Gene, 115 (1992) 49-54 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00
49
GENE06Ol
Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity* (DNA amplification; deletion; spiramycin production; mutator genes)
Jean-Marc Simonet, Dominique Schneider, Jean-Nicolas Voiff, Annie Dary and Bernard Decaris Laboratoire de G$n$tique et Microbiologie, Facult$ des Sciences, Universit~ de Nancy L F-54506 Vandoeuvre-lbs-Nancy C~dex (France) Received by J.C. Ensign: 19 August 1991 Accepted: 30 December 1991 Received at publishers: 17 February 1992
SUMMARY DNA amplification and deletions occur at high frequency in unstable regions localized on the Streptomyces ambofaciens chromosome. The structure of these regions was investigated, leading to the identification of internal reiterations which could play a role in the deletion and/or amplification mechanism(s). UV irradiation and treatments with mitomycin C, oxolinic acid and novobiocin were shown to efficiently induce genetic instability. Finally, mutator strains were isolated, in which genetic instability was dramatically increased. The involvement of an SOS-like response in genetic instability in S. ambofaciens is proposed.
INTRODUCTION Many Streptomyces species exhibit a very high degree of genetic instability. Molecular analyses ofthe mutant strains arising from spontaneous or treatment-increased instability often reveal genome rearrangements, which are mainly large deletions and/or amplifications (according to the nomenclature proposed by Fishman and Hershberger, 1983;
Correspondence to: Dr. J.M. Simonet, Laboratoire de G~n6tique et Microbiologie, Facult~ des Sciences B.P. 239, F-54506 Vandoeuvre-I~sNancy C~dex (France) Tel. (33-83)912204; Fax (33-83)901511. * Presented at the International Symposiumon Biologyof Actinomycetes, University of Wisconsin, Madison, WI (USA) 11-16 August 1991. Abbreviations: aa, amino acid(s); ADS, amplifiedDNA sequence; Amc, aerial mycelium;A UD, amplifiableunit of DNA; bp, base pair(s); ccc, covalentlyclosed circular; EtdBr, ethidiumbromide; HT, Hickey-Tresner (medium);IHS, internalhomologoussequence(see Fig. 1); kb, kilobase(s) or 1000bp; MC, mitomycin C; nt, nucleotide(s); ORF, open reading frame; Pig,colonypigmentation;oc, open circular; PFGE, pulsed-fieldgel electrophoresis; S., Streptomyces; Sp, spiramycin; UV, ultraviolet; wt, wild type.
ADS stands for amplified DNA sequence; A UD stands for amplifiable unit of DNA; reviewed by Leblond et al., 1990a; Birch et al., 1990). In S. ambofaciens DSM40697, two types of genetic instability were previously described: (/) a basic genetic instability, commonly reported, and (ii)hypervariability, which is a new aspect of instability closely associated with DNA amplification and deletion (Leblond et al., 1989). Most amplifications were located in two amplifiable regions, one of which, A UD6, was shown to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied possessed DNA amplifications, deletions or both at the A UD6 locus. Deletions occurred exclusively to one side of the ADS and removed part of the proximal c,opy of this A D S leading to the conclusion that multiple rearrangements can occur at this A UD locus. Moreover, one of the endpoints of the A UD6 shows homology with an internal sequence called IHS (Demuyter et al., 1991). The genomes of four S. ambofaciens strains from different geographical origins, including strain ATCC15154 (a close derivative of ATCC23877), and strain DSM40697 were analysed by PFGE. The analysis of the resulting macrorestriction patterns using rare-cutting enzymes indicated that S. ambofaciens ATCC15154 exhibited a restric-
50 tion pattern different from that of strain DSM40697. The total genome sizes of S. ambofaciens ATCC 15154 and DSM40697 were estimated to be about 6500 and 7800 kb, respectively (Leblond et al., 1990b). Using the same technique, the amplifiable loci A UD6 and A UD90 of S. ambofaciens DSM40697 were mapped within two adjacent Asel restriction fragments. The genetic instability and the formation of very large deletions were strictly correlated since all the mutants tested so far (30/30) presented deletions within the DNA region overlapping both amplifiable loci. The deletion size was estimated to range from 250 kb to more than 2000 kb. PFGE also permitted the localization of the ADS and their structure to be established revealing that amplification takes place at the A UD locus as a tandem array of the wt A UD sequence (Leblond et al., 1991). DNA analysis of the spontaneous mutant strain NSA420 revealed the presence of an ADS, which localized on the chromosome within the A UD6 region, and the presence of extrachromosomal multimeric ccc DNA molecules homologous to the ADS (Simonet et al., 1990). These extrachromosomal forms, originating from the A UD6 region, could correspond to a form of ADS elimination through recombination events between the reiterated sequences. The hypotheses proposed to explain the occurrence of deletions in the unstable regions involve recombination events taking place between reiterations. Such reiterations have been sought in both A UD6 and A UD90 regions. With the aim of extending our observations, the genetic instability of S. ambofaciens ATCC23877, a strain from which both ATCC15154 and RPI81110 were derived, was also studied. The presence of A UD regions homologous to the AUD6 and AUDgO of strain DSM40697 was investigated. In the strain RPI81110 lineage, the presence of the ADS205 (A UDgO family) was associated with the loss of Sp production. Furthermore, in the progeny of one of the amplified strains, the loss of the ADS was correlated with the restoration of Sp production (Dary et al., 1992). Finally, two other kinds of experiments were performed: (i) to modify the level of genetic instability by treatments which possess specificity of action in other bacteria, and (ii) to isolate mutants affected in genetic instability.
etal., 1992). Deletions were also detected in strains NSA207, NSA208 and NSA210 and were localized on the same side of the amplifiable region. Hybridization experiments revealed strong homologies between A UD205 and A UD90 in strain DSM40697. Similar experiments carried out with a probe specific to A UD6 gave identical hybridization patterns with DNA of both ATCC23877 and DSM40697 strains. Thus, the same unstable regions exist in two strains of different geographical origin, in spite of the fact that the genome size of these strains differed by 1300 kb (Leblond et al., 1990b). Moreover, it can be seen that at least one of them (AUDgO/AUD205) might be amplified and/or deleted in both lineages, either spontaneously (in strain DSM40697) or after treatment with EtdBr (in strain RPI81110).
RESULTS AND DISCUSSION
(b) The amplification of a particular nt sequence reversibly prevents Sp production Since strain RPI81110 synthesizes the macrolide antibiotic Sp, production was also tested in the amplified mutants NSA205 to NSA210, which indicated that they were all nonproducers (Sp-). Strain NSA205, which contained the largest ADS (89 kb), was examined by subcloning to investigate a possible correlation between this ADS and the Sp- phenotype. The ADS205 was unstable and lost with a high frequency (9/67). In all cases (9/9) the absence of the ADS205 was correlated with a return of Sp production. In one case (1/9) the ADS205 was lost, and a novel amplification (ADS213) was found. This indicated that only the amplification of a particular nt sequence was correlated with the absence of Sp production. As the Sp- phenotype was reversible, it was concluded that the genes involved in Sp biosynthesis were not deleted, a hypothesis which was confirmed by hybridization between the three resistance determinants and the known biosynthetic genes cloned by Richardson et al. (1990) and various DNA restriction fragments of RP181110 and mutant strains, although it is not yet known whether all of these genes are normally expressed in the Sp- ADS205 + strains. A variety of hypotheses such as the altered expression of one of the resistance genes, the presence of an inhibitor gene present on A UD205 and its over-expression in the amplified strain, as in the case of S. tendae (Koller and Riess, 1989), or the non-expression of a gene involved in Sp production may explain this reversible inhibition of Sp production.
