Patent Application: US-66165896-A

Abstract:
a process for replacing a nucleotide sequence in the genome of a mycobacterium strain comprises the steps of : a ) providing a vector containing sacb gene coding for levane saccharase enzyme and a nucleotide sequence of interest ; b ) transfecting the mycobacterium strain with the vector ; c ) selecting clones of the resulting transfected mycobacteria for replacement of the nucleotide sequence of interest by propagating the transfected clones in a culture medium supplemented with sucrose ; and d ) isolating the recombinant strain . the process is useful for positive selection of allelic exchange mutants , such as in mycobacterium tuberculosis complex .

Description:
it has been demonstrated that the bacillus subtilis sacb gene , encoding the secreted enzyme levansucrase ( sucrose : 2 , 6 - β - o - fructan 6 - β - o - fructosyltransferase ; ec 2 . 4 . 1 . 10 ), confers sucrose sensitivity to mycobacteria on 10 % sucrose ( pelicic et al ., 1996 ). the efficiency of selection , 5 × 10 - 4 for m . smegmatis and 10 - 6 for mycobacterium bovis bcg , is comparable to that for other bacteria where sacb has been used for positive selection of allelic exchange ( cai and wolk , 1990 ; cianciotto et al ., 1988 ; kaniga et al ., 1991 ; kamoun et al ., 1992 ; schafer et al ., 1994 ; schweizer , 1992 ). the possibility of using sacb as a positive - selection system for gene - replacement events in mycobacteria was tested . the pyrf gene of m . smegmatis was chosen as a model because it has previously been used in homologous recombination experiments in this species . pyrf mutants are uracil auxotrophs and can thus be easily screened . it was found that pyrf mutants resulting from allelic - exchange events were readily identified by either a one - step or a two - step selection on sucrose . using a two - step selection , uracil auxotrophic mutants were isolated with an unmarked , defined mutation in the pyrf gene . these results illustrate how sacb can be used to facilitate experiments involving allelic - exchange events in mycobacteria . pjq200 is a cloning vector carrying a gentamycin - resistance gene active mycobacteria , and the sacb gene of b . subtilis ( quandt and hynes , 1993 ). the pyrf gene disrupted by the aph cassette , conferring resistance to kanamycin , ( pyrf :: km ), was obtained from py6002 ( husson et al ., 1990 ) and inserted in pjq200 to give ppr26 . ppr26 was used to transform m . smegmatis by electroporation . the transformants resulting from homologous integration were selected on luria - bertani ( l ) medium supplemented with uracil ( lu ), and containing either ( i ) kanamycin ( luk ) as a classical test for the frequency of recombination or ( ii ) kanamycin and sucrose ( luks ) to assess the possibility of sucrose selection . the electroporation of 1 μg of vector resulted in approx . 100 transformants on luk and only approx . 5 on luks plates , a 20 - fold decrease . in the same conditions , py6002 , which lacks the sacb gene , gave the same number of colonies on luk and luks plates ( data not shown ). thus sacb , present on ppr26 , is responsible for the death of the majority of the transformants when selection is performed on sucrose . the phenotypes of the transformants were characterized by replica plating on different selective media . most of the clones ( 95 %) selected on luk plates were kanamycin resistant ( km r ), gentamicin resistant ( gm r ), sucrose sensitive ( suc s ), uracil positive ( ura - ) and resulted from single recombination events in the pyrf gene ( table 1 ). table 1______________________________________effects of the presence of sucrose on the proportion ofpyrf allelic - exchange mutants recovered from a one - step selectionafter transformation with ppr26 . percent of total clones (%). sup . a ura . sup .-, km . sup . r , gm . sup . s ura . sup .-, km . sup . r , gm . sup . rgrowth medium ( allelic exchange ) ( simple recombination ) ______________________________________luk 5 95luks 100 0______________________________________ . sup . a mean value of two independent experiments . genomic dna from these transformants was analyzed by southern blotting with a pyrf probe . these clones contained a complete copy of ppr26 inserted into the pyrf gene , thus possessing both an intact ( 2 . 5 kbp ) and an inactivated copy of pyrf ( 3 . 8 kbp ) ( fig1 ). the remaining clones 5 % were true uracil auxotrophs , with the phenotype km r , gm s , suc r , ura - . the endogenous allele of the pyrf gene , corresponding to a sphl fragment of 2 . 5 kbp on a southern blot , had been replaced by the inactivated copy corresponding to a fragment of 3 . 8 kbp ( fig1 ). the increase of 1 . 