Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot

Molecular Microbiology (1993) 9(6), 1239-1246

Molecular basis of streptomycin resistance inMycobacterium tuberculosis: alterations of theribosomal protein S12 gene and point mutations withina functional 16S ribosomal RNA pseudoknot

Marion Finken, Philip Kirschner, Albrecht Meier,Annette Wrede and Erik C. Bottger'Institut fur Medizinische Mikrobiologie. MedizinischeHochschule Harinover. Konstanty-Gutschow-Strasse 8,30623 Hannover, Germany.

Summary

Multidrug-resistant strains of Mycobacterium tuber-culosis have resulted in several recent outbreaks.Recognition of drug resistance is important both fortreatment and to prevent further transmission. Herewe use molecular biology techniques to study thebasis of streptomycin resistance in single and mul-tidrug-resistant M. tuberculosis. We demonstrate thatstreptomycin resistance is associated with mutationsimplicated in ribosomal resistance. The mutationsfound either lead to amino acid changes in ribosomalprotein SI 2 or alter the primary structure of the 16SrRNA. The 16S rRNA region mutated perturbs a pseu-doknot structure in a region which has been linked toribosomal S12 protein.

Introduction

Little is known about the mechanisms by which antibioticseffectively kill non-resistant Mycobacterium tuberculosisstrains. Isoniazid is thought to block synthesis of mycolicacids in the cell wall (Winder. 1982); more recent dataindicate a key role of the catalase-peroxidase gene in theaction of isoniazid (Zhang et at.. 1992). Interference withRNA polymerase is offered as the mechanism by whichrifampicin exerts its effects (Winder, 1982).

A wide range of antibiotics act by inhibiting protein syn-thesis and the majority of these drugs interact directly withribosomes. In early studies on antibiotic resistance muta-tions that affected ribosomes, attention was drawn to theribosomal proteins as possible target sites for antibioticinteraction (Cundliffe, 1981). More recently it has beenfound that many antibiotics protect specific nucleotides in

Received 19 April, 1993; revised and accepted 4 June. 1993. "For corre-spondence. Tel. (511) 532 4348; Fax (511) 532 4366.

the 16S and/or 23S rRNA from chemical probes whenthey bind to ribosomes (Moazed and Noller, 1987; Noileref al., 1990). The protected nucleotides are, in mostcases, identical to or located adjacent to bases that havebeen implicated in those functional processes known tobe affected by such drugs. Accordingly, it has been sug-gested that the mode of action of such drugs may be tointerfere directly with the function of highly conservedsites in rRNA (Noller et al., 1990). The 530 loop of 16Sribosomal RNA is one of the most highly conservedsequences in the entire 16S rRNA gene (Guteil ef al..1985; Woese ef al.. 1975). This strong conservationargues for an important role for this segment of RNA intranslation. The information presently in hand Indicatesthat this region of the 16S rRNA molecule is involved insome aspect of A-site tRNA ribosome interaction, i.e. thedecoding process (Moazed and Noller, 1986).

Streptomycin is an aminocyclitol glycoside antibioticwhich interferes with prokaryotic protein synthesis. Itsmain effects are induction of misreading of the geneticcode and inhibition of translational initiation (Pestka,1977). Streptomycin-induced miscoding is believed to bethe result of interference with a proof-reading step intranslation (Ruusala and Kurland, 1984). Mutant bacteriawith altered responses to streptomycin were discoveredas soon as this bacteriocidal antibiotic was used clinically(Miller and Bohnhoff, 1974). Aminoglycoside-modifyingenzymes are a frequent cause of streptomycin resistancein eubacteria (Benveniste and Davies, 1973). In addition,mutations in ribosomal protein S12 have been shown toconfer streptomycin resistance. Analysis of Sm" mutantsof Escherichia coti has identified four different allelic sub-classes {strAI, strA2, strA40and strA60) based on theirinfluences on phenotypic suppression of nonsense muta-tions (Breckenridge and Gorini, 1970); each of theseclasses has been identified with substitutions at differentamino acid positions in ribosomal protein SI 2 (FunatsuandWittmann, 1972).

