CN1061624A

CN1061624A - The integron of bar shaped bacteria is with the method for said integron conversion bar shaped bacteria and by it bar shaped bacteria that obtains

Info

Publication number: CN1061624A
Application number: CN91108861A
Authority: CN
Inventors: A·盖杨瓦基; O·R·阿瓦尔多; J·拉巴瑞; C·博纳米; G·莱博恩
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 1990-08-08
Filing date: 1991-08-08
Publication date: 1992-06-03
Also published as: WO1992002627A1; AU646886B2; PT98602B; AU8442391A; IE912791A1; EP0495078A1; FI921527A; ZA916216B; KR920702423A; HUT63656A; IL99126A0; PT98602A; BR9105857A; MX9100560A; JPH05502797A; CA2067240A1; FR2665711B1; FR2665711A1; NZ239309A; FI921527A0

Abstract

The present invention relates to the integron of bar shaped bacteria, be characterised in that it comprises: guarantee in said bar shaped bacteria the genomic homologous sequence of the gene effectively selected and said coryneform bacteria, wherein said sequence is suitable for said bacterium.

When said integron contains coding useful proteins sequence, its be particularly suitable for through cultivate by the bar shaped bacteria of the conversion white matter of laying eggs next life.

Description

The integron of bar shaped bacteria is with the method for said integron conversion bar shaped bacteria and by it bar shaped bacteria that obtains

The present invention relates to the integration of dna sequence dna predetermined in the genes of corynebacteria group.

This integration has two main purposes: the one, and can produce by corynebacterium strain and a kind ofly original should not express the specified protein that produces by it, perhaps make it certain homologous protein of excessive generation.Another purpose then may be by interference base because of validity block it and express, thereby cause losing gathering of reaction substrate in corresponding enzymic activity and the cell.

Except that corynebacterium and brevibacterium sp bacterium, general excellent bacillus all is to be considered to very thorny in damage so far;

-at first because there is not proper method to make it to transform,

-be owing to exist very big restrictive barriers in addition, be invalid mostly when causing the DNA conversion with other bacterial origins.

Proved already recently that electroporation method can be used for transforming excellent bacillus.Yet, if this transformation technology to the self-replication carrier is significant, then be preferably in industrial use no matter those can promptly count or its location those bacterial strains that all show very stablely this moment by the bacterium of the conversion of the integration in the karyomit(e) with regard to copying of its integrated element.

The object of the present invention is to provide the integration system of in coryneform bacteria, integrating.

The present invention relates to coryneform bacteria integron (integrons), it is characterized in that it comprises:

-guarantee that the gene selected, this gene are effectively in described coryneform bacteria, and hereinafter be referred to as " selection gene ",

The genomic homologous sequence of-described coryneform bacteria, it is adapted to said bacterium especially.

Said " integron " is meant the carrier that can have the not reproducible of integrating character in the genes of corynebacteria group, and this integron can be linearity or cyclic.

But in general, this integron can come from the plasmid of self-replication, can allow in different hosts such as intestinal bacteria synthetic.Before integration step, preferably remove the DNA that selects the non-coryneform bacteria of gene all traces in addition source, particularly those participate in the sequence of reproduction process.

The effective choice gene is in said coryneform bacteria:

-at predetermined substance, particularly antibiotic resistant gene,

-gene of phenotype, color and/or the compensating action that obviously can identify can be provided.

Antibiotics resistance selective marker more preferably wherein, can utilize in the case:

-give the gene A ph III of kalamycin resistance, note is made Km ^R,

-give the gene C at of chlorampenicol resistant, note is made Cm ^RBut also can utilize other genes, particularly erythromycin resistance gene.

" homologous sequence " is meant and is present in by the sequence in the coryneform bacteria of conversions accordingly or the sequence of at least 80% homology is arranged that it may relate to identical type or different types of sequence, and these sequences can be synthetic.

These sequences will adapt to or not fit into following some method, promptly must consider the restricted barrier problem that exists in the coryneform bacteria in following method.

This integron preferably provides as corresponding plasmid form, wherein except that integron, also comprise and duplicate part, wherein integron can be positioned at the both sides of the restriction site that allows cutting, and preferably comprises not the opposite tumor-necrosis factor glycoproteins corresponding to the restriction site in the integron such as Nat I, BstXl or Sac I.Like this, when the enzymic digestion of using at the known limitation site, can directly obtain integron; Because the integron end is a complementary, before being used to transform bar shaped bacteria, can make it to connect into ring-type as required like this.

In system of the present invention, integron is preferably as an entrained part of plasmid, and this plasmid vector can duplicate by replication origin or the replicon that duplicates in the part.According to being present in the character that this duplicates the replicon in the magazine, if plasmid be by bar shaped bacteria the unique endogenous replicon that has constitute, this complex plasmid just can only duplicate in bar shaped bacteria, but when two replicons different, that can both duplicate in its intrinsic host separately combined with plasmid, then it can duplicate in bar shaped bacteria and external host such as intestinal bacteria simultaneously.

In this case, can make up plasmid in being different from the system of corynebacterium, this is useful for the plasmid that is difficult to make up in corynebacterium and brevibacterium sp.

Owing to have homologous sequence simultaneously in integron and the genome, so must integron be inserted in the karyomit(e) by regrouping process.Like this, in first transformant, be cloned at first that the gene in the cloning site can duplicate (seeing accompanying drawing 2a) in this bacterial chromosome more than the integron.