3 kbp corresponds to the size of the kanamycin - resistance gene which was used to inactivate pyrf . moreover , no hybridization signal was detected in the auxotrophs when the vector pjq200 was used as a probe ( data not shown ), indicating that the vector sequence had been lost . all the transformants ( 100 %) selected on the luks plates exhibited the km r , gm s , suc r , ura - phenotype and were uracil auxotrophs generated by allelic exchange at the pyrf gene ( table 1 ). this was confirmed by southern - blot analysis , which gave hybridization patterns indistinguishable from those observed for the auxotrophs obtained on luk plates . see ( fig1 ). one - step selection on sucrose was 100 % efficient , because all of the clones corresponding to single recombination events ( incorporation of the entire vector bearing the sacb gene ) was eliminated . thus , when a suicide vector bearing the sacb gene is used for the homologous recombination experiments , mutants resulting from an allelic exchange can be directly selected by plating on sucrose . the possibility of selecting allelic - exchange events in two steps was next investigated . this procedure might be useful if the frequency of gene exchange for a gene of interest is too low to recover mutants in one step . for the first step , an individual clone was chosen at random from those obtained on a luk plate in the previous experiment . it was verified that this clone corresponded to a single recombination event in the pyrf gene by phenotypic ( km r , gm r , suc s , ura + ) and southern - blot analysis . the clone was propagated overnight in 7h9 medium ( difco ) supplemented with uracil ( without antibiotic selection ) to allow a deletion - recombination event to occur at the pyrf locus . any such event could either eliminate the wild - type or the interrupted allele of the pyrf gene . the culture was spread on luks plates ( step 2 ) to select cells which had lost the sacb gene . several thousands of colonies were obtained and approx . 200 clones were analyzed . two - thirds were uracil auxotrophs as verified by phenotypic analysis ( table 2 ) and southern hybridization ( fig1 ). the remaining colonies ( 1 / 3 ), through suc r , were not uracil auxotrophs ( km r , gm r , suc r , ura + ) and presumably corresponded to clones with a mutation in the sacb gene . table 2______________________________________effects of the presence of sucrose on the proportion ofpyrf allelic - exchange mutants recovered from a two - step selection . percent of total clones (%). sup . b ura . sup .-, gm . sup . s ura . sup .+, gm . sup . rtransforming growth suc . sup . r ( allelic suc . sup . r ( sacb ura . sup .+ dna medium exchange ) mutants ( revertants ) ______________________________________ppr28 luks 66 34 ndppr26 lus 35 7 58ppr34 lus 27 4 69______________________________________ . sup . a medium used in the second step of selection . . sup . b at least 192 clones were analyzed in each experiment . nd , not detectable genomic dna was probed with pjq200 by southern blotting : this showed that the sacb mutations were either point mutations ( no change of vector size ) or 2 kbp insertions possibly resulting from the insertion of a transposable element ( data not shown ). it is possible that the trapped sequence corresponds to is 1096 , which was discovered in m . smegmatis using a similar methodology with β - galactosidase as a reporter gene ( cirillo et al ., 1991 ). this suggests that sacb can be used as a transposon trap in m . smegmatis , as it has been used in escherichia coli ( gay et al ., 1985 ). the experiment was repeated , but with the second selection step on sucrose plates without kanamycin ( lus ). three times more colonies were obtained at the same dilution than by selection on luks plates . thus , the majority of the clones on lus plates were expected to be sensitive to kanamycin and to have resulted from loss of the interrupted allele of pyrf ( pyrf :: km ), and thus the km cassette . this was verified by phenotypic ( km s , gm s , suc r , ura + ) ( table 2 ) and southern - blot analysis ( fig1 ). a small proportion of the clones ( 7 %) resulted from mutations in the sacb gene . but a significant proportion of the clones obtained were uraxil auxotrophs ( 1 in 3 ), and corresponded to allelic - exchange mutants . therefore , positive selection of allelic - exchange events is possible on sucrose using a two - step methodology , even when no antibiotic selection was applied during the second step of selection . the feasibility of a two - step selection , in the absence of antibiotic selection during the second step , suggested that it might be possible to generate unmarked mutants as described for other bacteria ( donnenberg et al ., 1991 ; ried and colimer , 1987 ; schafer et al ., 1994 ; soupene et al ., 1995 ). the aph gene was excised with bamhl from the mutated allele pyrf :: km ( husson et al ., 