Several lines of evidence link the ribosomal S12 proteinto the 530 region of the 16S rRNA and to the selection ofcognate tRNAs at the ribosomal A site. Specific residuesin the 530 stem and loop are protected by SI 2 in chemical

1240 M.Finker)e\a\.

Fig. 1. Southern biol analysis of the strA gene in M. tuberculosis.Geromic DNAs (3.0 [ig] from M. tuberculosis (lane 2) and E. coli (lane 1)were digested with EcoRI, separated on an agarose gel (0.7 %),transferred to nitrocellulose membrane and hybndized with a ^^P-labe(ledprobe spanning the strA gene from Micrococcus luteus. Molecular weightmarkers are indicated.

protection experiments (Stern etai., 1988). Both phyloge-netic and mutational analyses have helped to elucidatethe structure of the 530 region. Based on comparativesequence analysis an unusual higher-order structuralinteraction involving the 530 loop was proposed (Woeseand Gutell, 1989). and subsequently confirmed by muta-tional analysis (Powers and Nolter. 1991). This interactioninvolves base pairing between residues 524-526 in the530 region hairpin loop with residues 505-507 in theadjoining 510 region bulge loop (see Fig. 3 later). Theresulting structure is an example of the class of RNAstructures called pseudoknots (Pleij et ai., 1985), Thispseudoknot is thought to be stabilized by ribosomal pro-tein S12 (Moazed and Noiler, 1986; Powers and Noller.1991; Stern ef ai., 1988). Although there are few exam-ples so far, pseudoknot structures have been found infunctionally important regions of different classes of RNAand represent Instrumental signals in such diverse phe-nomena as self-splicing of introns. autoregulated mRNAexpression, frame shifting and stop codon read-through(for a review see ten Dam ef ai., 1992).

Most strains of M. tubercuiosis are highly susceptible tostreptomycin (Sm), the earliest antibiotic effective againsttuberculosis and still a first-line agent in treatment(Roberts ef ai.. 1991). Early application of effective treat-ment is the key to curing and blocking transmission oftuberculosis. The strategy used to control tuberculosis inindustrialized nations has been based on a two-prongedapproach: identifying and treating persons with active

disease (to cure them and prevent further transmission)and identifying and treating those with subclinical infec-tion (to prevent the development of the disease). Thisstrategy generally has proven successful. However, on aworld-wide scale tuberculosis still remains the largestcause of death from a single organism (Murray ef ai..1990), and is an increasing cause of morbidity amongpersons with human immunodeficiency virus infection(Barnes et al.. 1991). Several outbreaks of multi-drugresistant tuberculosis among hospitalized patients withthe acquired immunodeficiency syndrome have recentlybeen described (Ediin et ai.. 1992; Snider and Rooper,1992). Mortality rates from disease of this type rangedfrom 70 to 90% with a median of 4 to 16 weeks from diag-nosis to death (Snider and Rooper, 1992). Unrecognizeddrug resistance may increase the risk of transmissionboth in this community and in the hospital if patients havethis infectious disease for weeks or months while theyreceive ineffective therapy pending the results of drug-susceptibility tests, since conventional drug testing of M.tubercuiosis requires several weeks (Roberts ef al..1991), Identification of the molecular mechanisms under-lying drug resistance may offer the possibility for rapidrecognition of drug-resistant strains and initiation of effec-tive chemotherapy.

In this study we investigated the role of the 530 loopand the 524-526, 505-507 pseudoknot in conferringstreptomycin resistance both at the level of primary rRNAsequence and at the level of the S12 gene.