(a) Characterization of an amplifiable region in the genome of strain RPI81110 Amplified mutant strains (NSA205 to NSA210) were isolated following treatment of strain RP181110 with EtdBr. The ADS of these strains were localized in an amplifiable region, AUD205, in the RP181110 genome (Dary
(c) Reiterated sequences in AUD6 To study the A UD6 structure, the isolation of this chromosomal region from the wt strain DSM40697 was undertaken. A genomic library of S. ambofaciens strain DSM40697 was constructed in E. coil and hybridizations, using the cloned S 1 fragment (Fig. 1) generated by the re-
51
~t3
....
•
!.J ....... L..! ........ ! ........ J.............................. 1.3 3.1
1.9
3.5
4.0
12.0
AUD6 (24.8 kb) SIL
LI .....................................
0.6
='~ IHS
15.0
~
I
,...
SIR
Fig. 1. Restriction map of the A UD6 locus of S. ambofaciens D SM40697. S 1 corresponds to the Barn H 1 fragment generated by the amplification ofA UD6. SIR and Sit_ are the right and the left parts of Sl, respectively. IHS is a DNA fragment which is homologous to SIR. The underlined 1.9-kb BamHl fragment was subcloned and its nt sequence determined (see section e and Fig. 2). Restriction fragment sizes are given in kb.
vealed its size to be 1842 bp and that it contained 6 9 ~ G+C, which is in good agreement with the high G+C content of the Streptomyces genome (70-74°,~,). The study of this sequence revealed one O R F which showed codon usage typical of Streptomyces (70, 50 and 9070 G+C in the first, second and third position of the codons, respectively). In addition, there is a sequence resembling the Streptomyces consensus promoter sequence immediately upstream from this O R F (Fig. 2). Comparison of the predicted aa sequence of the O R F with the GenBank database revealed homology (23.8?/o in 227 aa) with the gntR gene product of Bacillus subtilis (Fujita and Miwa, 1989), which is the repressor of the gluconate operon in that organism (Fig. 3). The analysis of the structure of this putative protein showed some features
iteration of AUD6 as probe (Demuyter et al., 1991), revealed a clone containing a cosmid with a 40-kb insert which belonged to the A UD6 region. Subsequently, the 1.9-kb B a m H l fragment was subcloned (Fig. 1). Hybridizations with S 1 (Fig. 1) allowed us to detect fragments external to the A UD (one of which could be a junction fragment). These signals were present only in the amplified strain but not in the wt strain, indicating that these reiterations could be involved in the amplification process. To determine which part of the 1.9-kb B a m H l fragment was reiterated, hybridizations were carried out using different regions of this D N A as probes against total D N A of the mutant strain amplified for A UD6. All the subclones gave the same pattern indicating that the entire 1.9-kb fragment at least was reiterated. The nt sequence analysis re-
A
B
I 10 I 20 I GGATCCGGTC CGC~CCTGGT 81 TGGTGCGCAC GTAGTCGATG 161 ATCGGTGTCG TGGTGTGCTG 241 CCGGGAGGCC TCGACGCTCT 321 AGCCGAAGCC GARF.~GTTCC 401 TCGTCGGGCA GTTCGGAGGC 481 CGCCTGCGCC CGTACCGCCT 561 TGGCGTGCGA GTAGACCAGT 641 &GGATGTCCC ACAGGGCGGT "/21 G ~ e T G C CACGTGTCCT 801 CGACGGCCAG TTCCCGGCCG 881 ARGGTCCGGC CGGGGGAGGC 961 T A T ~ T ~ T A T C G C lO41 CGG~CG~ace GGCGCGACAT 1121 GGAGAACGAA CTeGCCC.~GT 1201 TCGTGCAGGT CTTCCCCAN~ 1281 GAGGeGGTGG AI~CTCGGTTC 1361 ~GTGCGGCAG GGGCGCARGG 1441 ~GI~--r-C-C-r2-~-~ -~--,CGGGA~,GTC 1521 GAGAP,CGTGT eCCCGGCCGT 1601 CCGTACCGCC &TGCGTACAC 1681 CCACCGACGC CGCGACCGTG 1761 CGTCGGCCGC TGGTCCCCGC 1841 CC I 10 I 20
TTGaca
-
TTGgtc -
18 17
bp, bp -
I 30 I 40 GG~GCGGC GAGGTCGA~ AGCTGTTCGG TGATGAC~TG CCGGATGAGC CGGAAGGACT TGCC~G GGCCGCCTCG CGTACCGCTT CCAGGTACCT GGCCGGeTCG TAGACCCTGC TGTAGCCCAG CTCCAC~TGG .&CGCCGTCGC GCGAGCGGCC GTCGACGGCG GCG&TCGCCG CGATACGGGC GGGGTCCeTG TTGRF.~GTGG CGTCGCCGAG GACGARGACC TCGI~CGCTTT AGCACGGCAA TACTTGGCTC eT~ceT~ CTGC.CCCAGA CGATGGGGGT CAGCCGCACG ATCGGGTCGT TCGTCTCeCG CCTGGACGCT CTGCCCG~GG ACCTCGACCT GGAGGAGTTC ,,TGG&CC~CCG TCGCCGCGGC GTTCGTeGCC CAGCACCGCG ACCTGCGAGC CGTCTTCGAC CCCGT&CGAC GCAACATCGT TeGTCCGGCG TGTGCCCGGG
I
30
I
40
I 80 I 70 GCCGCCCGCG TGGACGACGG 80 TCATCACCTC GCCGACCC.-,CG 160 240 GGGTCCTCCA T C ~ CACGTCGTGC AGGAGGTGC~ 320 CCTCGGTGTC CCAGGGC'JL'GT 400 CCCCCGACGC CGGGeACGGC 480 CGRGC~TACG TCGGTGCCGC 560 GCAGGCCGGC GGTCTTGCCC 640 CGCCAGTAGG CGCCCTTGTA 720 GTGGTCGCGC AGATAGeTGG 800 CAGCGTGAF..G 880 ~ ~ G ~ 960 C ~ G G C T C A AACGAA£GGG 1040 CCGCCCTCTC 1120 GCTCCGGGCG CCTGTTGGCC CAGGAGGGCC 1200 CCGACGCGCA GTTCCTCCGG 1280 GGGGAGCTGC GGGACAN~C~ 1360 CTG&TGCGC~ 1440 CCAC~ CCCGCCGCCT CGGCCTCCAC 1520 TCCCCCTCGC 1600 ~ G GCACTCACCC GAACTGTTCG 1680 T~I~CCC~C G ~ . ~ G ~ C GGGGTCCTTC 1760 CGGCGCGG&T 1840 GGCGTCCCCG T ~ C G C G !842 I 8O 50 I 60 I 70
I 50 ATCCGGCGCA CTGGACGTCC CCTGGTTCTC TTCGGGGTGA GGGCGCGAAG CCTTGCGCAC CGeTCGACGT G C C ~ TCATCGTGAC CCGATCAGCA GeCGG,CACC CGATCCTGCT GCGCTCTACG TGGTCCTGAC CCGGTACGGG GGTCG&TCCG AGCTCGACCC TTCGGTCTGG C ~ T &G&TCTTCGA GACATCGAAC GGTCTGGGAG TGeGGCGTGC I
I 60 GATGGGTGAT CAGATCGAGT GGCC~TC GCCGGTGGTG TCGAGGTAGG CCCGTACGTG CGTCGAGAAG TGGTGGACGG GGGTCCGCGC GCGGGACCAC CCGTCCGTGG ~ T TI~:GATGTGC CCTCGAACTC AGAGCCTG~T GeTCGGG,TCG CGCGGTGGTG ACGAGGCGTT CTGGACCGC~ CGCGGTCACu GCATCCGCGC
tAGgaT aAGcaT
Fig. 2. Nucleotidesequence analysisof the 1.9-kbBamH! fragmentofA UD6. The nt sequence was determined usingthe Promega(Madison, WI) TaqTrack sequencing system and the Pharmacia double-stranded nested deletion kit which produces progressive deletions into the sequenced fragment. (A) The nt sequence. ORF is delimited by brackets. The putative protnoter (with the' -35' and" - 10' regions bo~ed at nt 943-948 and 967-971, respectively)is shown. The sequence resembling the operator of the gntR of the B. subtilis gluconate operon is overlined with a horizontal bracket. The sequence homologous to the 930-bp fragment of A uDgo is boxed with a thick line (see section d). (B) Comparison of the putative promoter located upstream from the ORF (bottom line) with the consensus sequence (top line) established for Streptomyces (Hopwood et ai., 1986).Upper-case letters indicate the occurrence of those particular nt in over 750o of the examples and lower-case letters indicate the occurrence of those nt in 50-75°0 of the examples.