1990 ) and a frameshift mutation was generated by blunting the ends with t4 dna polymerase and religating . this unmarked mutated copy of the pyrf gene ( pyrf + ) was inserted into pjq200 to generate ppr34 ( fig2 ). ppr34 was used to transform m . smegmatis by electroporation , and clones resulting from a single homologous recombination event were selected on lu supplemented with gentamicin ( lug ): the pjq200 vector carries the saccl gene conferring gentamicin resistance ( quandt and hynes , 1993 ). the phenotype and southern - blot analyzes confirmed that the selected clones resulted from a single cross - over in the pyrf gene ( data not shown ). as described above , the second step of selection was performed on lus medium . the clones were replica plated onto m63 minimal medium . approximately 1 / 3 of the clones were unable to grow on minimal medium and were thus uracil auxotrophs ( table 2 ). this is consistent with the results of the previous experiment with ppr26 , with no selective pressure for the antibiotic resistance during the second step ( table 2 ). as the frameshift mutation introduced into the pyrf gene destroyed a bamhl site , allelic - exchange mutants identified by their auxotrophic phenotype should have a different hybridization pattern in a southern - blot analysis with bamhl - cut dna . several ura + revertants and ura - mutants were analyzed by southern blotting . as expected , the revertant clones presented two fragments of 2 . 5 kbp and 5 kbp hybridizing to the pyrf probe . the unmarked auxotrophic mutants gave a single hybridizing bamhl fragment of 7 . 5 kbp , confirming the loss of the central bamhl site ( fig3 ). thus , a two - step selection with sucrose selection in the second step enabled us to isolate unmarked mutants carrying a known , specific lesion in the pyrf gene . in summary , a double - selection strategy was recently adapted to m . smegmatis using the rpsl gene as a counter - selectable marker ( sander et al ., 1995 ). however , its applicability to slow - growing mycobacteria has not been described . rspl is a dominant marker conferring susceptibility to streptomycin to a streptomycin - resistant strain which carries a mutation in the endogenous rpsl allele . when used as a counter - selectable marker , rpsl leads to allelic - exchange mutants resistant to streptomycin , an antibiotic , which is still a common component of several combined antituberculosis chemotherapeutic regimens ( who , 1991 ). sacb presents several interesting characteristics making it a useful counter - selectable marker for the positive selection of gene - exchange events in mycobacteria . first , sacb does not require an antibiotic - resistant strain to induce lethality . second , sacb can be used in two - step selection strategies . this approach will prove useful in cases where the frequency of allelic exchange for the gene of interest is too low for one - step selection . third , the efficiency of selection on sucrose in a two - step strategy is sufficiently high to detect rare deletions that give rise to unmarked defined mutants . although antibiotic markers are useful for genetic studies , an unmarked mutant is necessary when generating strains for use as new vaccines . moreover , in the construction of strains with multiple mutations there is no need for a corresponding number of antibiotic - resistance markers . the expression of sacb , the bacilus subtilis gene encoding levansucrase , is lethal to mycobacteria in the presence of 10 % sucrose . the use of sacb as a marker for positive selection of gene - replacement events into mycobacterium smegmatis is herein described . a sucrose counter - selectable suicide plasmid was used to deliver an inactivated copy of the pyrf gene ( pyrf :: km ) into the m . smegmatis genome . only uracil auxotroph clones , resulting from replacement of the endogenous pyrf allele , survived in a one - step selection on plates containing kanamycin and 10 % sucrose . this demonstrated that selection on sucrose against the maintenance of the vector bearing the sacb gene is 100 % efficient , enabling the positive selection of allelic - exchange mutants . two - step selection is also feasible ; it was used to construct unmarked pyrf mutants in which the gene was inactivated by a frameshift mutation . this method of generating unmarked , directed mutations is rapid and simple , making it a powerful tool for the genetic characterization of mycobacteria . this invention will be described in more detail in the following examples . the bacterial strains and plasmids used in this study are listed in table 3 . e . coli was routinely grown on liquid or solid luria - bertanl ( l ) medium , and kanamycin and gentamicin were used at 20 μg ml - 1 . m . smegmatis was grown on liquid middlebrook 7h9 medium ( difco ) supplemented with 0 . 