Results

We initally attempted cloning of the ribosomal S12 proteinof M. tubercuiosis using the strA gene from E. coli asprobe. However, owing to a very weak hybridization sig-nal even under low stringency conditions this attempt wasunsuccessful (data not shown). In view of the differentgenomic G+C content of these organisms this result wasnot unexpected. We therefore reasoned that an organismmore phylogenetically related to M. tubercuiosis and witha similar genomic G+C content might be more appropri-ate. Polymerase chain reaction (PCR) primers for amplifi-cation of the protein S12 gene from Micrococcus iuteuswere deduced using the published M. iuteus strA genesequence (Ohama ef ai, 1987). This probe hybridizedwith a single 4 kb fragment of EcoRI-restricted M. tuber-cuiosis genomic DNA (Fig. 1). Standard molecular biologytechniques were used to clone the M. tubercuiosis strAgene in a phage vector using the heterologous PCRderived strA gene from Micrococcus luteus as probe.

Table 1 lists the strains which were investigated in thisstudy. Nucleic acids extracted from streptomycin-resis-tant M. tubercuiosis sUalns and control streptomycin-sen-sitive strains were used as template in PCR to amplify the

Streptomycin resistarice in Mycobacterium tuberculosis 1241

16S rRNA 1

pos. 513 pos. 516

G A T C

Sm

G A T C G A T C G A T C

Fig. 2. Nucleotide sequence of a short region of the 16S rRNA gene demonstrating ihe mutations associated with streptomycin resistance. The wild-typesequence (Sm^) is shown along with those of the resistant (Sm*̂ ) isolates. Mutated bases are indicaled by asterisks, and the location of the mutation ismarked by an arrow.

genes coding for 16S rRNA and ribosomal protein S12.The nucleotide sequences of the S12 gene and the rele-vant regions of the 16S rRNA were determined by directsequencing.

For all control strains the bases involved in the pseudo-knot structure read 5'-GGC-3' (M. tuberculosis 16S rRNAposition 495 to 497 (Suzuki etai., 1987), corresponding toE. coii position 505-507) and 5'-GCC-3' {M. tubercuiosis16S rRNA position 514-516. corresponding to E. coiiposition 524-526). In contrast, three streptomycin-resis-tant isolates including two multidrug-resistant strains

show a C to T transition at M. tubercuiosis 16S rRNAposition 516 (see Fig.2 and Table 1). Using site-directedmutagenesis Powers and Noller have recently demon-strated that appropriate base pairing between residues524-526 and residues 505-507 is essential for ribosomefunction (Powers and Noller, 1991). Moreover, certainmild perturbations of the pseudoknot structure, i.e.creation of G-U wobble pairs between residues 524/507and 525/506, were shown to generate resistance to strep-tomycin. As a C to U transition at M. tubercuiosis 16SrRNA position 516 (corresponding to £. co//position 526)

Table 1. List of strains investigated in this study.

Number ot isolatesinvestigated

23 multidrug-

15 single drug-resistant isolates

35 controlisolates

Number ofisolates

32

9

45

31524

35

Drug sensitivity

Sm" (multidrug resistant)Sm" (multidrug resistant)

Sm*̂ (multidrug resistant)

Sm" (multidrug resistant)Sm" (multidrug resistant)

Sm" (single drug resistant)Sm"̂ (single drug resistant)Sm" (single drug resistant)Sm'' (single drug resistant)Sm" (single drug resistant)

Sm^

16SrBNA Mutations

Position513

A ^ C-

-

--

----

-

Position516

-

C->T

-

--

C-.T--—

-

Type and Location of Amino Acid Exchanges inRibosomal Protein 12

Aminoacidposition

-

-

43

88-

-4388—

-

Aminoacidexchange

-

-Lys^ArgLys->ThrLys-*Arg

—

-Lys-*ArgLys-»Arg—

-

CodonalteratiCHi

-

-AAG->AGGAAG->ACGAAG-*AGG

—

--AAG-^AGGAAG^AGG

-

Type otmutation

Transversion

TransitionTransitionTransversionTransition

Transversion

TransitionTransitionTransition

-

a. One of the three strains has an A to T transversion at this position.b. Multidrug resistance is defined by resistance to at least two standard anti-tuberculosis drugs.