52 1O 20 30 40 MAQTNGRTNRRDZYLKLR~VLTLELAPGAALSENELAASY~'VSRTPVR .... :. : : .:: . . . . . . . :. : : : : . . : : . . . : : : . : : :
&UDSE0 GntR
IILDSKDLLYpAI~4LSKASTGVRVAY- E L R M R Z V S G L I E S G T Z L S E N T Z R ~ E F S V S R S P V R 10 20 30 40 50
50 60 70 80 90 100 AUI)SE~ E S L Z L I d ~ E G L ~ V F P K X G S F V S R V D P ~ R V A D A Q F L R E A V E L G S L D A L P A E L D P A V V G E L :.: .:: : . . . . . . :. : .... :. .: .: .. : ..: .: GntR EALKILASEKI ZR-LERMGAVVIGLTEI(KZAE I~DVPT.LrRTFVFERLVKXDZEPLVKDL 60 70 80 90 100 110
II0 120 130 140 150 160 ~$1DSEQ R D - - N L ~ Q G R I t D L D L E E F F G L D E A F H Q G L M R L S G H G - - - N ~ T 4 T T V - A A A O G H L D R A R R L ....... : : .:: : :: .... : .:. .: . . . . . . . . . . :. GntR SKZIJUOIKVSZKTEDADEFSFQDVLFHET Z ZRAZDH S¥IQMZWNNLKPVMESF ILLSMRV 120 130 140 150 160 170 170 180 190 200 210 220 AUDSEQ GLIIENVSPAVFVAQHRE ZF-DAVTEGDVP LARTAMRTHLRAVFDD ZERZ RAHSP ELFATD 1.:. . . . . . ,1.. .1... : ." ... .. .: :..: .. : .... 1.. GntR RIJgJIKYEDFTRZ L D N H Z L Y Z Q & £ K T K D R A L M Z Q S L I I Q N F D D V Q D K V E D L W - L S g Q M L A K G 180 190 200 210 220 230
&UDSEQ AATVPVRRNIV~,~Z . Gn~R
~YNND 240
l"
-~ • TACTTGTAT&CAAGTATACT
gntR operator
*** * * * * * * * * * * ** ** TACATGTATACAAGCAT-CT . . ~ ~ . ..
AU~
Fig. 3. Analysisof the S. ambofiwiensdeduced aa sequence of the A UD6 ORF. (A) Comparison of the deduced aa sequence(AUD SEQ) with the B. subtilisg, tR gene product. Colons represent identical aa, whereas full stops represent aa conservativechanges as defined by Lipman and Pearson (1985). The single-letter code was used for the aa. Last digits of numerals are aligned with the corresponding aa. (B) Comparison of the operator sequence recognized by GntR (top line) with the sequence of A UD6 (bottom line and overlined in Fig. 2). The convergent arrows indicate a dyad symmetry.The asterisks indicate the conserved nt.
of DNA-binding proteins, i.e,, helix-turn-helix domains. Furthermore, the operator region (located immediately upstream from the gntR start codon) recognized by GntR in B. subtilis showed strong homologies (17/20 nt) with a sequence located immediately upstream from the start codon of the A U D 6 0 R F (Fig. 3).
(d) Reiterations in the AUDgO region In S. ambofaciens DSM40697, all mutant strains studied so far showed rearrangements in the A UD90 region. To study the deletions in the mutant strains carrying amplifications in this A UD region, hybridization experiments were carried out with a cloned ADS fragment as probe. This probe revealed several signals: (i) as in A UD6, faint signals appeared which could correspond to homologies external to the A UD, and (ii)signals corresponding to amplified fragments within the A UD90, suggesting the presence of internal reiterations. These reiterated sequences could thus be involved in the amplification and/or deletion processes.
(e) Spontaneous genetic instability
in Streptomyces umbofaciens ATCC23877 Genetic instability of S. ambofaciens ATCC23877 exhibits the same features as those described for strain DSM40497: Pig- mutants arise at high frequency (0.68 %) in the progenies of wt colonies. In contrast, the frequency of total auxotrophs was estimated as 0.007 %, which indi-
cates that genetic instability preferentially affects some traits. Inability to produce aerial mycelium ( A m c - ) could be associated with loss of pigmentation. This phenomenon was not observed among more than 35000 pigmented colonies. In addition, whereas 116 Pig + clones were antibiotic producers, about 11% (13/119) of Pig- mutants did not show any detectable antibiotic production. Characteristics of differentiation in Streptomycetes, such as colony pigmentation, aerial mycelium formation and antibiotic production, are affected by genetic instability and these mutants are more frequently found among Pig- than among wt phenotypes. At the molecular level, ADS have been detected in about 9% (5/55) of Pig- mutants. Using a specific probe of AUDgO we have found that about 60% (33/55) of Pigmutants, including amplified strains, presented either partial or total deletions at this locus. By contrast, no deletion was found in 13 wt clones:, l'herefore, the genetic instability of S. ambofaciens ATCC23877 was related to genome rearrangements as in strain DSM40697.
(f) Induction of genetic instability UV light and MC are known to induce both SOS functions and deletions in bacteria. Since genetic instability is related to deletions in strain ATCC23877, we have investigated the possible effect of both treatments on the frequency of Pig- mutants. Even at a high survival rate, UV light was able to increase the Pig- frequency almost to 30?/0 (Fig. 4). At each dose, the number of induced Pig- mutants was considerably higher than the total number of induced auxotrophs. Moreover, photoreactivation, which is known as an errorfree DNA repair system, has no influence on the increase in Pig- frequency (data not shown). These results suggest that this phenomenon is not only the result of classical UV-induced mutagenesis. Similar results have been obtained after treatment with MC (data not shown). At the molecular level, induced Pig- mutants were shown to harbour ADS (2/29, 7%) and partial or total deletions of the AUD90 locus (22/29, 76%). Therefore, both spontaneous and induced Pig- mutants presented the same features at the phenotypical as well as at the molecular level, suggesting that UV light and MC could induce genetic instability in S. ambofaciens. Oxolinic acid, an antibiotic related to nalidixic acid, inhibits DNA replication by acting on the A subunit of DNA gyrase and is an inducer of the SOS response in E. coil Spores orS. ambofaciens ATCC23877 were grown on complete medium containing oxolinic acid at a sub-inhibitory concentration (10 #g/ml). After 15 days of growth at 30 ° C, whereas the frequency of Pig- mutants was estimated as 0.7% for the control, this frequency rose to almost 100%
53 10°
~
10-1
i
(n 10.2
u>' 10-3
~" 104 =l
E
10 -s 0
I
I
100
200
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I
300
400
UV (J/m 2) Fig. 4. Effect of UV irradiation on the survival of spores ofS. ambofitciens ATCC23877 and on mutant production. I-I, survival fraction; l , Pigmutants; O, total auxotroph mutants. Methods. Spores were suspended in 5 ml of sterile water containing SDS (0. ! % w/v). They were swirled gently in a petri dish and irradiated at 100-400 J/m-" with two UV lamps (GTE Sylvania). Experiments were carried out in very dim light to avoid photoreactivation. After irradiation, spores were plated on HT medium (Pridham et al., 1957) and were incubated at 30cC in the dark. After ten clays of growth, the survival fraction was estimated as the ratio of the number of surviving colonies after irradiation to the number of colonies without irradiation. The Pig- mutant frequency was determined among the survivors. The total auxotroph frequency among the surviving bacteria was established by replica-plating the colonies on both minimal medium (Smokvina et ai., 1988) and HT medium. Auxotrophic mutants were defined as colonies growing on HT medium but not on minimal medium.