A kind of like this copy mode has useful technology and is worth in detection, this moment, gene was multiplied, and caused the activity by this genes encoding, and particularly corresponding enzymic activity significantly increases.

In view of the structure of first integrated part is equivalent to select the direct series connection repetition of gene homologous sequence on every side, so might develop first integrated part and this structure that increases in advance based on selection allowing detect on the substratum of expressing the selection gene.Therefore, when selecting gene to be antibiotics resistance gene, can select the bacterial strain of maximum resistance by antibiotic content in the increase substratum, be that those should be able to cross the bacterial strain of expressing this corresponding gene, said gene then is equivalent to homologous sequence, and is equivalent to insert all genes or dna sequence dna in the integron.

Certainly, integron preferably comprise except that one or more homologous sequences, be the sequence of certain useful sequence encoding, sequence in particular for certain useful peptide or protein coding, said sequence may come from or come from bar shaped bacteria together, perhaps, be that homologous but is not eukaryote or synthetic source promptly from the bacterium of other strains equally.

These sequences preferably comprise can guarantee the element of expressing in bar shaped bacteria, or is preferably in the stage that the Expression element that is suitable for host bacteria expresses and inserts it.

Under the situation of using the corynebacterium host, obtain crossing of some enzyme and express, particularly the expression of crossing of gltA or gdhA is highly significant.

Just as noted, might use integron of the present invention, utilize this system to guarantee to destroy gene.For destroying or replacing, just as shown in Figure 26, corresponding gene is a non-activity, thereby causes the excessive generation by the substrate of the enzyme of corresponding gene coding.

In general, integron exists to integrate the magazine form, that is to say except that selecting gene and homologous sequence, also can be mixed with the sequence that a plurality of clonal selection site can be provided under some situation, thereby allows to insert any dna sequence dna and/or gene.

In all cases, limit in order to carry out gene, people also attempt to dope the integron of the repetition DNA that does not have other strains.

Equally also might dope more complicated integration system, particularly include the integration system of the sequence of transposable element in addition.The sequence of transposable element can by beyond the protein coding sequence of bar shaped bacteria, can guarantee that the sequence assembling of transposition forms, and may relate to the transposition of non-translocase encoding sequence.

In many transposable elements, should should be mentioned that phage Mu, particularly the transposable element of phage miniMu form.Under the situation of use as the transposable element of phage miniMu form, can utilize the mark that may become phage part or different initial point part as described below, mark for example develops the color.

The invention still further relates to and utilize the integron that derives from different bar shaped bacterias, the particularly transposable element of corynebacterium bacterium (I SaB1 especially as shown in Figure 9).

Provided the characteristic of I SaB1 element among the

embodiment

10,11 of embodiment have provided and have allowed to select and the general method of identifying this type transposable element.

The invention still further relates to comprise and come from transposable element, especially the encode integron of translocase and/or transposition repressor sequence.

Integron of the present invention may comprise all or part of sequence, particularly corresponding to the sequence of I SaB1.

The such integrated structure that comprises phage miniMu fragment, homologous DNA sequence and selection gene is expressed as can obtain crossing of specific gene by above-described structure, perhaps then can destroy gene where necessary.

Described these structures in back are different from the structure of foregoing abbreviating as " integron ".

The invention still further relates to by aforementioned integron through integrating transformation, particularly through the resulting bar shaped bacteria bacterial strain of electric transformation introducing integron.

In the available bar shaped bacteria bacterial strain, be particularly suitable for industrially be:

Brevibacterium lactofermentus (Blactofermentum)

Brevibacterium flavum (Bflavum)

Corynebacterium glutamicum (Cglrtamicum)

The molasses coryneform bacteria of dwelling (Cmelassecola)

Be different from the bar shaped bacteria host in utilization and carry out as the final host under the situation of clonal selection, can in bar shaped bacteria, make up the possible character of duplicating, so that integron is adapted to bar shaped bacteria.Can in this bacterial strain, introduce except that containing integron for this reason, also contain the plasmid that duplicates part, or integrate, then through extracting plasmid, and can discharge the enzymic digestion of integron with one or more, to reclaim the integron adapted to, can connect or not connect the fragment of purifying, and will connect product and be used for integrating conversion; In this case, the restriction barrier does not just reconstruct too much difficulty.

In some cases, be useful by the transmission of bar shaped bacteria intermediate, particularly derive under the colibacillary situation at DNA, before integron is adapted to dwell the molasses coryneform bacteria, may be favourable suitably in Brevibacterium lactofermentus.

At last, the invention still further relates to the present invention's the application of bar shaped bacteria in industrial production, particularly relate to the application of integron of the present invention aspect preparation protein or meta-bolites.

The following example will further confirm advantage of the present invention.

Fig. 1 is the schema that is begun to prepare integron by plasmid replication,

Fig. 2 shows the insertion of integron:

Through single reorganization (a)

Through dual reorganization (b),

Fig. 3 shows the structure of pCGL519,

Fig. 4 shows that two have transformed the kalamycin resistance percentage ratio of bacterial strain and the funtcional relationship of time,

Fig. 5 shows the structure of miniMu,

·MudⅢ 1681，

·MudⅢ 1681-Cat，

Fig. 6 shows plasmid pCGL107 and pCGL107 ∷ Mud+,

Fig. 7 shows containing or do not contain the integration of the integron of miniMu,

Fig. 8 show by 3 of lac manipulations ' end insert district's beginning, Brevibacterium lactofermentus CGL2005(B115) the restriction figure of clone's insertion section,

Fig. 9 shows I SaB1 sequence,

Figure 10 is the restriction figure of I SaB1,

Figure 11 is the restriction figure of plasmid pCGL330,

Figure 12 is the restriction figure of plasmid pCGL331.