1242 W. F/n/cenetal.

530loop ?

cG

520-AC

c

510

A ACC

G G

3 C <

Gu —G —c -AU —

c —C -c -A —c —G -

= G

ACGGAG-GGUGC

CGG-530U

A

540

B530loop

520-•530

-540

Ffg. 3. A.Secondary structure of the 530 stem-loop region in 16S rRNA from M. tuberculosis(numbering is according to E. coli. for thecorresponding M. tut}erculosis numberingsubtract 10), The site of Ihe 513 mutarion(oorresponding to E.co//position 523) is indicated.B. Secondary structure of the 530 stem-loop toillustrate the interaction between positions505-507 and 524-526. The site of the 516mutation {corresponding to £ composition 526) isindicated.

500

results in a G-U wobble pair between residues 526 and505 (see Fig. 3), we conclude that this mutation confersstreptomycin resistance (Sm").

Five Sm^ isolates show an A to C transversion at invari-ant pos. 513 (see Figs 2 and 3; note that one Sm" strainshows an A to T tranversion at this position, see Table 1).By genetic engineering techniques an A to C transversionat £. coli position 523 (corresponding to M. tuberculosisposition 513) has been demonstrated to confer strepto-mycin resistance in E. co//(Melancon etal.. 1988). It isthus likely that in a subset of streptomycin-resistant M.tuberculosis isolates the loss of streptomycin sensitivity iscaused by point mutation within the 16S rRNA gene atInvariant position 513. No mutations were found in 16SrRNA positions involved In streptomycin binding (Moazedand Noller, 1987) i.e. E. co//16S rRNA positions 909-915,1413, 1487 and 1494 or in any other ribosomal site impli-cated in antibiotic binding. Sites conferring drug resis-tance to the aminoglycosides kanamycin, tobramycin and

gentamicin (De Stasio et al., 1989) were unaffected instreptomycin-resistant M. tuberculosis stra\T)s.

The ribosomal protein S12 gene strA was isolated fromcontrol and streptomycin-resistant M. tuberculosisstrains. Analysis of the amino acid sequence deducedfrom the strA nucleotide sequence revealed that a sub-group of the resistant isolates had a single amino acidreplacement In one of two positions: the lysine residue inposition 43 is replaced by arginine or threonine (13 iso-lates showed a replacement by arginine; in one isolatethe codon was changed to ACG resulting in a threonine)and at position 88 lysine is mutated to arginine (see Figs 4and 5 and Table 1). Our finding that there is a strongrestriction of the amino acid positions which can bereplaced in streptomycin-resistant M. tuberculosis agreesvery well with the genetic mapping and location of aminoacid replacements in protein S12 in E. co//mutants resis-tant to streptomycin. Mutations leading to streptomycinresistance were found to map at one of two sites in the

aa43

protefn S12

aa43 aa 88

Sm"

G A T C

Sm*

G A T C

Sm Sm'

Fig. 4. Nucleotide sequence ot a short region of the strA gene demonstrating the mutations associated witti streptomycin resistance For details see thelegend to Fig. 2.

Streptomycin resistance in Mycobacterium tuberculosis 1243

M.tb. Sm''M. tb . Sm̂E .c .

M.tb. Sm'M.tb. Sm̂E . c .

10 20 30

MPTIQQLVRKGRRDKISKVKTAALKGSPQRRGVCTRVYTTTPKKPNSALRA-VN P-AR-VA-SNVP--EAC--K

70 80

R / T 50 Fig. 5. Alignment of the amino acid sequences of- t S12 ribosomal proteins from streplomycin-

resistant M. tuberculosis (M.tb. SM* )̂,streptomycin-sensitive M. tuberculosis (M.tb.Sm^) and E. coli (E.c.)., — denotes identicalmatches. The Lys-43 and Lys-6a positions, whichare mutated In Sm" M. tuberculosis isolates areIndicated.