harboured a number of Pig- papillae which was 15-fold higher than the number of papillae observed in wt colonies. Furthermore, Pig- frequency was higher in the progenies of these mutants than in the wt progeny. These mutants, which harboured a high number of papillae and presented an increased level of genetic instability, were called mutators (Mut-). The fact that the three M u t - strains (rout-l, mut-2 and rout-3) exhibited the same sensitivity to UV light as the wt strain (data not shown) indicates that the mutator phenotype is not due to a deficiency in D N A repair. Furthermore, one of the Mut- strains (rnut-2) was resistant to MC (Fig. 5). The Mut- phenotype is reversible, and Mut ÷ mutants arose at high frequency (about 1~o) in the progenies of the two mutator strains where it has been tested (rout-2, rout-3). This novel kind o f ( M u t - , Uvr ÷ )mutants resembles the bglY mutants of E. coil, in which the frequency of deletions was preferentially increased and the expression of other genes was modified (Bertin et al., 1990; Lejeune and Danchin, 1990).
(h) Conclusions An unstable region, A UD205, has been described in the genome of S. ambofaciens RPI81110. A UD205 was shown to be homologous to A UD90 (described in strain DSM40697). Similar experiments demonstrated the existence in strain RPI81110 of an AUD homologous to AUD6. Despite their difference in genome size, both strains con-
10 2,"
on oxolinic acid-containing medium. In addition, most of the induced Pig- mutants (94~o) exhibited an hypervariable progeny, and 3/18 of these mutants presented ADS. Novobiocin acts on the B subunit of the DNA gyrase, and could also induce the SOS response by inhibiting DNA replication. At a sub-inhibitory concentration of 1 #g/ml, numerous mutant colonies were observed, which were phenotypically heterogeneous and exhibited numerous Pigsectors. These colonies gave rise to hypervariable progeny. In contrast, rifampicin, an antibiotic which inhibits transcription, was shown to have no influence on genetic instability. The fact that UV light, MC, oxolinic acid and novobiocin, that are inhibitors of DNA replication and inducers of the SOS response in E. coil, are able to induce genetic instability in S. ambofaciens, suggests that an SOS-like response could be involved to account for this phenomenon in Streptomycetes.
(g) Isolation of mutator (Mut-) mutants Three colonies harbouring a high number of Pig- papillae were independently isolated. Offspring of these colonies
10 1.
10 O, (n
#
10-1
10-2
0
1
2 3 MC (pg/ml)
4
Fig. 5. Effect of MC on the survival of wt spores and of Mut- mutants of S. ambofaciens ATCC23877. Survival fraction of the wt strain (1"1), mut-I ( 1 ) ; rout-2 (O); and rout-3 (•). Methods. Spores were treated for 3 h at 30cC in 5 ml of sterile water which contained MC at concentrations ranging from 0 to 5/~g/ml. The treatment was performed in very dim light. MC was eliminated after two centrifugations (3500 rpm; 15 min). Aliquots of the mixture were taken and diluted in sterile water. Spores were plated on HT medium and incubated at 30°C.
54 tained homologous A UD. The conservation of these unstable regions in otherwise different strains suggests that they could play an important role, structurally and/or functionaUy. Sp is not produced by strains in which a particular nt sequence is amplified. The loss of this amplification (ADS205) is correlated with the restoration of antibiotic production. The absence of Sp production cannot be explained by a deletion of the Sp production and/or resistance genes. The mechanism by which Sp production is inhibited in amplified Sp- strains has not yet been elucidated. The reversible inhibition of Sp production can be explained by various hypotheses such as the altered expression of one of the resistance genes, or the overexpression of a repressor gene present in the amplified DNA. A reiterated sequence external to the A UD was found in A UD6, whereas A UD90 contained a repetitive sequence external and internal to the A UD. Furthermore, the external signals appeared in the amplified strains but not in the wt strain, indicating that these sequences could be involved in the rearrangement process. These reiterations were found in both S. ambofaciens DSM40697 and S. ambofaciens ATCC23877. Analysis of the A UD6 sequence revealed the presence of an ORF. The deduced aa sequence of this ORF presented homology with that of the B. subtilis GntR regulatory protein. The fact that reiteraled sequences were found in both A UD6 and A UD90 suggested that these structures could be involved in the rearrangements associated with genetic instability in S. ambqfaciens. The effects of UV ir,'adiation, MC, oxolinic acid and novobiocin on genetic instability were investigated, leading to the conclusion that these treatments induced genetic instability in S. ambofaciens. All these antibiotics, in addition to the UV treatment, are inhibitors of DNA replication and could be inducers of an SOS-like response in streptomycetes. These facts suggest that such a phenomenon could be involved in genetic instability in S. ambofaciens.
ACKNOWLEDG EM ENTS
This work was supported by grants from the CNRS, the Universit6 Nancy I, the Minist6re de la Recherche et de la Technologic, and from the R6gion Lorraine. We are most grateful to Drs. D. Holmes and P. Kaiser for their help in writing the manuscript. We thank the Ligue Nationale Franqaise Contre le Cancer for its financial support to one of us (D.S.).
