Mud, pCGL229::Mud-, in which the genes for the transposases A and B were deleted by PstI digestion, was prepared from this vector.

EXAMPLE 6 Efficacy of electrotransformation of the constructs and their transfer into Brevibacterium lactofermentum An E. coli (DH5alpha) strain and two Brevibacterium lactofermentum strains CGL2002 and CGL2005 (B115) were electrotransformed with the vectors previously described. These two strains are partially permissive towards the DNA obtained from E. coli. The experiments gave the results presented in Table 2. The following commenjs may be made: The transformation efficiencyis substantially reduced in E. coli, be it in the case of the shuttle vector pCGL229 or in the case of the vector pCGL107 (which can replicate therein), when Mud+ is present in the vector, which is not the case when the transposition genes have been deleted. This phenomenon which is typical of replicative plasmids bearing active Mu derivatives may be attributed to a very high t Ll.

14 expression of the Mu transposase inside its natural host. This shows that in the constructs presented above, the Mud+ transposons used (MudII 1681 and MudII 1681-Cat) are effectively active.

It is not possible to obtain transformants with the shuttle vectors pCGL229::Mud+ or (or with the suicide vector pEV11::Mud) in any of the Corynebacterium strains tested. In the case of the pCGL229 derivatives, it is possible that the pBL1 replicon was inactivated during minimuduction into E. coli Rec+, which would explain the inability of the vector to multiply inside B. lactofermentum.

Transformants were obtained in B. lactofermentum CGL2005 (B115) with the integrative vector pCGL107 and its derivatives. The transformation efficiency obtained with pCGL107::Mud+ and pCGL107::Mud- is similar, which shows that miniMu MudII 1681-Cat A+B+ is not transposed with a high efficiency when it is introduced into B. lactofermentum using an integrative vector.

The decrease observed (factor of 20) in the transformation efficiency of the two pCGL107 derivatives compared to pCGL107 itself is probably due to the increase in the size of the transformant plasmid (10 kb in the case of pCGL107, 26.7 kb in the case of pCGL107::Mud+ and 22.1 kb in the case of pCGL107::Mud-).

EXAMPLE 7 Study of the events for the integration of pCGL107::Mud+ inside B. lactofermentum CGL2005 (B115) Transformation of the strain CGL2005(B115) with pCGL107::Mud+ (hereafter named pCGL320) has made it possible to obtain 147 kanamycin-resistant clones Ag/ml), but no transformant was obtained when selecting for resistance to chloramphenicol (5 pg/ml).

However, 103 of these clones are resistant to chloramphenicol after replicating on chloramphenicol. It therefore appears that a single copy of the Tn9 cat gene is not sufficiently expressed in order to allow primary selection of colonies; in contrast, this expression is 15 sufficient for determining a resistant phenotype by subsequent tests using streaks. All the clones obtained are of the phenotype lac- which corresponds to the phenotype observed in E. coli.

The two types of integrants obtained (the type 1 transformants, KmRCms and the type 2 transformants, KmRCmR) may correspond respectively to the substitution of the gdhA gene by a double "crossing over" event and to the integration of the complete plasmid inside the gdhA site by a single "crossing over" event (Figure The observations in favour of this interpretation are as follows: Glutamate dehydrogenase assay (according to the method of Meers et al., 1970) in the type 1 and 2 transformants (Table 5 out of 7 type 1 transformants (for example K2 and K3, Table 3) have no gdh activity, which is consistent with the substitution of the gdhA gene by the interrupted gene.

4 of the 5 type 2 transformants have a gdh activity which is similar to the control (KC2 and KC4 for example, Table 3).

Southern blot molecular analyses corresponding to BamHI digestion of the type 1 transformants (K2) and the type 2 transformants (KC2 and KC4) and detection of the characteristic bands (shown in Figure 7) by the plasmid probe pCGL107 (results not presented).

They indicate that the molecular structure of the gdh- transformants (K2) conforms to a gene substitution. They also confirm that the gdh+ transformants (type 2 events (KC2 and KC4) and some type 1 events are obtained by integration of the plasmid in the gdhA site.

EXAMPLE 8 Selection of possible transpositions The type 2 transformants (Km R and CmR) do not sufficiently express the chloramphenicol resistance gene in order to obtain growth of isolated colonies in the presence of 5 ig/ml of chloramphenicol. We have therefore ATL looked for the subclones CmR in these transfoimants with ''Cc 16 the hope of selecting transpositions of Mud (which bears the cat gene). These subclones have been obtained at a frequency close to 1 for 10 5 cells. After replicating on Xgal, these clones exhibit a whole graduation of colours from white to blue (30% are clearly blue). This result is confirmed by the measurement of beta-galactosidase activities (Table It may indicate a transposition of Mud, amplifying the resistance to chloramphenicol and giving rise to beta-galactosidase activities by protein fusion in the insertion site. Indeed, the same experiments performed with pCGL107::Mud- produced the same result indicating that these events are not due to transposition events.

