Replication of the linear chromosomal DNA from the centrally located oriC of Streptomyces ambofaciens revealed by PFGE gene dosage analysis Gilles Fischer, Anne-Catherine Holl, Jean Nicolas Volff, Dominique Vandewiele, Bernard Decaris, Pierre Leblond To cite this version: Gilles Fischer, Anne-Catherine Holl, Jean Nicolas Volff, Dominique Vandewiele, Bernard Decaris, et al.. Replication of the linear chromosomal DNA from the centrally located oriC of Streptomyces ambofaciens revealed by PFGE gene dosage analysis. Research in Microbiology, Elsevier, 1998, 149 (3), pp.203-210. 10.1016/S0923-2508(98)80080-6. hal-01658988 HAL Id: hal-01658988 https://hal.univ-lorraine.fr/hal-01658988 Submitted on 8 Dec 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. INSTITUT PASTEU LSEVlER ris 1998 G. Fischer (I), AC. 011 t2), J.N. Vol . Vandewiele (4), ecari s ( ’ ) and Leblond ( 1)(*) (‘kaboratoire de Genetique et Microbiologic, Unite’associee INRA 952, Faculte’ des Sciences, Universite Henri Poincare, Nancy I, B. P. 239, F-54506 Vandowvre-&Nancy (France), (2)Laboratoire de GtMtique et evolution des populutions, Universite’ des Sciences et Technologies de Lille, Btitiment SN2, 59655 Villeneuve d’Ascq cedex (France), ““Physiological Chemistry I, Theodor Boveri Institute for Biosciences (B&enter), University of Wuerzburg, Am erzburg (Germany), und “)Building 6, Room lA15, NICHD, NIH, 9000 Rockville Pike, Bethesda, MD 20892-2723 (USA) SU ARY Key-words: Streptomyces ambofaciens, OK; Linear chromosome, Replication, PFGE. INT ciated with proteins. This kind of structure has been called an “invertron” by Sakaguchi (1990) A physical map of the linear chromosome of and seems to be typical of Streptomyces genomes three strains of Streptomyces ambofaciens has (Lezhava et al,, 1995; Lin et al., 1993; Pandza et recently been constructed (Leblond et al., 1996). al., 1997 ; Redenbach et al, 1996). Two different The DNA sequences of the chromosome ends are replication mechanisms have been demonstrated inversely repeated over 210 kb in strain for invertrons. (1) Replication of DSM40697, an unusually long distance. The 5’ srrbtilis, by a bacteriophage-encoded DNA poly- ends of the chromosome are covalently asso- merase, is primed at both ends by the terminal Submitted October 21, 1997, accepted December 9, 1997. (*) Corresponding author. 204 G. FISCHER ET AL. proteins; it proceeds over the whole length of the yeast extract malt extract (YEME) medium and phage DNA, resulting in semi-conservative repli- transformed according to Hopwood et al. (1985). cation without formation of Okazaki fragments (reviewed in Salas, 1991). (2) The linear plasmid DNA purification, Southern analysis pSLA2 of Streptomyces rochei has a functional centrally located, bidirectional replication origin; Plasmid extractions were performed according to Sambrook et al. (1989) for E. coli. For Strepto- most of pSLA2 is replicated bidirectionally from myces, plasmid extractions and subsequent purifica- the central origin. Protein-primed DNA synthesis tions by caesium chloride-ethidium bromide gra- contributes only about 280 nucleotides at the end dients were carded out according to Hopwood et al. of the lagging strands (Chang and Cohen, 1994). (1985) and Sambrook et al. (1989), respectively. Genomic DNA extractions and restriction analyses The chromosome replication origins (oriC) of were carried out as previously described (Demuyter Streptomyces lividans 66 and Streptomyces coeli- et al., 1988 ; Leblond et al., 1996). Restriction color A3(2) were mapped close to the middle of enzymes and molecular biology reagents were pur- the chromosome associated with dnaA and gyrB chased from New England Biolabs (USA) and Boehringer Mannheim (Mannheim, Germany). genes (Musialowski et al., 1994). In the latter species, gene dosage experiments indicated that For Southern transfers, "Hybond-N" membranes (Amersham, UK) and either the "Vacugene" system oriC is active during vegetative growth (Calcutt (LKB, Sweden) or the Smith and Summers method and Schmidt, 1992; Musialowski et al., 1994; (1980) were used. For colony transfers, "Qiabrane" Zakrzewska-Czerwinska and Schrempf, 1992). membranes (Qiagen, Germany) were used according Here we show that S. ambofaciens also has an to the instructions of the supplier. DNA (purified or oriC locus near the middle of the chromosome in agarose) was labelled with digoxigenin (Dig)- and that replication proceeds from this locus by a labelled dUTP, and specific hybrids were detected using the "DIG DNA" labelling