REFERENCES Bertin, P., Lejeune, P., Laurent-Wintcr, C. and Danchin, A.: Mutations in bglY, the structural gene for the DNA-binding protein H 1, affect expression of several Escherichia t'oli genes. Biochimie 72 (1990) 889891. Birch, A., Hatisler, A. and Hiltter, R.: Genome rearrangement and genetic instability in Streptomyces spp. J. Bacteriol. 1",2 (1990)4138-4142. Dary, A., Bourget, N., Simonet, J.M. and Decaris, B.: The amplification of a specific DNA sequence reversibly prevents spiramycin production in Streptomyces ambqfaciens RPI81110. Res. Microbiol. 143 (1992) 99-112. Demuyter, P., Schneider, D., Leblond, P., Simonet, J.M. and Decaris, B.: A chromosomal hotspot for multiple rearrangements associated with genetic instability of StrelmmO'ces ambofat'iens DSM40697. J. Gen. Microbiol. 137 (19911 491-499. Fishman, S.E. and Hershberger, C.L.: Amplified DNA in Strepmmj'ces ji'adiae. J. Bacteriol. 155 (19831 459-466. Fujita, Y. and Miwa, Y.: Identification of an operator sequence for the" Bacillus suhtilis grit operon. J. Biol. Chem. 264 (1989) 4201-4206. Hopwood, D.A., Bibb, M.J., Chatcr, K.F., Janssen, G.R., Malpartida, F. and Smith, C.P.: Regulation ofgene expression in antibiotic-producing Streptomyces. In: Booth, !. and Higgins, C. (Eds.), Regulation of Gent Expression. Society for General Microbiology, Cambridge, UK, 1986, pp. 251-276. Koller, K.P. and Riess, G.: Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence in Streptom.vces temhte. J. Bacteriol. ! 71 (1989) 4953-4957. Leblond, P., Demuyter, P., Moutier, L., Laakel, M., Decaris, B. and Simonet, J.M.: Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptooo'ces amhqfacieos. J. Bacteriol. 171 (19891 419-423. Leblond, P., Demuyter, P., Simonel, J.M. and Decaris, B.: Genetic instability and hypervariability in Streptooo'ces amhq]'aciens: towards an understanding of a mechanism of genome plasticity. Mol. Microbiol. 4 (1990a) 707-714. Leblond, P., Francou, F.X., Simonet, J.M. and Dccaris, B.: Pulsed-field gel analysis of the genome of StrelmmO't'es amhohtcieos strains. FEMS Microbiol. Lett. 72 (1990b) 79-88. Leblond. P., Demuyter, P., Simonet, J.M. and Decaris, B.: Genetic instability and associated genomic plasticity in StrelmmO'ces ambt!facieos: PFG E evidence for large alterations in a limited genomic region. J. Bacteriol. 173 (19911 4229-4233. Lejeune, P. and Danchin, A.: Mutations in the hglY gene increase the fi'equency of spontaneous deletions in b.~'cherichia coil K-12. Proc. Natl. Acad. Sci. USA 87 (19901 36(I-363. Lipman, D.J. and Pearson, W.R.: Rapid and sensitive protein similarity searches. Science 227 (1985) 1435-1441. Pridham, T.G., Anderson, P., Foley, C., Lindenfelser, L.A., Hesseltine, C,W, and Benedict, R.C.: A selection of media for maintenance and taxonomic study of Streptoooz'es. Antibiotic Annual 1956-1957, 947953. Richardson, M.A., Kuhstoss, S., Huber, M.L.B., Ford, L., Godfrey, O., Turner, J.R. and Rao, R.N.: Cloning of spiramycin biosynthetic genes and their use in constructuing Sn'ept,mn'ces ambq]'aciens mutants defective in spiramycin biosynthesis. J. Bacterioi. 172 (1990) 3790-3798. Simonet, B., Dary, A., Decaris, B. and Simonet, J.M.: Characterization of a family of multimcric ccc molecules of amplified chromosomal DNA in Strepmoo'ces ambqhtcieos DSM40697. FEMS Microbiol. Lett. 78 (1991) 25-32. Smokvina, T., Francou. F. and Luzzati, M.: Genetic analysis in Strepwno'ces ambofaciens. J. Gen. Microbiol. 134 (1988) 395-402.
49
GENE06Ol
Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity* (DNA amplification; deletion; spiramycin production; mutator genes)
Jean-Marc Simonet, Dominique Schneider, Jean-Nicolas Voiff, Annie Dary and Bernard Decaris Laboratoire de G$n$tique et Microbiologie, Facult$ des Sciences, Universit~ de Nancy L F-54506 Vandoeuvre-lbs-Nancy C~dex (France) Received by J.C. Ensign: 19 August 1991 Accepted: 30 December 1991 Received at publishers: 17 February 1992
SUMMARY DNA amplification and deletions occur at high frequency in unstable regions localized on the Streptomyces ambofaciens chromosome. The structure of these regions was investigated, leading to the identification of internal reiterations which could play a role in the deletion and/or amplification mechanism(s). UV irradiation and treatments with mitomycin C, oxolinic acid and novobiocin were shown to efficiently induce genetic instability. Finally, mutator strains were isolated, in which genetic instability was dramatically increased. The involvement of an SOS-like response in genetic instability in S. ambofaciens is proposed.
INTRODUCTION Many Streptomyces species exhibit a very high degree of genetic instability. Molecular analyses ofthe mutant strains arising from spontaneous or treatment-increased instability often reveal genome rearrangements, which are mainly large deletions and/or amplifications (according to the nomenclature proposed by Fishman and Hershberger, 1983;
Correspondence to: Dr. J.M. Simonet, Laboratoire de G~n6tique et Microbiologie, Facult~ des Sciences B.P. 239, F-54506 Vandoeuvre-I~sNancy C~dex (France) Tel. (33-83)912204; Fax (33-83)901511. * Presented at the International Symposiumon Biologyof Actinomycetes, University of Wisconsin, Madison, WI (USA) 11-16 August 1991. Abbreviations: aa, amino acid(s); ADS, amplifiedDNA sequence; Amc, aerial mycelium;A UD, amplifiableunit of DNA; bp, base pair(s); ccc, covalentlyclosed circular; EtdBr, ethidiumbromide; HT, Hickey-Tresner (medium);IHS, internalhomologoussequence(see Fig. 1); kb, kilobase(s) or 1000bp; MC, mitomycin C; nt, nucleotide(s); ORF, open reading frame; Pig,colonypigmentation;oc, open circular; PFGE, pulsed-fieldgel electrophoresis; S., Streptomyces; Sp, spiramycin; UV, ultraviolet; wt, wild type.
ADS stands for amplified DNA sequence; A UD stands for amplifiable unit of DNA; reviewed by Leblond et al., 1990a; Birch et al., 1990). In S. ambofaciens DSM40697, two types of genetic instability were previously described: (/) a basic genetic instability, commonly reported, and (ii)hypervariability, which is a new aspect of instability closely associated with DNA amplification and deletion (Leblond et al., 1989). Most amplifications were located in two amplifiable regions, one of which, A UD6, was shown to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied possessed DNA amplifications, deletions or both at the A UD6 locus. Deletions occurred exclusively to one side of the ADS and removed part of the proximal c,opy of this A D S leading to the conclusion that multiple rearrangements can occur at this A UD locus. Moreover, one of the endpoints of the A UD6 shows homology with an internal sequence called IHS (Demuyter et al., 1991). The genomes of four S. ambofaciens strains from different geographical origins, including strain ATCC15154 (a close derivative of ATCC23877), and strain DSM40697 were analysed by PFGE. The analysis of the resulting macrorestriction patterns using rare-cutting enzymes indicated that S. ambofaciens ATCC15154 exhibited a restric-
50 tion pattern different from that of strain DSM40697. The total genome sizes of S. ambofaciens ATCC 15154 and DSM40697 were estimated to be about 6500 and 7800 kb, respectively (Leblond et al., 1990b). Using the same technique, the amplifiable loci A UD6 and A UD90 of S. ambofaciens DSM40697 were mapped within two adjacent Asel restriction fragments. The genetic instability and the formation of very large deletions were strictly correlated since all the mutants tested so far (30/30) presented deletions within the DNA region overlapping both amplifiable loci. The deletion size was estimated to range from 250 kb to more than 2000 kb. PFGE also permitted the localization of the ADS and their structure to be established revealing that amplification takes place at the A UD locus as a tandem array of the wt A UD sequence (Leblond et al., 1991). DNA analysis of the spontaneous mutant strain NSA420 revealed the presence of an ADS, which localized on the chromosome within the A UD6 region, and the presence of extrachromosomal multimeric ccc DNA molecules homologous to the ADS (Simonet et al., 1990). These extrachromosomal forms, originating from the A UD6 region, could correspond to a form of ADS elimination through recombination events between the reiterated sequences. The hypotheses proposed to explain the occurrence of deletions in the unstable regions involve recombination events taking place between reiterations. Such reiterations have been sought in both A UD6 and A UD90 regions. With the aim of extending our observations, the genetic instability of S. ambofaciens ATCC23877, a strain from which both ATCC15154 and RPI81110 were derived, was also studied. The presence of A UD regions homologous to the AUD6 and AUDgO of strain DSM40697 was investigated. In the strain RPI81110 lineage, the presence of the ADS205 (A UDgO family) was associated with the loss of Sp production. Furthermore, in the progeny of one of the amplified strains, the loss of the ADS was correlated with the restoration of Sp production (Dary et al., 1992). Finally, two other kinds of experiments were performed: (i) to modify the level of genetic instability by treatments which possess specificity of action in other bacteria, and (ii) to isolate mutants affected in genetic instability.
etal., 1992). Deletions were also detected in strains NSA207, NSA208 and NSA210 and were localized on the same side of the amplifiable region. Hybridization experiments revealed strong homologies between A UD205 and A UD90 in strain DSM40697. Similar experiments carried out with a probe specific to A UD6 gave identical hybridization patterns with DNA of both ATCC23877 and DSM40697 strains. Thus, the same unstable regions exist in two strains of different geographical origin, in spite of the fact that the genome size of these strains differed by 1300 kb (Leblond et al., 1990b). Moreover, it can be seen that at least one of them (AUDgO/AUD205) might be amplified and/or deleted in both lineages, either spontaneously (in strain DSM40697) or after treatment with EtdBr (in strain RPI81110).