100

KVARVKLTSQVEVTAYIPGEGHNLQEHSMVLVRGGRVKDLPGVRYKIIRG- - -C-R- -NGF- - - S - - G V I - I HTV--

110 120H . t b . SmM . c b . Sm*E . c .

SLDTQGVKNRKQARSRYGAKKEKGA - - C S - - - D K--V-RP-A

StrA gene, the distance between the two sites was 0.3map units (Breckenridge and Gorini, 1970). The geneticdata were corroborated by protein-chemistry results as bypeptide mapping (Funatsu and Wittmann, 1972) the twosites in the S12 protein conferring streptomycin resis-tance were identified as amino acid positions 42 (replace-ment of lysine by asparagine, threonine, arginine) and 87(replacement of lysine by arginine), which correspond toM. tuberculosis S12 positions 43 and 88 respectively (seeFig. 5).

Discussion

In this report we have investigated the molecular mecha-nisms underlying streptomycin resistance in single andmultidrug-resistant M. tuberculosis. In general, resistanceis conferred either by an altered structure of the drug tar-get, or by an efficient drug degradation system or perme-ability barrier. Mutations in the ribosomal protein SI 2involving two conserved amino acids were identified in 20of 38 streptomycin-resistant isolates, but in none of the 35streptomycin-sensitive isolates. The mutations resulted ina replacement of lysine by arginine or threonine at aminoacids 43 or 88 respectively. In nine of the 18 strepto-mycin-resistant strains with a wild-type strA gene, but innone of the control strains, the small subunit rRNA wasfound to be mutated at invariant positions 513 or 516. Themajority of acquired resistance to streptomycin in singleas well as in multidrug-resistant M. tuberculosis isolates isthus associated with single mutations in ribosomal proteinS12 or within the 530 loop of the 16S rRNA. As the corre-sponding mutations have been shown to confer astreptomycin-resistant phenotype in E. coli (Funatsu andWittmann, 1972; Melancon et al.. 1988; Powers andNolter, 1991), we conclude that the mutations found areresponsible for streptomycin resistance in M. tuberculosis.

Streptomycin is known to selectively perturb the proof-reading process. Streptomycin-induced misreading of the

genetic code is suppressed by mutations in the ribosomalS12 protein (Breckenridge and Gorini, 1970; Funatsu andWittmann, 1972). It has been known for some time thatcertain Sm" mutants exhibit an enhanced accuracy oftranslation over that of wild-type ribosomes (Allen andNoller, 1989; Brouadlon et at., 1983; Gorini 1971;Ruusala etal., 1984). The normal role of the S12 proteinseems to maintain some slight imprecisions in decoding,as mutations In the strA gene have been shown to restricttranslational leakiness, the mutant Si 2 protein giving aribosome that more rigorously permits only the use ofaminoacyl-tRNA appropriate to each codon, thus enforc-ing a more precise expression of each codon in a mRNA(Bohmann etal., 1984). With a number of such sfrA muta-tions, streptomycin antagonizes the effect of the changein protein SI 2. restoring the ribosome to its nomnal situa-tion of permitting a low level of inaccuracy in translation.Streptomycin dependence appears to be an extreme ver-sion of translational restriction, in which mutated versionsof protein S12 cause the ribosome to become toodemanding in its recognition requirements of aminoacyl-tRNA species (Birge and Kurland, 1969; Ozaki ef al.,1969). Our results that mutations within the 530 loop areassociated with streptomycin resistance point to theinvolvement of the 530 loop in the control of translationaccuracy and are in accordance with a previous sugges-tion that the proof-reading control may depend upon theconformation of the 530 loop (Moazed and Noller, 1986;Powers and Noller, 1991).