EXAMPLE 9 Demonstration of tandem amplification in pCGL107::Mud+ plasmid chromosome It appears that almost all these Lac+ clones previously isolated (with the exception of KC3T4) also have an amplified gdh activity (Table Moreover, besides one exception (KC3T4), the beta-galactosidase (assayed according to Miller, 1972) and gdh activities are almost proportional. The same remark may be made concerning the assay of chloramphenicol acetyl transferase (according to the method of Shaw, 1975, Table This result is inconsistent with the transposition of Mud which never carries adjacent sequences during its transposition. Rather, it indicates a tandem amplification in the chromosome of the repeat unit pUN-Mud-gdh which is delimited by sequence homologies. This poin.'. has been confirmed by digestion of the genomic DNA of the strains KC3, KC3T1 and KC3T3 with BamHI, NotI and Xbal. Not only is the BamHI band inside Mud (and equal to 7 kb) amplified, but also the bands containing the ends of Mud (11 kb and 2.4 kb), which conforms to a tandem amplification and which prevents a transposition event.

Moreover, the NotI digestion (which cuts only once inside MudII 1681-Cat) and the XbaI digestion (which cuts only once inside gdh') substantially amplify the 24 kb band of the repeat unit.

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17 This amplification appears to be relatively stable given that the beta-galactosidase activity remains at a level of 70% of the initial activity after generations without selection pressure. The Cat and Gdh activities also exhibit a loss of 30% after generations. This tandem amplification is similar to the results of Albertini et al. (1985) and of Janniere et al.

(1985) in Bacillus subtilis. The beta-galactosidase activity of the amplified clones is attributed to an amplified parasite translation (outside the reading frame of the ampicillin resistance gene where the lac operon is fused) existing in B. lactofermentum and undetectable in E. coli.

EXAMPLE Study and characterisation of the insertion element ISaB1 An insertion element was isolated by recovering plasmids in E.coli DH5alpha from the amplified DNA of KC3T4.

The genomic DNA of the B. lactofermentum strain CGL2005 (B115), of some derivatives and of other strains of corynebacteria, has been probed using a probe containing the insertion element previously isolated. Firstly, the 3.5 kb PvuII fragment inside Mu, arising from the amplified DNA in KC3T4 and containing the insertion element, was used to probe the initial blot which contains the BamHI-digested DNAs from integrants and various amplified strains (including KC3T4). As the insert does not contain a BamHI site, this experiment makes it possible to reveal the BamHI genomic fragments containing at least one insert or one insertion fragment.

For the Kl- strain (which corresponds to a type 1 substituted integrant obtained from pCGL107:Mud-), five bands appear, revealing the presence of several copies (intact or not) of the insert.

The BamHI digestion of genomic DNAs obtained from B. lactofermentum CGL2005 (B115) showed four bands which are also observed for Kl- (18 kb, 5.9 kb, 5 kb and 4.5 kb in size respectively); the fifth band which is observed for the other strains Kl-, KC1- and KC3 (6.5 kb in size) 18 may indicate a transposition in a strain CGL2005 (B115) segregant from which these strains were derived.

Two bands, 18 kb and 4.5 kb in size, appear which are common to two different brevibacteria lines, (i) Brevibacterium lactofermentum CGL2005 (B115) and (ii) Brevibacterium lactofermentum CGL2002; in contrast, the strain CGL2002 does not show other bands. This indicates mobility of the element. The Corynebacterium melassecola strains produce faint hybridisation signals with the insert indicating the existence in this species of other different but related sequences.

A first mobile insertion element specific for B. lactofermentum has been identified and cloned; it is named ISaBi and several copies (2 to 5 copies) may be present in the genome. It is capable of being transposed several times in different sites in an amplified region.

Its detailed restriction map is known. Different but related sequences exist in the genome of other corynebacteria.

ISaB1 consists of 1288 base pairs; the ends may be identified because ISaBi is inserted in a fragment which corresponds to the terminal region of the lac operon which was sequenced by Hediger et al.

(Biochemistry Proc. Natl. Acad. Sci. USA 82, 1985). ISaBl was inserted between the nucleotides 5575 and 5576, duplicating a 5 bp target sequence (CCGAT) (Figure 8).

