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Gene, 87 (1990) 45-51 Elsevier 45 GENE 03399 Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids (Recombinant DNA; palindrome; ~-galactosidase; shuttle vector; gene bank; trpE) Michael Hunger, Robert Sehmueker, VeerabrahmaKishan and Wolfgang Hillen Lehrstuhlfiir Milcrobiologie. Institutflir Milo'obiologie und Biochemie der Friedrich-Alexander Universit~t Erlangen-Narnberg. Staudtstr. 5.8520 Erlangen (F.R. G.) Received by T.A. Bickle: 4 September 1989 Revised: 23 September 1989 Accepted: 25 September 1989 SUMMARY A shuttle plasmid for Acinetobacter calcoaceticus and Escher/chia coli has been constructed from a cryptic A. calcoaceticus Iwoffl plasmid and pBR322. It is transformed to A. calcoaceticus BD413 by natural competency, yielding about 106 transformants per/~g of plasmid DNA. The Apa and Tca genes of pBR322 are functional in A. calcoaceticus. A gene bank was constructed from chromosomal A. calcoaceticus DNA and the shuttle plasmid. Direct transformation to A. calcoaceticus yielded about 95% recombinants, indicating a sixfold enrichment of recombinant plasmids compared to E. coll. One clone complementing a trpE mutation carried a 20-kb insertion and transformed with a 30-fold higher efficiency when compared to the vector. A deletion analysis of the shuttle plasmid indicates that 2.2 kb is necessary for autonomous replication and stable maintenance in A. calcoaceticus. No rearrangements of the DNA or loss of plasmids are found in that organism, even in the absence of selective pressure, when this sequence is present. A further insertionai inactivation analysis creating lacZ transcriptional fusions suggests that the origin of replication (or/) is contained within about 1350 bp. Analysis of ~-galactosidase production in A. calcoaceticus indicates that only a weak promoter activity is directed out of one end of this or/. Its sequence contains A + T-rich regions, an 18-bp element with nearly perfect palindromic symmetry and eleven repeats of the consensus sequence, AAAAAATAT, eight of which are clustered within 360 bp. However, no open reading frames or significant homologies to other ori were found. INTRODUCTION Members of the genus Acinetobacter are widely distributed, versatile bacteria with the ability to degrade many organic carbon sources (Juni, 1978). A number of genes Correspondence to: Dr. W. Hillen, Lehrstuhl flir Mikrobiologie, Staudtstrasse 5, D8520 Erlangen (F.R.G.) Tel. (913 I) 858081; Fax (9131)858082. Abbreviations: A., Acinetobacter; Ap, ampicillin; pGal, ~.galactosidase; bp, base pair(s): cfu, colony forming units; kb, kilobase(s) or 1000 bp; Kin, kanamycin; LB, Luria-Bertani broth; nt, nucleotide(s); ORF, open reading flame; ori, origin(s) of DNA replication; R, resistance; Tc, tetracycline; wt, wild type. 03"/8-1119/90/$03.50© 1990ElsevierScience PublishersB.V.(BiomedicalDivision) from Acinetobacter species have been recently cloned and characterized mainly by mating procedures (Goosen et al., 1987; Cleton-Jansen et al., 1988). This involves primary cloning in a suitable host strain followed by mobilization of the recombinant plasmids. In this article we describe the isolation of a plasmid from A. calcoaceticus Iwoffi and the construction of shuttle vectors by fusions with pBR322. The resulting shuttle plasmids were stable, small and could be used for direct cloning of gene banks from A. calcoaceticus in that organism. This resulted in enrichment of recombinant plasmids and preference of large insertions, which facilitates cloning experiments considerably in comparison to mating procedures. Furthermore, no cloning in intermediate hosts with poten- 46 tial instability ofgenes is necessary and the shuttle plasmids can be prepared from E. coil in high yields. The sequence required for stable maintenance in A. calcoaceticus is defined by deletion and insertion analyses, its promoters determined by the