2 % glycerol and 0 . 05 % tween , or on solid l medium . when required , antibiotics were included at the following concentrations : kanamycin , 20 μg ml - 1 ; and gentamicin , 5 μg ml - 1 . table 3______________________________________bacterial plasmids and strains used in this study . strain / plasmid relevant characteristics source / reference______________________________________straine . coli dh5α φ80dlaczδm15 reca1 enda1 gibco brl hsdr17 ( r . sub .. sup .-, m . sub . k . sup .-) m . smegmatis highly transformable mutant snapper et al . mc 155 ( 1990 ) plasmidpy6001 puc19 with the pyrf gene of husson et al . m . smegmatis on a sau3a1 ( 1990 ) fragmentpy6002 insertion of the aph cassette husson et al . in bamhl of py6001 resulting ( 1990 ) in pyrf :: kmpjq200 cloning vector with sacb quandt and hynes and aacc1 ( 1993 ) ppr2b pjq200 with pyrf :: km on this work an xbal - mlul fragment of py6002ppr33 excision of the aph gene this work from py6002 and blunting resulting in pyrf . sup .+ ppr34 pjq200 with pyrf . sup .+ on an this work xbal - mlul fragment of ppr33______________________________________ transformants were selected on lu medium ( l medium with 0 . 2 mm uracil ) supplemented with kanamycin ( luk ) or gentamicin ( lug ). 10 % sucrose was added where indicated ( lus or luks plates ). uracil auxotrophs were identified by their inability to grow on m63 minimal medium with glucose as the sole carbon source . restriction enzymes , t4 dna polymerase , and t4 dna ligase were purchased from boehringer mannheim , amersham , and gibco brl , respectively . all enzymes were used in accordance with the manufacturer &# 39 ; s recommendations . plasmid dna was isolated by qiagen preparation ( qiagen inc .). dna fragments used in the cloning procedures and for radiolabelling were gel purified using the geneclean ii kit ( bio 101 inc .). electrocompetent bacteria were prepared by the method of sander et al . ( 1995 ) with minor modifications . bacteria were grown in 400 ml of l ( e . coli ) or 7h9 medium ( m . smegmatis ) to an od 600 of ) 0 . 4 . after three washings in 10 % glycerol , the cells were resuspended in 1 ml 10 % glycerol . aliquots ( 100 μl ) of freshly prepared mc 2 155 cells were electroporated with 1 μg of vector dna in 0 . 2 cm cuvettes ( bio - rad ) with a single pulse ( 2 . 5 kv , 25 μf , 200 ohms ). after 3 - 4 days of incubation , single colonies were picked and resuspended in 7h9 medium aliquoted in 96 - well microtiter plates . forty - eight clones were replicated simultaneously on different selective media using a replica plater ( sigma ). mycobacterial genomic dna was isolated as follows : cells from a 5 ml culture were pelleted by centrifugation ( 15 min , 5000 × g ). the pellet was resuspended in 250 μl of solution 1 ( 25 % sucrose , 50 mm tris - hcl ph 8 . 0 , 50 mm edta , 500 μg ml - 1 lysozyme ) and incubated overnight at 37 ° c . two hundred and fifty microliters of solution ii ( 100 mm tris - hcl ph 8 . 0 , 1 % sds , 400 μg ml - 1 proteinase k ) was then added and the samples incubated for 4h at 55 ° c . dna was then extracted twice with phenol - chloroform , and concentrated by ethanol precipitation . one microgram of genomic dna was digested overnight with an excess of restriction enzyme ( 30 u ) and separated by electrophoresis using 0 . 7 % agarose gels . southern blotting was performed in 20 × sspe ( 150 mm nacl , 8 . 8 mm nah 2 po 4 , 1 mm edta , ph 7 . 4 ) using hybond - n + nylon membranes ( amersham ) with standard methods ( sambrook et al ., 1989 ). the megaprime random - primed labelling kit ( amersham ) and 5 μmcl of α - 32 p !- dctp were used to label probes . non - incorporated label was removed by filtration through a nick column ( pharmacia ). prehybridization and hybridization were performed at 65 ° c . using rh buffer ( amersham ), as recommended by the manufacturer , serial 15 min washings were performed at 65 ° c . as follows : two washes with ( 2 × sspe , 0 . 1 % sds ), one wash with ( 1 × sspe , 0 . 1 % sds ) and two washes with ( 0 . 7 × sspe , 0 . 1 % sds ). blots were exposed overnight to x - omat ar x - ray film ( kodak ) at - 80 ° c . ppr26 was constructed by inserting the blunt - ended xbal - mlul fragment ( 5 . 9 kbp ), containing the pyrf :: km allele excised from py6002 ( husson et al ., 1990 ), into the smalcut pjo200 vector ( quandt and hynes , 1993 ). the aph cassette used to inactivate the pyrf gene was excised from py6002 by bamhl digestion , and a frameshift mutation was introduced in the pyrf gene ( pyrf + ) by blunting with the t4 dna polymerese and religation , resulting in ppr33 . ppr34 was constructed by cloning a blunt - ended xbal - mlul fragment ( 4 . 