Our data provide genetic evidence supporting the ideathat resistance against multiple unrelated drugs in M.tuberculosis is not caused by a single event involvingresistance to a panel of antibiotics, e.g. an altered perme-ability barrier, but rather conferred by an accumulation ofmultiple mutational events specific for each drug's targetrespectively as demonstrated for streptomycin. Ulti-mately, identification of the molecular mechanisms under-lying drug-resistance may offer the possibility for rapid

1244 M. Finkeneta\.

Table 2. NucleoKde sequences of theoligonucleotides used. Primer

S5S7S13S16S11SI 7248254259260261264278285289

CGTGGCGTGTGCACCCGTGGACGTCGACGGGCACCTGGTGGCCGACAAACAGAACGTGTTCACCAACTGGGTGACGTCGAGGTCACGGCGTACCGCGTGTACACCACCACTGTGTGGGTTTCCTTCCTTGGCCAAGGAAGGAAACCCACACTTTGACGAACAACGCGACAAGTCCTG TGGATGTCAAACCCAAGGAGGTGATCCAGCCGCATGGACAGAGGCGACAAGGGAGAGGTGAAGTCATCATGCCCGAGAGTTTGATCCTGGCTCAGAAGTCGGAGTCGCTAGTAAT

Ta/get

Si2gene(M,lut,)S12gene(M.lul.)S12 9ene(M.tb.)S12 9ene(M.tb.}S12 9ene(M,tb.)S12gene(M.tb.)16SrDNAleSfDNA16SrDNA16SfDNA16SrDNA16SrDNAleSrDNA16SrDNA16SrDNA

Direction

ForwardReverseForwardReverseForwardForwardForwardReverseReverseReverseReverseReverseForwardForwardForward

Position

93-111**276-257'5' non-coding"S7 gene"181-198"106-123"830-850*^84a-831'^609-590^^1007-988'̂1542-1523'=1046-1027'=1190-1209'7-28°1332-1351'=

a. Micrococcus luteus (M. lut.).b. M. tuberculosis (M.tb.).c. Corresponding E. co//position.

recognition of drug-resistant M. tuberculosis strains bynucteic acid-based assays and may altow prompt initia-tion of effective chemotherapy. The results presented inthis paper are a first step towards this goal.

Our findings go beyond the mere etucidation of drugresistance in M. tuberculosis. A wide range of antibioticsact by inhibiting protein synthesis and the majority ofthese drugs interact directly with ribosomes (Moazed andNotlor, 1987). Such antibiotics have been instrumentat inanalysing the mechanism of translation and the detaits ofribosome function both directty and indirectly (e.g. resis-tant mutants). Mutations in rRNA operons conferringantibiotic resistance, however, could not be isotated byclassicat genetics in eubacteria because of the multiplecopy number of these operons and the observation thatsensitivity to antibiotics that act by inhibiting protein syn-thesis is frequently dominant over antibiotic resistance(Lederberg, 1951). Ribosomat resistance to antibioticsbased on mutated rRNA genes in eubacteria was ontypossible to demonstrate by in vitro molecutar biologytechniques using high-copy-number ptasmids bearing themutated rRNA operon in question (De Stasio etal., 1989,Metancon etal., 1988). In contrast, slow-growing myco-bacteria contain a singte rRNA operon (Su2ut<i ef al.,1987). Thus, rRNA mutations which are normally reces-sive in eubacteria can be expected to confer a dominantphenotype in mycobacteria.

This report is the first demonstration of acquired drugresistance in eubacteria associated with a single pointmutation in a rRNA gene. Surprisingty, 16S rRNAnucleotides protected by streptomycin in vitro (Moazedand Notler, 1987), were not involved in mutations confer-ring acquired streptomycin resistance. The mutated posi-tions 513 and 516 represent two of the few characteristicinvahant bases in the small subunit rRNAs of eubacteria.

chtoroptasts and mitochondria. Mutation of thesenucleotides does not prevent the binding of streptomycin,thus, it has been hypothesized that they might interferewith the conformational perturbations that account forstreptomycin's action on the ribosome (Dahlberg, 1989).Phytogenetic, genetic and biochemicat evidence indi-cated that the 530 stem loop is folded into a pseudoknot-tlke structure invotving 16S rRNA position 516 and thatthe assembly of this structure is influenced by ribosomalprotein S12. Our resutts demonstrate the feasibility ofdrug-resistant mycobacterium isotates to dissect themechanisms of ribosomal resistance: the importance of a16S rRNA pseudoknot in conferring streptomycin resis-tance was demonstrated both at the level of primary 16SrRNA sequence and at the tevet of the stabitizing S12 pro-tein.