Embodiment 1: the structure of the chromosomal dna library of the molasses corynebacteria A of dwelling TCC17965 and the clone of gltA gene

The chromosomal DNA for preparing the molasses corynebacteria A TCC17965 bacterial strain of dwelling according to the method for people such as Ausubel (1987) proposition.With the above-mentioned DNA of restriction endonuclease Mbo I (Boehringer) digestion 10 μ g.Press the described method of people such as Ausubel (1987) on saccharose gradient according to each dna fragmentation of size separation.The fragment of the about 6-15kb of reservation size is used for the constructed dna storehouse.

According to Birnboim and Doly(1979) method by people such as the intestinal bacteria GM2199 bacterial strain that arbitrarily obtains preparation clone pUN121(Nilsson, 1983) plasmid.Cut this plasmid with restriction endonuclease Bcl I (Boehringer), make it linearizing.

Under the described condition of people such as Ausubel (1987), connect 1 μ g through linearizing plasmid pUN121 of Bcl I and the above-mentioned 6-15kb dna fragmentation of 2 μ g, with the constructed dna storehouse with the D4 dna ligase.According to the described program of people such as Dower (1988), will connect mixture with electroporation and introduce in the e.colistraindh5.On the LB substratum that contains 10 μ g/ml tsiklomitsins, directly select the clone who has recombinant plasmid, can grow.By Birmboim and Dloy(1979) method preparation the clone's of tetracyclin resistance plasmid is all arranged.The set of these plasmids promptly is equivalent to the DNA library.

DNA library with the molasses corynebacteria A TCC17965 of dwelling transforms the active intestinal bacteria W620 of shortage Oxalacetic transacetase bacterial strain.Containing the intestinal bacteria W620 transformant clone that selection can be grown on the basic selection substratum of tsiklomitsin.This clone carries recombinant plasmid pCGL508.In the 3.5kb dna fragmentation that the feasible molasses coryneform bacteria dna fragmentation of dwelling that carries complete gltA gene of difference during clonal selection is confined to be stipulated by two Hind III sites.

Embodiment 2: integron pCGL519

The flow process of preparation integron as shown in Figure 1.

Selected genes aph III is as selecting gene; When integrating copy in karyomit(e), its resistance of giving can be up to 600 μ g/ml kantlex.Generalized case is 25 μ g/ml.Initial that select to make among the embodiment 1, have coding and dwell the 3.5kb Hind III fragment of structure gene gltA of molasses coryneform bacteria Oxalacetic transacetase as the genomic homologous dna fragment of corynebacterium, and be inserted in unique site in a plurality of cloning sites (Hind III site).Expectation is the sequence of N ot I site that is equivalent to 8 Nucleotide for integrated strategy restriction site the most useful, that be on the integron edge, and Bst X I site.But Restriction Enzyme Bst X I recognition sequence (CCAN ₅NTGG); Therefore it also usually cuts 6 nucleotide sequences and may produce a plurality of fragments.But the fragment that is discharged can finally cause the single reorganization of isolated fragment according to the character recombine of different B st X I site interior segments.

Plasmid pCGL519(Fig. 3) is a example to the plasmid that produces the integron sensitivity.Be the coryneform integration test of molasses of in the integration magazine of escherichia coli plasmid, dwelling at first.PCGL519 has Not I site, fragment reproducible and that integrate to constitute by two edges.The segmental integron of homology of chromosome Hind III that first fragment is equivalent to contain multiple clone site, selection gene aph III and carries the gltA gene of coding Oxalacetic transacetase.Second fragment contains duplicating part (the Ssp I of 3kb-Hpa I fragment), can duplicate the replication origin of the phage M13 that duplicates part, orip15A and reverse complemental of first plasmid pACY184 in intestinal bacteria of the plasmid pBL I that can duplicate in bar shaped bacteria.The Hind III fragment that will contain the 3.5kb of gltA gene is inserted among the interior carrier pCGL243 of intestinal bacteria, is built into plasmid pCGL159.

As shown in Figure 1, after clonal selection, transform Brevibacterium lactofermentus with pCGL519.When pCGL519 transfers among the Brevibacterium lactofermentus CGL2002,, replaced so contain the Nat I fragment of replication origin because the part of duplicating of pBL I loses activity in intestinal bacteria.This has shown a compensation advantage of magazine structure.

Embodiment 3: integrate

Plasmid extract by Brevibacterium lactofermentus begins, and pCGL519 is transferred to respect to intestinal bacteria to be had among the molasses corynebacteria A TCC17965 of dwelling of considerable restraint.The system that is proposed allows by separation quality grain pCGL519 in the molasses corynebacteria A TCC17965 bacterial strain of dwelling, and said bacterial strain is restrictive fully with respect to intestinal bacteria, and then just part is restrictive with respect to Brevibacterium lactofermentus.So just make the integration magazine that F-strain is had modification.