expression of i a c Z fusions in A. calcoaceticus and the nt sequence of the replicon is presented. MATERIALS AND METHODS Restriction endonucleases were purchased from Boehringer, Mannheim. Bethesda Research Laboratories, Eggenstein and N.E. Biolabs, Schwalbach. T4 DNA ligase and calf intestine phosphatase were from Boehringer, Mannheim. All reactions and polyacrylamide and agarose gel electrophoresis were done as described previously (Hillen et al., 1982). Plasmid DNA from E. coli and A. calcoaceticus grown in LB broth (Table I) was isolated on a small scale by the method of Birnboim and Doly (1979) and in large amounts as described (Hillen et al., 1981) and purified by CsCI density-gradient centrifugation. RESULTS AND DISCUSSION (a) Construction of a shuttle plasmid for Acinetobacter calcoaceticus and Escherichia coli A cryptic plasmid, pWH 1277, about 4.5 kb, was isolated from A. calcoaceticus lwoffi, linearized with P m l l and ligated with Pmll-cleaved pBR322. The ligation products were transformed to E. coil R R I and the resulting plasmid was called p W H 1266. The physical structure of p W H 1266 is shown in Fig. 1. As derived from a restriction analysis the entire sequence of the cryptic A . calcoaceticus plasmid is present in p W H 1266. Purified D N A prepared from E. coil was used to transform A. calcoaceticus BD413. Transformants were selected on LB plates containing Ap or Tc indicating that both pBR322-derived resistance genes are expressed and functional in A . calcoaceticus. Plasmid TABLE I Bacterial strains and plasmids Strain Genetic features Reference or source -- Juni (1972) Juni (1972) Legler (1970) A. calcoaceticus a BD4 (DSM586) BD413 (DSM588) Iwo~ (DSM30013) trpE -- £. coil b RRI Plasmlds pBR322 pWH 1266 pWHI267 pWH1268 pWH1269 pWH 1270 pWHI271 pWHI272 pWHI273 pWHI274 pWH 1281-1295 pWH 1296 pWHI297 F - , hsdS 20, (r~ mg ) reed +, ara.14, proA 2, lacY l , gaIK2, rpsL20 (Sma), xyl. 5, mtl.I, supE44 Apa, Tca Apa, Tca Apa Apa, Tca ApR, Toa Apa, TcR Apn, Tca Apa, Tca Apa, Tca Apa,Tcn Apa, TcR, Kina TcR ApR, TcR Fusion of pBR322 and pWHI277 at/hull Deletion of the BamHI.BgllI fragment from pWHI266 Deletion of the NdeI fragment from pWH1266 Hincll.BgllI A from pWH1277 in/~ull of pBR322 Hincll-Bglll B from pWHI277 in/h,ulI of pBR322 AccI C from pWHI277 in Pvull of pBR322 Accl A from pWHI277 in Pmll of pBR322 Accl B from pWHI277 in Pmll of pBR322 Substitution of NdeI B of pWH 1273 by Ndel B of pWHI270 Insertion and replacement derivatives of pWHI266 creating lacZ fusions EcoRl deletion of pWHI281 Insertion element deleted from pWHI289 Bolivar et al. (1977) Bolivar et al. (1977) This work, Fig. 1 This work, Fig. 2 This work, Fig. 2 This work, Fig. 2 This work, Fig. 2 This work, Fig. 2 This work, Fig. 2 This work, Fig. 2 This work, Fig. 2 This work, Fig. 3 This work, Fig. 3 This work, Fig. 3 a A. calcoaceticusBD4 was used as a donor of chromosomal DNA with the wt t~'p£ gen# (Juni, 1972).A. cal¢oaceticus BD413 carries the trp£ marker and exhibits natural competency0uni, 1972).It was used as a recipient for cloningexperiments in A¢inetobacter.A. cal¢oaceticuslwojO/iturned out to contain a cryptic plasmid which was used to construct the shuttle vector. A. calcoaceticu~strains were grown in Erlenmeyer flasks at 30°C on a New Brunswick Gyrotory Shaker (350 rpm) in LB (I0 mg peptone/5 rag yeast extract/10 mg NaCI, all per ml, pH 7A). Ap (I00/~8/ml) and Tc (I0/Ag/ml) were added for selection. Transformants of A. oakoaceticus complemented by the gene bank were selected on minimal medium containing: 15 mg agar/6 mg Na2HPO4.2H20/3 mg KH2PO41~).9mg NaCI/I mg NH4CI/0.25 mg MgSO4.7 H20/2 mg glucose/100 #g Ap, all per ml. DSM, Deutsche Sammlungder Mikroorganismen. b E. coli RRI was generally used for transformations as described (Cohen et al., 1972). 