and detection kit bidirectional mechanism. (Boehringer Mannheim). Alternatively, probes were labelled with 32p according to Demuyter et al., (1988) using the "Multiprime" labelling system (Amersham). Hybridizations were performed at MATERIALS AND METHODS 68°C corresponding to a stringency consistent with the detection of 100 % homologues. Bacterial strains S. ambofaciens DSM40697 (HOtter, 1967), Gene bank preparation S. ambofaciens ATCC 15154 (Pinnert-Sindico et al., 1955), and S. lividans TK21 (Hopwood et al., 1985) An S. ambofaciens cosmid gene library was con- were the Streptomyces strains used in this work. structed using the "Packagene" kit (Promega, USA). Escherichia coli HB 101 (Boyer and Roulland-Dus- DNA was partially Pstl-digested, ligated into the Pstl soix, 1969; Boehringer Mannheim, Germany) and site of pHC79 (Hohn and Collins, 1980; Boehringer E. coli GM33 (Yanish-Perron et al., 1985; Stratagene, Mannheim) and introduced into E. coli HB 101. USA) were used as hosts for the cloning experiments. Cloning of oriC from S. ambofaciens Media, culture conditions and transformation The non-replicating 1.1-kb Bcll fragment of plas- E. coli cells were grown in Luria-Bertani mid plJ702 (Hopwood et al., 1985) containing the medium, according to Sambrook et al. (1989) and thiostrepton resistance gene (tsr) was purified from transformed by electroporation (Biorad, Hercules, an agarose gel using the Geneclean TM procedure (Bio Calif., USA). Streptomyces were grown at 30°C in 101, La Jolla, CA, USA) and ligated to Be/l-digested ATCC = American Type Culture Collection. Mb = megabase. DSM = Deutsche Sammlung yon Mikroorganismen. PFGE = pulsed-field gel electrophoresis. kb = kilobase. YEME = yeast extract malt extract. CHROMOSOME REPLICATION IN STREPTOMYCES AM~OFACIENS 205 DNA of pNSA2001, a cosmid harbouring the Isolation of or/C of So ambofaciens on a 2.7-kb S. ambofaciens oriC region. Plasmid pNSA2001 was self-rep|icating DNA fragment isolated from E. coli GM33 [Dam-] to allow cleavage with Bcfl restriction. The ligatior, mixture was intro- duced by transformation into S. lividans TK21 proto- Since oriC of S. ambofaciens was expected plasts, and thiostrepton-resistant colonies were to be close to dnaA, within pNSA2001, we selected (50 ~tg/ml; Hopwood et al., 1985). undertook the subcloning of pNSA2001 recom binant cosmid (inserted DNA circa 35 kb) with PFGE and gene dosage analysis Bcll in order to isolate the DNA fragment con- ferring autonomous replication. Plasmid Pulsed-field gel electrophoresis (PFGE) experi- pNSA2001 BclI fragments were ligated to ments were carried out according to Leblond et al. the I.l-kb Bcll tsr (thiostrepton resistance) (1993). Electrophoresis was performed in a contour- clamped homogeneous electric fields system (CHEF, fragment (see "Materials and methods"). Biorad; Chu et al., 1986). Gene dosage experiments The iigation mixture was introduced into were carried out at different growth times. After S. lividans TK21. Forty thiostrepton-resistant growth of S. ambofaciens DSM40697 in YEME clones were isolated, and for two of them the medium (Hopwood et al., 1985) growth curves were extrachromosomal content was studied by cae- calculated, followed by measuring of the DNA con- centrations as described by Burton (1956). For each sium-chloride gradient analysis. Both of them Asel fragment, the intensity was measured from a contained pNSA2004, a 3.8-kb circular mole- densitometric reading using the "GelDoc 1000" cule consisting of a 2.7-kb Bcl| fragment from system and the Molecular AnalystTM software (Bio- pNSA2001 and the 1.1-kb Bcll tsr fragment. rad), and was expressed relative to its molecular The 2.7-kb fragment is wholly ~ontained within weight. the 4.4 kb-BamHl fragment, as shown by hybridization of pNSA2004 DNA onto BamHl- RESULTS digested DNA of pNSA2001 (fig. 1). In addi- tion, the former fragment hybridized to the Cloning of the gyrAB-dnaA region of the dnaA probe pFF911 (Musialowski et al., 1994) S. ambofaciens DSM40697 and to pUOR2, which contains oriC of S. livi- dans (Zakrzewska-Czerwinska and Schrempf, A gene library was constructed from partially ! 992). Furthermore, plJ702 tsr DNA (1.1-kb Pstl-digested S. ambofaciens DSM40697 ligated Bcll fragment) was used as a probe onto South- to pHC79 and propagated into E. coli HB 101. ern blots of total DNA of one of the S. lividans Among 2,000 recombinant clones, 11 hybridized TK21 tsr-resistant transfornmnts digested with to the gyrB probe pLST182 (Thiara and Cund- restriction enzymes cutting