After with the plasmid pCGL519 of restriction endonuclease Not I (Boehringer) digestion, contain the integron of gltA gene and selection gene A ph III with low melting-point agarose gel separation and purifying from the molasses corynebacteria A TCC17965 of dwelling.Make the integron self of purifying like this be connected to become annular.Use electroporation people such as (, 1990) Bonamy then, this is connected mixture introduce in the molasses corynebacteria A TCC17965 bacterial strain of dwelling.Analysis presents the coryneform clone of the molasses of dwelling of resistance in 25 μ g/ml kantlex.

Obtain 500 transformant.Have 31 not have plasmid pCGL519 in 50 transformant having analyzed, with Xba I digestion back with wherein 20 of Southern blotting analysis.They all accord with the result who integrates through homologous recombination in the gltA zone.According to the character (cyclic monomer molecule or linearity or ring-type polymerizable molecular) that connects product, can explain first intasome (integrant) based on the result of simple or dual " exchange ".Through Not I digestion and with corresponding to the segmental probe hybridization of 3.5kb Hind III that contains the gltA gene after, in pulsed field, analyze.Analyze through this, further confirm to have only the copy of an integron to integrate.

The mode of duplicating of participating in wild-type gltA gene copy is: the enzymic activity that has predicted, multiply by factor 1.82, and this conforms to the engram analysis result, and shows that the sample of the copy of integrating is a non-activity.Wild type strain is come together in the table 1 with the result who is made comparisons by the bacterial strain of plasmid replication conversion.Detected the structural stability of integrating, the percentage ratio of kalamycin resistance cell and the enzymic activity of Oxalacetic transacetase are still stable after 30 generations of finding to go down to posterity.

Through containing 800 μ g/ml, be the excessive kantlex of 1000 μ g/ml then, and further contain on the incubation chamber of 1000 μ g/ml kantlex and Xin Meisu and select, the structure of having integrated with amplification.Obtain direct placed in-line amplified production.Though amplification structure and kalamycin resistance are stable, after this can not keep the high-level enzymic activity of the Oxalacetic transacetase that obtained originally.This might be the specificity inactivation that has produced the gltA gene.

Embodiment 4: based on the structure of miniMu

The Mu derivative of selecting for use in the present embodiment is that Mid II 1681 and Mud II 1681-Cat(are respectively with Km ^RAnd Cm ^RRepresentative), its size is respectively 14.8kb and 16.6kb(Fig. 5).MiniMu Mud II 1681-Cat is people such as transposon Mud II 1681(Castilho, 1984) derivative.They have the necessary factor of aforementioned transposition (except the HU), and the gene of heat sensitivity repressor C (regulating the gene of the expression of translocase A and B), antibiotics resistance gene (being representative with aph II and cat respectively) and lacA, lacY and lacZ ' gene.The latter is since the 8th codon, and is inserted into when reading in the sign indicating number as Mud, can predict proteinic transduction fusion.

These transposons are transferred in the coryneform bacteria.After being incorporated into transposon in the karyomit(e), the copy that can integrate by the method described in the embodiment 8 amplification.Relevant with this amplification is that in many cases, the integration resulting target gene (structure gene of glutamate dehydrogenase) that also can similarly increase increases up to 25 times corresponding enzymic activity.

Embodiment 5: the structure that is used for the carrier of miniMu transfer

(pCGL107 Fig. 6) contains by kalamycin resistance Km the carrier of integrating ^RPeople such as the gdhA gene (Gdh ') that sign (aph III) interrupts, pUN121(Nilsson, 1983) replicon (ori) and give tetracyclin resistance (Tet ^R) and amicillin resistance (Amp ^R) gene.This carrier can not duplicate in Brevibacterium lactofermentus, and integrates people such as (, 1990) Leblon on the homologous site of gdhA gene through simple " exchange ".

The intestinal bacteria OR1836 of Mud II 1681-Cat from the MC4100 bacterial strain introduces among the integrative vector pCGL107 through trace transduction (minimuduction).In resulting different the insertion, in certain a kind of insertion still keep colibacillary lac-phenotype (pCGL107:: Mud+, Fig. 6).Can remove pCGL107 by the preparation of same plasmid:: Mud(Fig. 6 of Mud-), and wherein the gene of translocase A and B through the digestion of Hind III and lack.

Also once tested other transfering strategy; Mud II 1681 is introduced in the bar shaped bacteria, transposon is positioned on two dissimilar carriers:

-suicide vector (non-duplicate, nonconformable) pEV11 ∷ Mud, it relates to the derivative of the gene pUC18 that does not introduce Mud II 1681.

-shuttle vectors (pCGL229), it has the Cat gene of replicon (Hind III-Hpa I fragment), p15A replicon and the Tn9 of pBL1.

In the Rec+ intestinal bacteria, Mud II 1681 is introduced in the shuttle vectors through the trace transduction.In the various insertions of gained, one of them still remains with Lac-phenotype (pCGL229 ∷ Mud+).Can remove the Mud of pCGL229 ∷ Mud-by this preparing carriers, and wherein lack the gene of translocase A and B through the digestion of Pst I.