Experimental procedures

Cloning of the strA gene

The SI 2 gene from M. luteus was amplified by polymerasechain reaction using primers {S5 and S7) deduced from thepublished sequence resutting in a 560 bp DNA fragment (seeTable 2). The amplified gene fragment was tabelled by randompriming and used in Southern Btot analysis. Genomic DNAfrom M. tuberculosis was prepared using a modifiedtysozyme/proteinase K procedure (Rogatt et al.. 1990),digested with fcoRt. subjected to agarose gel electrophoresison low-melting-point agarose, the appropriate region compris-ing 3.5 to 4.5 kb gene fragments excised, purified and ctonedinto XZAP II (Stratagene). Standard techniques {Sambrook etal., 1989) and the M. luteus strA gene probe were used to iden-tify and isotate recombinant ctones containing the S12 gene(EMBL accession number X70995). The sequence determinedwas used to derive primers for amplification and directsequencing.

Streptomycin resistance in Mycobacterium tuberculosis 1245

M. tuberculosis isolates

Drug sensitivity of the clinical isolates was determined usingthe proportion method (Roberts etai., 1991). Concentrationsused were streptomycin 6 .0mgr \ rifampicin 2 .0mgr \ etham-butol 7.5 mg I"'', pyrazinamide 100 mg r', isoniazid 1.0 mg 1"^.

Nucleic acid extraction, PCR ampiification and directsequencing

For nucleic acid extraction a small loopful of bacteria was dis-persed in 100 \i\ H2O, heated for lOmin at 80"C to inactivatethe mycobacteria and then transferred to a 1.5 ml screw-topplastic microfuge tube containing glass beads with a diameterof 100 (im (Sigma). A tissue disintegrator (H. Mickle) was usedto disrupt the celts. A 5M1 aliquot of the supernatant was usedin PCR, The 16S rRNA gene was amplified in two overlappingfragments (Edwards et al., 1989)- Primers 285 and 264 wereused to amplify a 1046bp fragment; primers 248 and 261directed the synthesis of a 712bp fragment. For sequencedetermination of the relevant regions (corresponding to E. coiipositions 400-970 and 1320-1510 respectively) primers 259,260, 254, 278 and 289 were used.

For amplification of the strA gene from M. tuberculosis,primers flanking the 5' end (S13) and the 3' end {S16) werechosen and resulted in a 501 bp fragment, which contains thecomplete SI 2 gene. The following primers were used forsequencing: S11, SI 3 and SI 7.

The nucleotide sequence of the amplified gene fragmentswas determined by direct sequencing. Typically, one of theprimers (285, 261 and SI 6) used in the amplification reactionwas biotinylated to allow ssDNA sequencing using streptavidin-coated paramagnetic beads (Dynabeads^", Dynai).

The primers used in this investigation are listed in Table 2.

Acknowledgements

We are indebted to Drs Max Salfinger, Gaby Pfyffer, Karl-HeinzSchroder, Sabine Rusch-Gerdes and Thomas Bodmer for pro-viding drug-resistant strains (clinical isolates). We thankSusanne Maibom for typing the manuscript, Dieter Bitter-Suer-mann for continuous encouragement and Matthias Frosch forcomments on the manuscript. This work was supported in partby the Niedersachsischer Verein zur Bekampfung derTuberkulose e.V. and by a grant from the Bundesministeriumfur Forschung und Technologie (01 Kl 89117).

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