The entire sequence of ISaBl is given in Figure 9 and the restriction map in Figure 10. Two open reading frames have been identified which may correspond, given the sequence analyses, to the transposase structural gene and to the transposition repressor gene.

EXAMPLE 11 Trapping vector for insertion elements and transposons The ISaBI insert has been obtained during gene amplification studies in the chromosome of B. lactofermentum CGL2005 (B115). In order to isolate all the insertion elements capable of being transposed in corynebacteria, special integrative vectors, pCGL330 and pCGL331, were constructed (Figures 11 and 12). These two I, i y 19 vectors consist of a first fragment derived from the pUN121 vector. The pUN121 plasmid is replicative in E. coli conferring the resistance to ampicillin; it bears a sequence which codes for the lambda phage cI repressor which inhibits the expression of an operon fusion between the lambda phage PL promoter and a tetracycline resistance gene. Insertion inside the cI gene will, as a result, inactivate the repressor which will thereby permit expression of the tetracycline resistance gene which is expressed in corynebacteria under the control of PL. The insertion events may thus be selected for the resistance to tetracycline. The vector pUN121 was linearised at the SspI site and fused with a second fragment obtained by digestion of the vector pCGL107 with EcoRI, filled using Klenow fragment to give, after transformation in E. coli DH5alpha, the vectors pCGL330 and pCGL331 which differ in their direction of cloning. The EcoRI fragment derived from pCGL107 contains a selection gene for the direct transformation of corynebacteria (the aphIII gene which confers the resistance to kanamycin) and a fragment containing a homologous region of the gdhA gene (glutamate dehydrogenase structural gene) which will serve as integration site inside the chromosome of corynebacteria.

The B. lactofermentum strains CGL2005 (B115) and CGL2002 were electrotransformed for resistance to kanamycin with the pCGL330 and pCGL331 plasmids. The transformation frequency was approximately 10 3 per pg in each case, which is compatible with an integration of the plasmids. The transformants (such as CGL2005::pCGL330 and CGL2005::pCGL331) are sensitive to tetracycline which confirms that the regulation of PL by cI functions well in the transformants. The frequency of tetracyclineresistant segregants was measured after about generations.

Mutations inactivating the cI gene appear in the strains possessing the integrated plasmids pCGL330 and pCGL331 at a frequency of 2 x 10-6 per generation.

Recovery of plasmid was achieved from genomic DNA

-I)

20 extracted from tetracycline-resistant segregants isolated from the strains CGL2005::pCGL331 and CGL2005::pCGL330 and digested by the PstI enzyme.

These digested DNAs were ligated and the product of the ligation was used to transform the strain of E. coli for the resistance to tetracycline. The recovered plasmids were analysed and in 7 out of 9 tetracycline-resistant clones, an insertion element was identified. In one case (arising from CGL2005::pCGL331), an insertion element identical to ISaB1 and located inside cI was identified (1.2 kb in size, possessing the unique AccI, EcoRV and XhoI sites). In another case (arising from CGL2005::pCGL330), an insertion element different from ISaBI (1.0 kb in size, possessing an AccI site but no EcoRV or XhoI site) was identified.

The transposon trapping vector is functional; in most cases, the mutation obtained is an insertion; the frequencies of resistance to tetracycline therefore fairly accurately measure the frequencies of transposition; the most mobile elements have therefore been identified; among them, ISaB1 has been reisolated and another insertion element different from ISaBI has been identified.

The strains mentioned have the following origins: Escherichia coli DH5alpha Gibco BRL MC4100 Casadaban (1976) OR1836 Reyes Brevibacterium lactofermentum CGL2002 Bonamy et al. (1990) CGL2005 (B115) Bonnassie et al. (1990) Corynebacterium melassecola ATCC17965 ORSAN ATCC17965::gltA (the present application) Four of these strains were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of Institut Pasteur (Paris) on 23 July 1991: Corynebacterium melassecola ATCC 17965::gltA n, I-1124

'A

21 Escherichia coli CR1836 no 1-1125 Brevibacterium lactofermentun CGL2005 (B115) no 1-1126 Brevibacterium lactofermentun CGL2002 no 1-1127 The strain DH5alpha is available in the Clontech Laboratories Cataloque, no. C1021-1, (Palo Alto, CA, USA) and the strain MC4100 at ATCC under the no. 35695.