RESULTS AND DISCUSSION
(b) The amplification of a particular nt sequence reversibly prevents Sp production Since strain RPI81110 synthesizes the macrolide antibiotic Sp, production was also tested in the amplified mutants NSA205 to NSA210, which indicated that they were all nonproducers (Sp-). Strain NSA205, which contained the largest ADS (89 kb), was examined by subcloning to investigate a possible correlation between this ADS and the Sp- phenotype. The ADS205 was unstable and lost with a high frequency (9/67). In all cases (9/9) the absence of the ADS205 was correlated with a return of Sp production. In one case (1/9) the ADS205 was lost, and a novel amplification (ADS213) was found. This indicated that only the amplification of a particular nt sequence was correlated with the absence of Sp production. As the Sp- phenotype was reversible, it was concluded that the genes involved in Sp biosynthesis were not deleted, a hypothesis which was confirmed by hybridization between the three resistance determinants and the known biosynthetic genes cloned by Richardson et al. (1990) and various DNA restriction fragments of RP181110 and mutant strains, although it is not yet known whether all of these genes are normally expressed in the Sp- ADS205 + strains. A variety of hypotheses such as the altered expression of one of the resistance genes, the presence of an inhibitor gene present on A UD205 and its over-expression in the amplified strain, as in the case of S. tendae (Koller and Riess, 1989), or the non-expression of a gene involved in Sp production may explain this reversible inhibition of Sp production.
(a) Characterization of an amplifiable region in the genome of strain RPI81110 Amplified mutant strains (NSA205 to NSA210) were isolated following treatment of strain RP181110 with EtdBr. The ADS of these strains were localized in an amplifiable region, AUD205, in the RP181110 genome (Dary
(c) Reiterated sequences in AUD6 To study the A UD6 structure, the isolation of this chromosomal region from the wt strain DSM40697 was undertaken. A genomic library of S. ambofaciens strain DSM40697 was constructed in E. coil and hybridizations, using the cloned S 1 fragment (Fig. 1) generated by the re-
51
~t3
....
•
!.J ....... L..! ........ ! ........ J.............................. 1.3 3.1
1.9
3.5
4.0
12.0
AUD6 (24.8 kb) SIL
LI .....................................
0.6
='~ IHS
15.0
~
I
,...
SIR
Fig. 1. Restriction map of the A UD6 locus of S. ambofaciens D SM40697. S 1 corresponds to the Barn H 1 fragment generated by the amplification ofA UD6. SIR and Sit_ are the right and the left parts of Sl, respectively. IHS is a DNA fragment which is homologous to SIR. The underlined 1.9-kb BamHl fragment was subcloned and its nt sequence determined (see section e and Fig. 2). Restriction fragment sizes are given in kb.
vealed its size to be 1842 bp and that it contained 6 9 ~ G+C, which is in good agreement with the high G+C content of the Streptomyces genome (70-74°,~,). The study of this sequence revealed one O R F which showed codon usage typical of Streptomyces (70, 50 and 9070 G+C in the first, second and third position of the codons, respectively). In addition, there is a sequence resembling the Streptomyces consensus promoter sequence immediately upstream from this O R F (Fig. 2). Comparison of the predicted aa sequence of the O R F with the GenBank database revealed homology (23.8?/o in 227 aa) with the gntR gene product of Bacillus subtilis (Fujita and Miwa, 1989), which is the repressor of the gluconate operon in that organism (Fig. 3). The analysis of the structure of this putative protein showed some features
iteration of AUD6 as probe (Demuyter et al., 1991), revealed a clone containing a cosmid with a 40-kb insert which belonged to the A UD6 region. Subsequently, the 1.9-kb B a m H l fragment was subcloned (Fig. 1). Hybridizations with S 1 (Fig. 1) allowed us to detect fragments external to the A UD (one of which could be a junction fragment). These signals were present only in the amplified strain but not in the wt strain, indicating that these reiterations could be involved in the amplification process. To determine which part of the 1.9-kb B a m H l fragment was reiterated, hybridizations were carried out using different regions of this D N A as probes against total D N A of the mutant strain amplified for A UD6. All the subclones gave the same pattern indicating that the entire 1.9-kb fragment at least was reiterated. The nt sequence analysis re-
A
B
I 10 I 20 I GGATCCGGTC CGC~CCTGGT 81 TGGTGCGCAC GTAGTCGATG 161 ATCGGTGTCG TGGTGTGCTG 241 CCGGGAGGCC TCGACGCTCT 321 AGCCGAAGCC GARF.~GTTCC 401 TCGTCGGGCA GTTCGGAGGC 481 CGCCTGCGCC CGTACCGCCT 561 TGGCGTGCGA GTAGACCAGT 641 &GGATGTCCC ACAGGGCGGT "/21 G ~ e T G C CACGTGTCCT 801 CGACGGCCAG TTCCCGGCCG 881 ARGGTCCGGC CGGGGGAGGC 961 T A T ~ T ~ T A T C G C lO41 CGG~CG~ace GGCGCGACAT 1121 GGAGAACGAA CTeGCCC.~GT 1201 TCGTGCAGGT CTTCCCCAN~ 1281 GAGGeGGTGG AI~CTCGGTTC 1361 ~GTGCGGCAG GGGCGCARGG 1441 ~GI~--r-C-C-r2-~-~ -~--,CGGGA~,GTC 1521 GAGAP,CGTGT eCCCGGCCGT 1601 CCGTACCGCC &TGCGTACAC 1681 CCACCGACGC CGCGACCGTG 1761 CGTCGGCCGC TGGTCCCCGC 1841 CC I 10 I 20
TTGaca
-
TTGgtc -
18 17
bp, bp -
I 30 I 40 GG~GCGGC GAGGTCGA~ AGCTGTTCGG TGATGAC~TG CCGGATGAGC CGGAAGGACT TGCC~G GGCCGCCTCG CGTACCGCTT CCAGGTACCT GGCCGGeTCG TAGACCCTGC TGTAGCCCAG CTCCAC~TGG .&CGCCGTCGC GCGAGCGGCC GTCGACGGCG GCG&TCGCCG CGATACGGGC GGGGTCCeTG TTGRF.~GTGG CGTCGCCGAG GACGARGACC TCGI~CGCTTT AGCACGGCAA TACTTGGCTC eT~ceT~ CTGC.CCCAGA CGATGGGGGT CAGCCGCACG ATCGGGTCGT TCGTCTCeCG CCTGGACGCT CTGCCCG~GG ACCTCGACCT GGAGGAGTTC ,,TGG&CC~CCG TCGCCGCGGC GTTCGTeGCC CAGCACCGCG ACCTGCGAGC CGTCTTCGAC CCCGT&CGAC GCAACATCGT TeGTCCGGCG TGTGCCCGGG
I
30
I
40
I 80 I 70 GCCGCCCGCG TGGACGACGG 80 TCATCACCTC GCCGACCC.-,CG 160 240 GGGTCCTCCA T C ~ CACGTCGTGC AGGAGGTGC~ 320 CCTCGGTGTC CCAGGGC'JL'GT 400 CCCCCGACGC CGGGeACGGC 480 CGRGC~TACG TCGGTGCCGC 560 GCAGGCCGGC GGTCTTGCCC 640 CGCCAGTAGG CGCCCTTGTA 720 GTGGTCGCGC AGATAGeTGG 800 CAGCGTGAF..G 880 ~ ~ G ~ 960 C ~ G G C T C A AACGAA£GGG 1040 CCGCCCTCTC 1120 GCTCCGGGCG CCTGTTGGCC CAGGAGGGCC 1200 CCGACGCGCA GTTCCTCCGG 1280 GGGGAGCTGC GGGACAN~C~ 1360 CTG&TGCGC~ 1440 CCAC~ CCCGCCGCCT CGGCCTCCAC 1520 TCCCCCTCGC 1600 ~ G GCACTCACCC GAACTGTTCG 1680 T~I~CCC~C G ~ . ~ G ~ C GGGGTCCTTC 1760 CGGCGCGG&T 1840 GGCGTCCCCG T ~ C G C G !842 I 8O 50 I 60 I 70