6 kbp ), containing the mutated copy pyrf + , into the smal site in pj0200 . py6001 ( husson et al ., 1990 ) was reconstructed during ppr33 construction , by omitting the blunting step before the religation . the 2 . 5 kbp sphl fragment of py6o01 corresponding to the pyrf gene was used as a probe in the southern - blot experiments . over the last decade , the genetic characterization of mycobacteria has greatly benefited from the development of efficient genetic systems , resulting in the identification of several genes that could play a role in virulence ( 5 ). however , the construction of defined mutants leading to a better understanding of the physiopathology of tuberculosis is still hampered by the great difficulty to perform allelic exchange ( 6 ). due to the rarity of double - crossover events and the high levels of illegitimate recombination , the isolation of a gene exchange mutant is always cumbersome , if possible at all . despite those difficulties , allelic exchange in the fast - growing m . smegmatis has early been achieved ( 4 ) using a traditional protocol of mutagenesis ( 17 ); a gene inactivated in vitro by the insertion of an antibiotic resistance marker is delivered on a suicide vector into the bacteria . anyhow , the proportion of allelic exchange mutants was variable and generally low , representing less than 10 % of the transformants ( 4 , 14 , 18 ). the ratio , double - crossover towards single recombination and illegitimate recombination , is even more unfavorable in slow - growing mycobacteria . this partly explains why the first attempts to perform allelic exchange in mycobacteria from the m . tuberculosis complex were unsuccessful ( 1 , 8 ). nevertheless , it has been demonstrated that allelic exchange was possible in m . bovis bcg using the urec gene coding for a subunit of the urease ( 16 ). reyrat et al . took advantage of the fact that urease activity can be monitored with a simple calorimetric test , which facilitated the otherwise time consuming screening step of transformants ( 16 ). ure clones represented only 4 % of the transformants . simultaneously , two other groups succeeded in identifying low - frequency allelic exchange in slow - growing mycobacteria ( 10 , 12 ). the overall proportion of double recombinants is thus generally lower than m . smegmatis and it depends essentially on the selected gene . moreover , the frequency of homologous recombination for several different genes leud ( 2 ), purc ( 7 ), is too low to enable the detection of double recombinants in a classical gene exchange experiment ( 17 ). expression of sacb is lethal to mycobacteria in the presence of sucrose sacb , making it a useful counter - selectable marker for positive selection of gene replacement events as demonstrated in the fast - growing mycobacterium smegmatis . following the same methodology , a sucrose counter - selectable vector was used to deliver , into the mycobacterium bovis bcg genome , an inactivated copy ( urec :: km ) of the urec gene encoding the mycobacterial urease . a two - step selection procedure on 2 % sucrose allowed the positive selection of gene exchange mutants . this technique should thus be an extremely useful facility for the genetic analysis of pathogenic mycobacteria . as for the experiments in m . smegmatis , the sucrose counter - selectable suicide vector we used was pjq200 ( 15 ). the urec :: km mutated allele was excised from pjδ64k ( 16 ) on a saci - kpni fragment , which was made blunt - ended and inserted into smai - cut pjq200 to give rise , following the orientation of the insert , to ppr24 or ppr25 . ppr24 was selected at random for the subsequent experiments and used to transform m . bovis bcg by electroporation . however , the results are similar when using ppr25 ( data not shown ). the transformants , resulting either from homologous or illegitimate recombination of ppr24 into the chromosome , were selected on 7h10 medium supplemented with kanamycin . the electroporation of one μg of closed circular ( cc ) ppr24 vector resulted in approximately 200 transformants , thus the frequency of transformation was similar to that when pjδ64k was used ( 16 ). transformant colonies were phenotypically characterized using the urea / indol colorimetric test ( 11 ): a red coloration was scored as ure + , whereas a yellow coloration is considered as ure - . only 4 % of the transformants were ure - and resulted thus from an allelic exchange event ( table 4 ). table 4______________________________________effects of the presence of 2 % sucrose on theproportion of urec allelic exchange mutants . percent of total clones (%). sup . bmedium . sup . a ure . sup .- c ure . sup .+ c______________________________________without sucrose 4 96 ( one step ) with 2 % sucrose 26 74 ( two - step ) ______________________________________ . sup . a the selective medium was 7h10 supplemented with kanamycin ( 20 μ · ml . sup .