*1 TABLE 1 SPECIFIC ACTIVITY OF CITRATE SYNTHASE STRAINS SPECIFIC ACTIVITY RELATIVE ACTIVITY ATCC17965 1.041 1 ATCC17965: :gltA 1.893 1.82 ATCC17965(pCGL519) 5.340 5.13 Specific activity of citrate synthase in 11mol CoASH/min/mg of proteins 23 TABLE 2: Efficiency of transformation of vectors hearing the miniMu transposons Bacterial strains E. coli DH5a B.lactofermentum CGL2 002 5 x 10' 0 2 x10' 0 pCGL229 pCGL229: :Iud+ pCGL2 29: :MudpCGLlO7 pCGL1O7: :Mud+ pCGL107: :Mud- B. lactofermentum CGL2005 (B115) 106 n.d.

n.d.

4 x 102 5 X 102 4 x10 10~5 5 x 102 10o 5 0 3 0 n. d.

24 TABLE 3: ASSAY OF GLUTAMATE TRANS FORMANTS Bacterial strain DEHYDROGENASE IN THE PRIMARY Specific activity of Gdh in pmol of NADPH 2 used/mmn/mg of protein Initial strain KmsCmS CGL2005 (B115) 2.18 transformant iRcmS 06 :s0. 06 O06 <O 06 2.18 06 trans formant KmRCmR KC2 KC 3 KC4 CC 5 KC7 2.24 2.12 2.18 3.45 2.06 25 TABLE 4: ASSAY OF -GALACTOSIDASE AND GLUTAMATE DEHYDROGENASE ACTIVITIES IN THE AN-1LIFIED INTEGRANTS Bacterial Strdin fi-galactosidase act- .vity Glutamate dehydrogenas e specific activity Initial strain Km SCM CGL2005 (B115) Primary integrants Km RCMR KC2 KC 3 Amplified integrants KG 3T13 KC 3T2 KC3T3 KC3T4 KG 3T 5 KC3T6 KC3T7 KC2 Ti 3.7 2.48 6.2 4.5 2 .24 2.06 27.9 20.7 48.2 32.2 19.7 19 .0 34.5 7.4 18.2 16.0 49.6 2 .24 8.55 11.9 28.0 2.73 26 TABLE 5: ASSAY OF CAT, #-GAl and

STRAINS

GDH ACTIVITIES IN THE AM4PLIFIED Bacterial strain pB-gal activity Cat specific activity Gdh specific activity S

S

Initial strain Km Cm CGL2005 (B115) Primary integrant KR mS K2 K3 Primary integrant Km RCMR KC 3 KC 7 Amplified inte~grants KC3T 1 KC3T3 KC3T4 3.7 :50.07 2.18 <0 .07 :50.07 -50.006 :50. 006 4.5 2.72 3.4 2.06 1.82 27.9 48. 2 32.2 19 .7 19.7 33.3 18.4 13.6 18.2 49.6 2.24 8.55

'I

.9 27

REFERENCES

Albertini A.M. and Galizzi A. (1985). Amplification of a chromosomal region in Bacillus subtilis; J. Bacteriol., 162:1203-1211 Ausubel Brent Kingston Moore D.D., Seidman Smith J.A. and Struhl K. (1987) in "Current protocols in Molecular Biology", Greene Publishing Associates and Wiley Interscience Birnboim H.C. and Doly (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7:1513-1523 Bonamy Guyonvarch Reyes David F. and Leblon G. (1990) Interspecies electro-transformation in Corynebacteria. FEMS Microbiology Letters 66:263-270 Bonnassie S. Oreglia J. Trautwetter A. and Sicard A.M.

(1990) Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermemtum, FEMS Microbiology Letters 72:143146.

Casadaban M.J. Transposition and fusion of the lac genes to selected promoters in E. coli using bacteriophages lambda and Mu. J. Mol. Biol. 104 (1976) 541-555 Castilho Olfson P. and Casabadan M.J. (1984) Plasmid insertion mutagenesis and lac gene fusion with miniMu bacteriophage transposons, J. Bacteriol. 158:488- 495 Dower Miller J.F. and Ragsdale High Efficiency transformation of E. coli by high voltage electroporation. Nucleic Acid Res. 16 (1988) 6127-6145 Janniere Niaudet Pierre Ehrlich S.D. (1985) Stable gene amplification in the chromosome of Bacillus subtilis, Gene 40:47-55 28 Maniatis Fritsch Ed. and Sambrook J. (1982).

Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Meers Tempest D.W. and Brown C.M. Glutamine (amide):2-Oxoglutarate Amino Transferase Oxido-reductase (NADP), an Enzyme Involved in the Synthesis of Glutamate by Some Bacteria J. General Microbiology 64 (1970) 187- 194 Miller J. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) p352- 355 Nilsson Uhlen Josephson Gatenbeck S. and Philipson 1983. An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nuc. Acid. Res. 11, 8019-8030 Shaw W.V. (1975) Chloramphenicol acetyl transferase from chloramphenicol resistant bacteria. Metho. in Enz. 43: 737-755

N

29 International Application No: PCTI FR91 00656

MICROORGANISMS

optional Sheet in connection with the microorganism referred to an page 1.0~ l ine-.. 2-6 of the desciptionI A. IDENTIFICATION OF DEPOSIT'I Further deposits are Identified on an additional sheet [a Name of depositary Institution COLLECTION NATIONALE DE CULTURES DE MICROORGANISMS INSTITUT PASTEUR Address of depositary Institution (inciuding postal code and country) rue du Docteur Roux 75724 PARIS CEDEX

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Claims (21)

1. Integron of Corynebacterium characterised in that it comprises: a gene ensuring an effective selection in said corynebacterium, a homologous sequence as hereinbefore defined of the genome of said corynebacterium, said sequences having been adapted to said bacterium.

2. Integron according to claim 1, characterised in that it is obtained from a plasmid which comprises a replicative region in addition to the integron, the integron being flanked with inverted repeat sequences corresponding to a restriction site not 1 o present in the integron.

3. Integron according to claim 2, characterised in that the replicative region comprises an origin of replication which is effective inside a bacterium which is not a Corynebacterium.

4. Integron according to claim 3, characterised in that the origin of replication is effective inside E. coli. Integron according to any one of claims 2 to 4, characterised in that the replicative region comprises an origin of replication which is effective in Corynebacteria.

6. Integron according to any one of claims 1 to 5, characterised in that it further comprises the sequences of a transposable element.

7. Integron according claim 6, characterised in that the transposable element comprises sequences obtained from a Corynebacterium.

8. Integron according to claim 7, characterised in that the sequence is obtained from Brevibacterium.

9. Integron according to claim 8, characterised in that the sequence is obtained from ISaB1 as described in Figure 9. Integron according to any one of claims 6 to 9, characterised in that it further comprises the sequences of a transposable element ensuring the transposition with the exception of the proteins encoded by the Corynebacterium.

11. Integron according to any one of claims 6 to 10, characterised in that the S" 30 sequences of the transposable element lack the sequences encoding transposases.

12. Integron according to any one of claims 1 to 11, characterised in that it comprises an effective sequence as hereinbefore defined.

13. Integron according to claim 12, characterised in that the effective sequence encodes an effective peptide or protein.

14. Integron according to claim 13, characterised in that the effective protein is an homologous protein. Integron according to claim 13, characterised in that the effective protein is an heterologous protein. Sj [N:\LIBAA00045:JJJ 32 of 2 0

16. Integron according to any one of claims 13 to 15, characterised in that the effective protein is an enzyme.

17. Integron according to claim 16, characterised in that the sequence encoding a effective protein is at least one of the genes gltA and gdhA, both genes being as hereinbefore defined.

18. Integron according to any one of claims 2 to 17, characterised in that the restriction site not present in the integron is at least one of NotI and BstXI as hereinbefore defined.

19. Integron according to any one of claims 1 to 15, characterised in that said sequences are adapted for a strain of Corynebacterium. Integron according to claim 19, characterised in that said sequences are transferred into a strain of Brevibacterium before being adapted in Corynebacterium.

21. Integron according to claim 20, characterised in that said sequences are amplified.

22. Amplified integron according to claim 21, characterised in that the amplified sequences are stable.

23. Integron according to claim 1, characterised in that it contains a homologous sequence of Corynebacterium melassecola which can be integrated inside the chromosome of Brevibacterium lactofermentum. 24 Integron of Corynebacterium as herein defined and substantially as hereinbefore described with reference to any one of the Examples or Figures. Process for transforming a strain of Corynebacterium with an integron according to any one of claims 1 to 24, characterised in that the integron is introduced into the said strain by electroporation.

26. Process for transforming a strain of Corynebacterium with an integron being substantially as hereinbefore described with reference to any one of the Examples or Figures.

27. Corynebacterium being the product of the process according to claim 25 or claim 26. 30 28. Corynebacterium according to claim 27, characterised in that it is a strain of: B. lactofennentum B. flavum C. glutamicum C. melassecola Dated 30 November, 1993 Centre National de la Recherche Scientifique (CNRS) Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 1N:\LIBAA00045:JJJ 33 of 2 ex