Embodiment 6: the electricity of structure transforms usefulness and the transfer in Brevibacterium lactofermentus thereof

Utilize aforementioned bearer transformed into escherichia coli bacterial strain (DH5 α) and two Brevibacterium lactofermentus bacterial strain CGL2002 and CGL2005(B115).With respect to e. coli dna, these two bacterial strains just partly allow.These experimental results are listed in the table 2, and can draw following observation conclusion thus:

-in intestinal bacteria, no matter be or it may duplicate in intestinal bacteria at carrier pCGL107(at shuttle vectors pCGL229) situation under, the net efficiency that transforms when having Mud+ on the carrier is very low, but then presents another kind of situation after having lacked transposable genetic.Carry this typical phenomenon of plasmid replication of the derivative of active Mu, may be very strong owing to the expression to the translocase of Mu in its natural host.This shows, in above-mentioned structure, Mud+(Mud II of being utilized 1681 and Mud II 1681-Cat) good active arranged.

-in any one bar shaped bacteria bacterial strain of being tested, all can not with shuttle vectors pCGL229 ∷ Mud+ or-(can not with suicide vector pEV11 ∷ Mud) make transformant.Under the situation of the derivative that uses pCGL229, in the time of might be in little intestinal bacteria Rec+ that transduces, its replicon pBL1 be a non-activity, and reason is this carrier ability that do not increase in Brevibacterium lactofermentus.

-at Brevibacterium lactofermentus CGL2005(B115) in, available integrative vector pCGL107 and derivative thereof make transformant.Is similar with pCGL107 ∷ Mud+ with the changing effect that pCGL107 ∷ Mud-obtains.This shows that its efficient is not high when introducing MiniMu Mud II 1681-CatA+B+ in the Brevibacterium lactofermentus by integrative vector.

Compare with pCGL107 itself, the transformation efficiency of viewed two kinds of pCGL107 derivatives reduces, really be because (length is 10kb under the situation of pCGL107 due to the length increase of transformant plasmid, under the situation of pCGL107 ∷ Mud+, be 26.7kb, and be 22.1kb under the situation of pCGL107 ∷ Mud-).

Embodiment 7: to pCGL107 ∷ Mud+ at Brevibacterium lactofermentus CGL2005(B115) in the research of integration

Call pCGL320 in the following text by pCGL107 ∷ Mud+() conversion bacterial strain CGL2005(B115) can obtain 147 kantlex (25 μ g/ml) resistance clone, but the none transformant is selected at paraxin (5 μ g/ml) resistance.Yet after selecting on paraxin, it is chloramphenicol resistances that 103 such clones are arranged.Thereby the Cat gene that shows Tn9 is not enough to express initial option is carried out in permission to the clone single copy; On the contrary, detect resistant phenotype, find that this expression is enough by later scratch test.Resulting clone presents corresponding to the lac-phenotype of seeing in intestinal bacteria.

(1 type transformant is Km to resulting two types intasome ^RCm ^S, 2 types are Km ^RCm ^R) may be equivalent to respectively replace gdhA gene and the integration (Fig. 7) of the complete plasmid on the gdhA site that obtains through single " exchange " via dual " exchange ".The relevant data of making this explanation is:

The dosage of glutamate dehydrogenase is (referring to people's such as Meers technology in-1 type and the 2 type transformant, 1970) (table 3): 5 (example K2 and K3 in 7 transformant of 1 type, table 3) do not have the gdh activity fully, this can be counted as owing to interruption gene substitution the gene of gdhA.There are 4 in 5 transformant of 2 types and are proved similar gdh activity (example KC2 and KC4, table 3).

-the Southern microsphere of the BamH I digestion product that is equivalent to 1 type (K2) and 2 types (KC2 and KC4) transformant is analyzed, and with the characteristic strip (result is not shown) of pCGL147 plasmid probe announcement.These results show, transformant gdh(K2) molecular structure further confirmed the replacement of gene.Also confirm simultaneously all can obtain gdh+ transformant (2 types (KC2 and KC4) and several 1 type transformant) at gdhA site digested plasmid.

Embodiment 8: to the selection of possibility transposition

In the presence of paraxin (5 μ g/ml), isolating bacterium colony still can be grown, as can be known 2 type transformant (Km ^RAnd Cm ^R) can not be enough to express chloramphenicol resistance gene.For this reason, we have studied Cm ^RThe transformant of subclone, and it carries the cat gene wish to select Mud() transposition.These subclones are with per 10 ⁵There is 1 frequency to obtain in the individual cell.After duplicating on the Xgal, these clones present the color gradient (clean 30% blue look) from white to orchid.These results β-gala glycosides sugar after measured are active and further confirmed (table 4).Chlorampenicol resistant has been amplified in this transposition that shows Mud, and the fusion of inserting the site because of protein presents betagalactosidase activity.In fact, the identical experiment of being done with pCGL107 ∷ Mud-draws equifinality, shows that these phenomenons are not because due to the result of transposition.

Embodiment 9: the evidence that plasmid pCGL107 ∷ Mud+ connects on karyomit(e) and increases

Confirmed the front isolating almost all clone (except the KC3T4) the gdh activity (table 4) of having amplified is all arranged.In addition, except that indivedual persons (KC3T4), beta-galactosidase enzymes (press Miller, 1972 described methods are measured) almost is proportional with the activity of gdh.Detection (pressing show, 1975 described methods, table 5) to E.C. 2.3.1.28 also obtains equifinality.This result contradicts with the transposition of Mud, because Mud never takes away adjacent sequence during transposition.Exactly, this shows the series connection amplification of repeating unit pUN-Mud-gdh on karyomit(e), makes its homology because of sequence constitute the edge.This point can be confirmed by the genomic dna of BamH I, Not I and Xba I digestion bacterial strain KC3, KC3T1 and KC3T3.The result that the BamH I band (equaling 7kb) of Mud inside not only, and the band (11kb and 2.4kb) that contains the Mud marginarium also increased, this situation confirm series connection amplification and transposition antithesis.In addition, through Not I (only in Mud II 1681-cat, cutting once) and Xba I (only once) digestion in the middle cutting of gdh ', the 2.4kb band of the repeating unit that increased significantly.