I 50 ATCCGGCGCA CTGGACGTCC CCTGGTTCTC TTCGGGGTGA GGGCGCGAAG CCTTGCGCAC CGeTCGACGT G C C ~ TCATCGTGAC CCGATCAGCA GeCGG,CACC CGATCCTGCT GCGCTCTACG TGGTCCTGAC CCGGTACGGG GGTCG&TCCG AGCTCGACCC TTCGGTCTGG C ~ T &G&TCTTCGA GACATCGAAC GGTCTGGGAG TGeGGCGTGC I
I 60 GATGGGTGAT CAGATCGAGT GGCC~TC GCCGGTGGTG TCGAGGTAGG CCCGTACGTG CGTCGAGAAG TGGTGGACGG GGGTCCGCGC GCGGGACCAC CCGTCCGTGG ~ T TI~:GATGTGC CCTCGAACTC AGAGCCTG~T GeTCGGG,TCG CGCGGTGGTG ACGAGGCGTT CTGGACCGC~ CGCGGTCACu GCATCCGCGC
tAGgaT aAGcaT
Fig. 2. Nucleotidesequence analysisof the 1.9-kbBamH! fragmentofA UD6. The nt sequence was determined usingthe Promega(Madison, WI) TaqTrack sequencing system and the Pharmacia double-stranded nested deletion kit which produces progressive deletions into the sequenced fragment. (A) The nt sequence. ORF is delimited by brackets. The putative protnoter (with the' -35' and" - 10' regions bo~ed at nt 943-948 and 967-971, respectively)is shown. The sequence resembling the operator of the gntR of the B. subtilis gluconate operon is overlined with a horizontal bracket. The sequence homologous to the 930-bp fragment of A uDgo is boxed with a thick line (see section d). (B) Comparison of the putative promoter located upstream from the ORF (bottom line) with the consensus sequence (top line) established for Streptomyces (Hopwood et ai., 1986).Upper-case letters indicate the occurrence of those particular nt in over 750o of the examples and lower-case letters indicate the occurrence of those nt in 50-75°0 of the examples.
52 1O 20 30 40 MAQTNGRTNRRDZYLKLR~VLTLELAPGAALSENELAASY~'VSRTPVR .... :. : : .:: . . . . . . . :. : : : : . . : : . . . : : : . : : :
&UDSE0 GntR
IILDSKDLLYpAI~4LSKASTGVRVAY- E L R M R Z V S G L I E S G T Z L S E N T Z R ~ E F S V S R S P V R 10 20 30 40 50
50 60 70 80 90 100 AUI)SE~ E S L Z L I d ~ E G L ~ V F P K X G S F V S R V D P ~ R V A D A Q F L R E A V E L G S L D A L P A E L D P A V V G E L :.: .:: : . . . . . . :. : .... :. .: .: .. : ..: .: GntR EALKILASEKI ZR-LERMGAVVIGLTEI(KZAE I~DVPT.LrRTFVFERLVKXDZEPLVKDL 60 70 80 90 100 110
II0 120 130 140 150 160 ~$1DSEQ R D - - N L ~ Q G R I t D L D L E E F F G L D E A F H Q G L M R L S G H G - - - N ~ T 4 T T V - A A A O G H L D R A R R L ....... : : .:: : :: .... : .:. .: . . . . . . . . . . :. GntR SKZIJUOIKVSZKTEDADEFSFQDVLFHET Z ZRAZDH S¥IQMZWNNLKPVMESF ILLSMRV 120 130 140 150 160 170 170 180 190 200 210 220 AUDSEQ GLIIENVSPAVFVAQHRE ZF-DAVTEGDVP LARTAMRTHLRAVFDD ZERZ RAHSP ELFATD 1.:. . . . . . ,1.. .1... : ." ... .. .: :..: .. : .... 1.. GntR RIJgJIKYEDFTRZ L D N H Z L Y Z Q & £ K T K D R A L M Z Q S L I I Q N F D D V Q D K V E D L W - L S g Q M L A K G 180 190 200 210 220 230
&UDSEQ AATVPVRRNIV~,~Z . Gn~R
~YNND 240
l"
-~ • TACTTGTAT&CAAGTATACT
gntR operator
*** * * * * * * * * * * ** ** TACATGTATACAAGCAT-CT . . ~ ~ . ..
AU~
Fig. 3. Analysisof the S. ambofiwiensdeduced aa sequence of the A UD6 ORF. (A) Comparison of the deduced aa sequence(AUD SEQ) with the B. subtilisg, tR gene product. Colons represent identical aa, whereas full stops represent aa conservativechanges as defined by Lipman and Pearson (1985). The single-letter code was used for the aa. Last digits of numerals are aligned with the corresponding aa. (B) Comparison of the operator sequence recognized by GntR (top line) with the sequence of A UD6 (bottom line and overlined in Fig. 2). The convergent arrows indicate a dyad symmetry.The asterisks indicate the conserved nt.
of DNA-binding proteins, i.e,, helix-turn-helix domains. Furthermore, the operator region (located immediately upstream from the gntR start codon) recognized by GntR in B. subtilis showed strong homologies (17/20 nt) with a sequence located immediately upstream from the start codon of the A U D 6 0 R F (Fig. 3).
(d) Reiterations in the AUDgO region In S. ambofaciens DSM40697, all mutant strains studied so far showed rearrangements in the A UD90 region. To study the deletions in the mutant strains carrying amplifications in this A UD region, hybridization experiments were carried out with a cloned ADS fragment as probe. This probe revealed several signals: (i) as in A UD6, faint signals appeared which could correspond to homologies external to the A UD, and (ii)signals corresponding to amplified fragments within the A UD90, suggesting the presence of internal reiterations. These reiterated sequences could thus be involved in the amplification and/or deletion processes.
(e) Spontaneous genetic instability
in Streptomyces umbofaciens ATCC23877 Genetic instability of S. ambofaciens ATCC23877 exhibits the same features as those described for strain DSM40497: Pig- mutants arise at high frequency (0.68 %) in the progenies of wt colonies. In contrast, the frequency of total auxotrophs was estimated as 0.007 %, which indi-
cates that genetic instability preferentially affects some traits. Inability to produce aerial mycelium ( A m c - ) could be associated with loss of pigmentation. This phenomenon was not observed among more than 35000 pigmented colonies. In addition, whereas 116 Pig + clones were antibiotic producers, about 11% (13/119) of Pig- mutants did not show any detectable antibiotic production. Characteristics of differentiation in Streptomycetes, such as colony pigmentation, aerial mycelium formation and antibiotic production, are affected by genetic instability and these mutants are more frequently found among Pig- than among wt phenotypes. At the molecular level, ADS have been detected in about 9% (5/55) of Pig- mutants. Using a specific probe of AUDgO we have found that about 60% (33/55) of Pigmutants, including amplified strains, presented either partial or total deletions at this locus. By contrast, no deletion was found in 13 wt clones:, l'herefore, the genetic instability of S. ambofaciens ATCC23877 was related to genome rearrangements as in strain DSM40697.