- 1 ). . sup . b 50 clones were analyzed . . sup . c ure . sup .- : no change of color ; ure . sup .+ : change of the color to red . the results were absolutely in accord with what was already described by reyrat et al . in the initial allelic exchange experiments ( 16 ). for a two - step selection on sucrose as already described for m . smegmatis ( 14 ), one need to select a clone corresponding to a single - recombination event in the target gene . five randomly chose ure + transformants were propagated in 7h9 until saturation . the genomic dna was extracted and analyzed by southern - blotting using the vector ppr24 as a probe . surprisingly , all the clones corresponded to single homologous recombination events and contained a complete copy of ppr24 inserted into the urec gene ( fig4 ). this is in contrast with what was described in previous allelic exchange attempts with other genes , where the vast majority of the analyzed clones , up to 80 %, resulted from illegitimate rather than homologous recombination ( 1 , 8 ). the differences could possibly be due to the length and the structure of the genes that have been employed . when propagated in 7h9 without antibiotic , the above described single - recombinants could undergo two changes that would render them km r , suc r . ( i ) cells may lose the sacb gene during a second cross - over , ( ii ) sacb may be inactivated by a point mutation , a deletion or an insertion ( fig5 ). the frequency of the second homologous recombination event is very low , and was estimated at 10 - 5 in m . smegmatis for the pyrf gene ( 4 ). thus the detection of an allelic exchange mutant , though possible for a gene with a defined and an easily screenable phenotype is virtually impossible for the vast majority of genes where the screening is based on a southern - blot experiment . cultures of the single recombinant clones were spread , at a 1 / 50 dilution , on 7h10 - km supplemented with 2 % sucrose . in contrast to our previous experiments ( 13 , 14 ), the concentration of sucrose was lowered to 2 % because the growth of untransformed m . bovis bcg is dramatically slowed down in the presence of 10 % sucrose ( 13 ). the efficiency of selection remained the same , whereas the growth rate was unaffected on 2 % sucrose ( 13 ). 500 suc r , km r colonies were obtained from one ml of culture , and 50 clones were analyzed by the phenotypic test . the proportion of ure - mutants was much higher than in a classical experiment presenting a 6 - fold increase ( table 4 ). approximately one in four colonies tested ( 26 %) corresponded to an allelic exchange mutant as also verified on a southern - blot ( fig4 ). the remaining clones ( 74 %) though suc r , were ure - and presumably corresponded to clones with a mutation in the sacb gene . their genomic dna was probed with ppr24 in a southern - blot experiment and this showed no apparent change of the vector size , suggesting that the sacb mutations were point mutations or micro - deletions ( fig4 ). unlike what was observed in m . smegmatis , with the pyrf gene , no mutants corresponding to the insertion of an is element were observed ( 14 ). in summary , it has been demonstrated that a two - step positive selection of allelic exchange mutants , using sacb as a counter - selectable marker , is possible and very efficient in the slow - growing m . bovis bcg . as for the results obtained in m . smegmatis using the same protocol ( 14 ), a high proportion of the clones selected on sucrose were allelic exchange mutants . in cases where the allelic exchange is possible using a classical protocol of mutagenesis , one - step selection on sucrose would greatly reduce the number of clones that have to be tested in order to isolate a mutant . in conclusion , a general protocol has been designed that should render the creation of defined mutants in bacteria of the m . tuberculosis complex much easier than it was until now , paving the way for further genetic characterization of this important pathogen . moreover , this protocol should also make possible the creation of unmarked mutants , when the antibiotic resistance cassette in the gene is replaced by a frameshift mutation and the second - step selection is performed on sucrose medium without antibiotic pressure ( 14 ). it was already demonstrated for m . smegmatis ( 14 ) and other bacteria where the sacb gene can be used as a counter - selectable marker ( 3 , 19 ). plasmids ppr23 , ppr24 , ppr25 , ppr26 , ppr27 , ppr34 , and ppr2 deposited under the provisions of the budapest treaty at the national collection of cultures of microorganisms ( c . n . c . m .) in paris on jun . 19 , 1996 , and assigned the following reference numbers : aldovini , a ., husson , r . n ., and young , r . a . 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