Under no selective pressure, record betagalactosidase activity after 15 generations and kept 70% of activity level, show that amplification is a quite stable.Cat and Gdh activity lose 30% equally after 25 generations.This series connection amplification situation makes the people associate people (1985) such as people such as Albertini (1985) and Janniere to result of experiment that subtilis does.Can be with the parasitism transduction (reading outside the sign indicating number zone of the blue or green plain resistant gene of the ammonia benzyl that has merged operon) of the clone's that increases betagalactosidase activity owing to the amplification that in Brevibacterium lactofermentus, exists but in intestinal bacteria, do not detect.

Embodiment 10: to the The Characteristic Study of the part I SaB1 of insertion

Through reclaiming the bacillus coli DH 5 alpha plasmid, begin to separate insertion element by the DNA of the amplification of KC3T4.Detect lactic fermentation bacillus CGL2005(B115 by the probe of the insertion element of having separated before containing), the genomic dna of some derive strain and other bar shaped bacteria bacterial strains.The 3.5kb Pvu II fragment of Mu inside comes from KC ₃T ₄The DNA of middle amplification also contains insertion element, and said fragment can be used for detecting the initial trace of the BamH I digestion product DNA that contains intasome and respectively increase bacterial strain (KC3T4).Because insertion portion does not contain BamH I site, contain an insertion at least or insert segmental BamH I genomic fragment so this experiment can demonstrate.Be equivalent to derive from pCGL107 at bacterial strain K1-(:: the replacement intasome of 1 type of Mid-), 5 bands occur, show the copy (integer or non-integer) that has a plurality of insertions.

From Brevibacterium lactofermentus CGL2005(B115) genomic dna, its BamH I digestion product demonstrates four bands (sizableness in 18kb, 5.9kb, 5kb and 4.5kb) total with K1-; Be present in the 5th band among other bacterial strains K1-, KC1-and the KC3 (size is 6.5kb) and show as bacterial strain CGL2005(B115 in these bacterial strain sources) segregant in transposition.

At (ⅰ) Brevibacterium lactofermentus CGL2005(B115) with these two different tyrothricin pedigrees of (ⅱ) Brevibacterium lactofermentus CGL2002 between, it is the common band of 18kb and 4.5kb that two sizes are arranged; On the contrary, bacterial strain CGL2002 does not then have other band.This has showed the transport property of these elements.The molasses corynebacterium strain of dwelling provide and insertion portion between very weak hybridization signal, be translated into be present in these bacterial classifications in other different sequences of person.

Identified and cloned Brevibacterium lactofermentus the migration of first specificity insertion element and name and be I SaB1, it may present a plurality of copies (2 in the genome is to 5 copies).But different loci is easy to carry out multiple transposition in amplification region.The sequence that has difference in other coryneform genomes but manifested.

I SaB1 is made of 1288 base pairs; Because being inserted into, I SaB1 is equivalent to measure in the fragment of 3 ' stub area of lac operon of sequence, so can identify its peripheral part by people such as Hediger (Biochemistry, Proc.Natl.Acad.Sci.USA, 82,1985).I SaB1 inserts between Nucleotide 5575 and 5576, and the target sequence (CCGAT) that duplicates a 5bp (Fig. 8).Provided the whole sequence of I SaB1 among Fig. 9, Figure 10 shows its restriction map.Identify two open phases reading the sign indicating number district,, show that it may be corresponding to the structure gene and the transposition repressor gene of translocase based on given The sequencing results.

Embodiment 11: insertion element and transposon capture carrier

At Brevibacterium lactofermentus CGL2005(B115) karyomit(e) in institute do in the gene amplification research process, obtain I SaB1 and insert.In order to separate all insertion elements that shifted in the coryneform bacteria, special integrative vector: pCGL330 and pCGL331(Figure 11 and 12 have been made up).These two carriers are to be made of first fragment that is derived from carrier pUN121.Plasmid pUN121 can duplicate in intestinal bacteria, and carries amicillin resistance; It has the sequence of the C I repressor of coding lambda particles phage, is stoped the expression that operon merges between lambda particles phage unit's PL promotor and the anti-bosom of tsiklomitsin gene.The intragenic insertion inactivation of Cl, so repressor can make tetracycline resistance gene be expressed in coryneform bacteria under the control of PL.Can also select to insert according to tetracyclin resistance.In Ssp I site pUN121 is cut into linearity, and with EcoR I digestion with the Klenow filling, resulting second fragment of pCGL107 carrier merges, and in order to obtain carrier pCGL330 and pCGL331 behind the transformed into escherichia coli DH5 α, both difference is its clone's direction.Contain the fragment (structure gene of glutamate dehydrogenase) that is suitable for directly transforming coryneform selection gene (giving the aph III gene of kalamycin resistance) and contains the source part of gdgdhA gene unit by pCGL107 deutero-EcoR I fragment, it can be used as integration points in chromosome of corynebacterium.