(f) Induction of genetic instability UV light and MC are known to induce both SOS functions and deletions in bacteria. Since genetic instability is related to deletions in strain ATCC23877, we have investigated the possible effect of both treatments on the frequency of Pig- mutants. Even at a high survival rate, UV light was able to increase the Pig- frequency almost to 30?/0 (Fig. 4). At each dose, the number of induced Pig- mutants was considerably higher than the total number of induced auxotrophs. Moreover, photoreactivation, which is known as an errorfree DNA repair system, has no influence on the increase in Pig- frequency (data not shown). These results suggest that this phenomenon is not only the result of classical UV-induced mutagenesis. Similar results have been obtained after treatment with MC (data not shown). At the molecular level, induced Pig- mutants were shown to harbour ADS (2/29, 7%) and partial or total deletions of the AUD90 locus (22/29, 76%). Therefore, both spontaneous and induced Pig- mutants presented the same features at the phenotypical as well as at the molecular level, suggesting that UV light and MC could induce genetic instability in S. ambofaciens. Oxolinic acid, an antibiotic related to nalidixic acid, inhibits DNA replication by acting on the A subunit of DNA gyrase and is an inducer of the SOS response in E. coil Spores orS. ambofaciens ATCC23877 were grown on complete medium containing oxolinic acid at a sub-inhibitory concentration (10 #g/ml). After 15 days of growth at 30 ° C, whereas the frequency of Pig- mutants was estimated as 0.7% for the control, this frequency rose to almost 100%
53 10°
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i
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I
100
200
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300
400
UV (J/m 2) Fig. 4. Effect of UV irradiation on the survival of spores ofS. ambofitciens ATCC23877 and on mutant production. I-I, survival fraction; l , Pigmutants; O, total auxotroph mutants. Methods. Spores were suspended in 5 ml of sterile water containing SDS (0. ! % w/v). They were swirled gently in a petri dish and irradiated at 100-400 J/m-" with two UV lamps (GTE Sylvania). Experiments were carried out in very dim light to avoid photoreactivation. After irradiation, spores were plated on HT medium (Pridham et al., 1957) and were incubated at 30cC in the dark. After ten clays of growth, the survival fraction was estimated as the ratio of the number of surviving colonies after irradiation to the number of colonies without irradiation. The Pig- mutant frequency was determined among the survivors. The total auxotroph frequency among the surviving bacteria was established by replica-plating the colonies on both minimal medium (Smokvina et ai., 1988) and HT medium. Auxotrophic mutants were defined as colonies growing on HT medium but not on minimal medium.
harboured a number of Pig- papillae which was 15-fold higher than the number of papillae observed in wt colonies. Furthermore, Pig- frequency was higher in the progenies of these mutants than in the wt progeny. These mutants, which harboured a high number of papillae and presented an increased level of genetic instability, were called mutators (Mut-). The fact that the three M u t - strains (rout-l, mut-2 and rout-3) exhibited the same sensitivity to UV light as the wt strain (data not shown) indicates that the mutator phenotype is not due to a deficiency in D N A repair. Furthermore, one of the Mut- strains (rnut-2) was resistant to MC (Fig. 5). The Mut- phenotype is reversible, and Mut ÷ mutants arose at high frequency (about 1~o) in the progenies of the two mutator strains where it has been tested (rout-2, rout-3). This novel kind o f ( M u t - , Uvr ÷ )mutants resembles the bglY mutants of E. coil, in which the frequency of deletions was preferentially increased and the expression of other genes was modified (Bertin et al., 1990; Lejeune and Danchin, 1990).
(h) Conclusions An unstable region, A UD205, has been described in the genome of S. ambofaciens RPI81110. A UD205 was shown to be homologous to A UD90 (described in strain DSM40697). Similar experiments demonstrated the existence in strain RPI81110 of an AUD homologous to AUD6. Despite their difference in genome size, both strains con-
10 2,"
on oxolinic acid-containing medium. In addition, most of the induced Pig- mutants (94~o) exhibited an hypervariable progeny, and 3/18 of these mutants presented ADS. Novobiocin acts on the B subunit of the DNA gyrase, and could also induce the SOS response by inhibiting DNA replication. At a sub-inhibitory concentration of 1 #g/ml, numerous mutant colonies were observed, which were phenotypically heterogeneous and exhibited numerous Pigsectors. These colonies gave rise to hypervariable progeny. In contrast, rifampicin, an antibiotic which inhibits transcription, was shown to have no influence on genetic instability. The fact that UV light, MC, oxolinic acid and novobiocin, that are inhibitors of DNA replication and inducers of the SOS response in E. coil, are able to induce genetic instability in S. ambofaciens, suggests that an SOS-like response could be involved to account for this phenomenon in Streptomycetes.
(g) Isolation of mutator (Mut-) mutants Three colonies harbouring a high number of Pig- papillae were independently isolated. Offspring of these colonies
10 1.
10 O, (n
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2 3 MC (pg/ml)
4
Fig. 5. Effect of MC on the survival of wt spores and of Mut- mutants of S. ambofaciens ATCC23877. Survival fraction of the wt strain (1"1), mut-I ( 1 ) ; rout-2 (O); and rout-3 (•). Methods. Spores were treated for 3 h at 30cC in 5 ml of sterile water which contained MC at concentrations ranging from 0 to 5/~g/ml. The treatment was performed in very dim light. MC was eliminated after two centrifugations (3500 rpm; 15 min). Aliquots of the mixture were taken and diluted in sterile water. Spores were plated on HT medium and incubated at 30°C.
54 tained homologous A UD. The conservation of these unstable regions in otherwise different strains suggests that they could play an important role, structurally and/or functionaUy. Sp is not produced by strains in which a particular nt sequence is amplified. The loss of this amplification (ADS205) is correlated with the restoration of antibiotic production. The absence of Sp production cannot be explained by a deletion of the Sp production and/or resistance genes. The mechanism by which Sp production is inhibited in amplified Sp- strains has not yet been elucidated. The reversible inhibition of Sp production can be explained by various hypotheses such as the altered expression of one of the resistance genes, or the overexpression of a repressor gene present in the amplified DNA. A reiterated sequence external to the A UD was found in A UD6, whereas A UD90 contained a repetitive sequence external and internal to the A UD. Furthermore, the external signals appeared in the amplified strains but not in the wt strain, indicating that these sequences could be involved in the rearrangement process. These reiterations were found in both S. ambofaciens DSM40697 and S. ambofaciens ATCC23877. Analysis of the A UD6 sequence revealed the presence of an ORF. The deduced aa sequence of this ORF presented homology with that of the B. subtilis GntR regulatory protein. The fact that reiteraled sequences were found in both A UD6 and A UD90 suggested that these structures could be involved in the rearrangements associated with genetic instability in S. ambqfaciens. The effects of UV ir,'adiation, MC, oxolinic acid and novobiocin on genetic instability were investigated, leading to the conclusion that these treatments induced genetic instability in S. ambofaciens. All these antibiotics, in addition to the UV treatment, are inhibitors of DNA replication and could be inducers of an SOS-like response in streptomycetes. These facts suggest that such a phenomenon could be involved in genetic instability in S. ambofaciens.
ACKNOWLEDG EM ENTS
This work was supported by grants from the CNRS, the Universit6 Nancy I, the Minist6re de la Recherche et de la Technologic, and from the R6gion Lorraine. We are most grateful to Drs. D. Holmes and P. Kaiser for their help in writing the manuscript. We thank the Ligue Nationale Franqaise Contre le Cancer for its financial support to one of us (D.S.).
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