For kalamycin resistance, available plasmid pCGL330 and pCGL331 electricity transforms Brevibacterium lactofermentus CGL2005(B115) and the bacterial strain of CGL2002.In each case, transformation frequency almost all is every microgram 10exp3, and this is that integration with plasmid adapts.(as CGL2005 ∷ pCGL330 and CGL2005 ∷ CGL331) is all very sensitive to tsiklomitsin for these transformant, proves that Cl is to P _LBe adjusted in and play good effect in the transformant.Frequency in 10 back detection tetracyclin resistance segregants that go down to posterity.

Making the frequency of occurrences of sudden change in the bacterial strain of plasmid pCGL330 with integration and pCGL331 of Cl gene inactivation is per generation 2 * 10exp-6.

From bacterial strain CGL2005 ∷ pCGL331 and CGL2005 ∷ pCGL330, separate and, therefrom extract genomic dna then to reclaim plasmid with Pst I digestion tetracyclin resistance segregant.

For obtaining tetracyclin resistance, connect the DNA that these have digested, and connect the DH5 α bacterial strain of product transformed into escherichia coli with this.The plasmid that analyze to reclaim has also been identified among 9 tetracyclin resistances clones 7 insertion element.(come from CGL2005 ∷ pCGL331) under a kind of situation,, from the I saB1 that identifies, identify insertion element (its size has unique Acc I, EcoRV and xho I site for 1.2kb) according in the location of Cl inside.(come from CGL2005 ∷ pCGL330) in another case and then identify the insertion element (its size is 1.0kb, has the Acc I, and does not have EcoRV and xho site) of different I SaB1.

Capture carrier and embodied the function of transposon; In most of the cases, the gained sudden change is a kind of insertion; Can detect the frequency of transposition according to the frequency of tetracyclin resistance quite exactly; Identified the element that maximum migration is arranged; Comprising separating I SaB1 again, and identify other insertion elements of different I SaB1.

The source of listed each bacterial strain is:

Intestinal bacteria

·DH5alpha ：GibcoBRL

·Mc4100 ：Casadabam（1976）

·OR1836 ：Reyes

Brevibacterium lactofermentus

CGL2002: people such as Bonamy (1990)

CGL2005(B115): people such as Bonnassie (1990)

The molasses coryneform bacteria of dwelling

·ATCC17965 ：ORSAN

ATCC17965 ∷ gltA:(the application)

Have four in these bacterial strains and be deposited with la Collection Na-tonale de Cultures de Microorganismes(CNCM on July 23rd, 1991) de l ' Institut Pastewr(Paris):

-corynebacterium melassecola ATCC17965 ∷ gltA:No. I-1124

-intestinal bacteria OR1836:No. I-1125

-Brevibacterium lactofermentus CGL2005(B115): No. I-1126

-Brevibacterium lactofermentus CGL2002:No. I-1127

Bacterial strain DH5alpha can find in the catalogue of Clontech Laburatories, and its registration number is NO.C1021-1(Palo Alto, CA, and USA), and bacterial strain MC4100 is No.35695 in the registration number of ATCC.

Table 1The specific activity of Oxalacetic transacetase

Bacterial strain	Specific activity	Relative reactivity
			ATCC17965	1,041	1
ATCC17965∷gltA	1,893	1,82
			ATCC17965(pCGL519)	5,340	5,13

Oxalacetic transacetase specific activity (micromole CoASH/ minute/mg protein)

Table 2: transformation efficiency with carrier of MiniMu

Bacterial isolates

CGL2002 CGL2005（B115）

pCGL229 5×10 ⁷10 ⁵10 ⁶

pCGL229∷Mud+ 2×10 ⁴0 n·d·

pCGL229∷Mud- 4×10 ⁶0 n·d·

pCGL107 10 ⁵3×10 ³10 ⁴

pCGL107∷Mud+ 5×10 ²0 4×10 ²

pCGL107∷Mud- 10 ⁵n·d· 5×10 ²

Table 3: the activity level of glutamate dehydrogenase in elementary conversion

Bacterial isolates Gdh ratio adds activity (micromole

NADPH ₂Consume

/ minute/mg protein)

Initial strain km ²Cm ²

CGL2005（B115） 2，18

Transformant km ²Cm ²

k1 2，0

k2 ≤0，06

k3 ≤0，06

k4 ≤0，06

k5 ≤0，06

k6 2，18

k7 ≤0，06

Transformant km ²Cm ²

KC2 2，24

KC3

2，12

KC4 2，18

KC5 3，45

KC7 2，06

Table 4: in the intasome of amplification, beta-galactosidase enzymes with

The activity level of glutamate dehydrogenase

Bacterial isolates betagalactosidase activity glutamate dehydrogenase specific activity

Initial strain

CGL205（B115） 3，7 2，48

KC2

6，2 2，24

KC3

4，5 2，06

The intasome of amplification

KC3T1 27，9 18，2

KC3T2

20，7 16，0

KC3T3 48，2 49，6

KC3T4

32，2 2，24

KC3T5 19，7 8，55

KC3T6 19，0 11，9

KC3T7 34，5 28，0

KC2T1

7，4 2，73

Table 5: CAT, β GA1 and the active level of GDH in the bacterial strain of amplification

The active eat specific activity of bacterial isolates β gal Gdh specific activity

Initial strain

CGL2005（B115） 3，7 ≤0，07 2，18

Elementary integration

K2 ≤0，07 ≤0，006

K3 ≤0，07 ≤0，006

Elementary integration

KC3

4，5 2，72 2，06

KC7

3，4 1，82

The integration of amplification

KC3T1 27，9 19，7 18，2

KC3T3 48，2 33，3 49，6

KC3T4

32，2 18，4 2，24

KC3T5 19，7 13，6 8，55

Reference

Albertini A.M.and Galizzi A.（1985）.Amplification of a chromosomal region in Bacillus subtilis;J.Bacteriol.，162：1203-1211）

Ausubel M.A.，Brent R.，Kingston R.E.，Moore D.D.，Seidman J.G.，Smith J.A.，and Struhl K.（1987）in"Current protocols in Molecular Biology"，Greene Publishing Associates and Wiley-Interscience

Birnboim H.C.and Doly J.，（1979）A rapid alkaline extraction procedure for screening recombinant plasmid DNA.Nucleic Acid Res.7：1513-1523

Bonamy C.，Guyonvarch A.，Reyes O.，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 lactofermentum，FEMS Microbiol.Letters，vol.72：143-146

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 B.A.，Olfson P.and Casabadan M.J.（1984）Plasmid insertion mutagenesis and lac gene fusion with miniMu bacteriophage transposons，J.Bacteriol.158：488-495

Dower W.J.，Miller J.F.and Ragsdale C.W.，High Efficiency transformation of E.coli by high voltage electroporation.Nucleic Acid Res.16（1988）6127-6145

Janniere L.，Niaudet B.，Pierre E.，Ehrlich S.D.（1985）Stable gene amplification in the chromosome of Bacillus subtilis，Gene 40：47-55

Maniatis T.，Fritsch Ed.and Sambrook J.（1982）.Molecular cloning：A laboratory manual.Cold Spring Harbor Laboratory Press，Cold Spring Harbor，NY

Meers J.L.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 B.，Uhlén M.，Josephson S.，Gatenbeck S.et Philipson L.，1983.An improved positive selection plasmid vector constructed by oligonucléotide 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

Claims

1, the integron of bar shaped bacteria is characterised in that it comprises

--guarantee effective gene of selecting in said bar shaped bacteria,

--the genomic homologous sequence of said bar shaped bacteria, wherein said sequence is adapted to said bacterium.

2, according to the integron of claim 1, the plasmid that is characterised in that its source also comprises and duplicates part except that containing integron, and this integron is arranged in the both sides of the opposite tumor-necrosis factor glycoproteins that is equivalent to integron unrestriction site.

3,, be characterised in that duplicating part is included in effective replication origin in the non-bar shaped bacteria according to the integron of claim 2.

4,, be characterised in that replication origin is effective in intestinal bacteria according to the integron of claim 3.

5,, be characterised in that duplicating part is included in the bar shaped bacteria effectively replication origin according to each integron among the claim 2-4.

6,, be characterised in that it also comprises the sequence of transposable element according to the integron of claim 1.

7, according to each integron among the claim 1-6, be characterised in that transposable element comprises the sequence from bar shaped bacteria.

8,, be characterised in that this sequence is from brevibacterium sp according to the integron of claim 7.

9, integron according to Claim 8 is characterised in that this sequence is from as shown in Figure 9 ISaB1.

10, according to each integron among the claim 6-9, be characterised in that it except that comprising protein coding sequence, also comprise the transposable element sequence of assurance by the coryneform bacteria transposition.

11, according to each integron among the claim 6-10, be characterised in that these transposable element sequences lack the sequence of the translocase of encoding.

12, according to each integron among the claim 1-11, be characterised in that it comprises a useful sequence.

13,, be characterised in that peptide or protein that said useful sequence encoding is useful according to the integron of claim 12.

14,, be characterised in that useful proteins matter is homologous protein according to the integron of claim 13.

15,, be characterised in that useful proteins matter is heterologous protein according to the integron of claim 14.

16, according to each integron among the claim 13-15, be characterised in that useful proteins matter is enzyme.

17,, be characterised in that coding useful proteins sequence is selected from following gene according to the integron of claim 16:

-gltA，

-gdhA。

18, according to each integron among the claim 2-17, be characterised in that non-existent restriction site is selected from the integron:

-NotⅠ，

-BstⅩⅠ。

19, according to each integron among the claim 1-15, be characterised in that said sequence is adapted to the bar shaped bacteria bacterial strain.

20,, be characterised in that said sequence was transferred in the brevibacterium sp bacterial strain before being adapted to corynebacterium according to the integron of claim 19.

21,, be characterised in that said preface is amplified according to the integron of claim 20.

22,, being characterised in that amplification produces the sequence of stable integration according to the integron of the amplification of claim 21.

23,, be characterised in that it contains the coryneform homologous sequence of integrating of the molasses of dwelling in Brevibacterium lactofermentus according to the integron of claim 1.

24, utilize the method for each integron conversion bar shaped bacteria bacterial strain among the claim 1-23, be characterised in that through electroporation integron is introduced in the said bacterial strain.

25, the bar shaped bacteria that can make by the method for claim 24.

26,, be characterised in that it relates to following bacterial strain according to the bar shaped bacteria of claim 25:

-Brevibacterium lactofermentus,

-brevibacterium flavum,

-corynebacterium glutamicum,

-molasses the coryneform bacteria of dwelling.