47 Barn HI Pvu II ( Nd.l~ Pvull ~ ~ Pvull Pvull 0 BamHI "°°'l I A©cl B9111 Ndel~[ 4 Hin©ll Acc I Fig. 1. Construction of the shuttle plasmid pWH1266 for "E. coil and A. calcoaceticus. The cryptic A. calcoaceticus-derived plasmid, pWH 1277, is shown with some relevant restriction sites, and pBR322 is displayed with the relevant genetic markers. The pWH 1266 shuttle vector resulted from fusion of both plasmids at their PvulI sites. The pWH 1277-derived portion is indicated by the heavy line. Only restriction sites relevant for experiments presented in this article are shown. pBR322, however, does not replicate in A. cakoaceticus. Plasmid DNA from twelve independent candidates was purified and digested with PvuII (data not shown). All of them turned out to be identical to the E. coli-derived pWHI266. The same result was obtained when plasmids from E. coil transformed with A. calcoaceticus-derived pWH1266 were analyzed. These results indicate that the shuttle plasmid pWH 1266 is able to transform both strains without undergoing frequent rearrangements. Further restriction mapping of pWH 1266 revealed the locations of cleavage sites indicated in Fig. 1. No cleavage sites were found for Xhol, KpnI, XbaI, and Sacl. In addition, pWH 1277 contains no recognition sites for BamHI, EcoRl, EcoRV, HindIll, PstI, ClaI, SalI, AatI, Smal, NruI, Nael, StuI, Hpall, HaeII, HaelIl and Thai. (b) Optimization of the transformation rates of Acinetobaeter calcoaeeticus for the shuttle plasmid A fresh overnight culture ofA. calcoaceticus BD413 was diluted 1000-fold in 20 ml LB and shaken at 30°C. Several times 0.2 ml of the culture were transformed with 500 ng pWH1266 and transformants were scored on LB plates containing 100/~gAp/ml. Competency is observed from the early log phase to the stationary phase. A maximum of competency is found in the late log phase of growth with about 6 × l0 s transformants per 500 ng of plasmid DNA. This procedure is slightly different from the reported protocol (Singer et al., 1985). 500ng of plasmid DNA proved to be saturating under these conditions, pWHI266 prepared from E. coil and A. calcoaceticus showed the same transformation efficiency.This indicates that no restriction barrier exists in A. calcoaceticus BD413 for E. coli-derived DNA. The yields of plasmid preparations from E. coli (up to 1/tg/ml) indicated that pWH 1266 has a high copy number, while it appears to be a low copy number plasmid in A. calcoaceticus (yielding about 10 ng/ml). (c) Molecular cloning of the trpE gene from Acinetobacter calcoaceticus Total DNA from A. calcoaceticus BD4 was prepared, 1/tg partially cleaved with Sau3A and ligated with 20 ng BamHI-finearized pWHI266. The figation products were transformed into E. coil and A. calcoaceticus BD413 .,rpE, and scored for Tc sensitivity. In E. coli about 16% and in A. calcoaceticus about 95~o of the transformants were Tc-sensitive. It is concluded that A. caicoaceticus shows a sixfold preference for these recombinant plasmids. The experiment yielded 2 x l0 4 primary transformants in A. calcoaceticus BD413 as determined on LB plates containing Ap. The bulk of transformed cells were selected on minimal medium plates with Ap, resulting in 20 colonies. Restreaking on minimal plates with Ap yielded 16 candidates. From these the plasmid DNA was prepared from 3 ml overnight cultures and retransformed to A. calcoaceticus BD413 trpE. The transformants were plated on LB-Ap plates and minimal medium Ap plates. Four plasmids yielded roughly the same number of transformants on both plates indicating that the trpE complementation was physically linked to Ap resistance. The sizes of the insertions varied between 10 and 30 kb. Restriction mapping indicated that the vector portion did not undergo any noticeable rearrangement during the cloning experiments. One candidate, pWHI275, contained a 20-kb insertion. The transformation efficiency of pWH1275 in A. calcoaceticus was compared to that ofpWH 1266 and turned out to be 30-fold higher (data not presented). This result, together with the high insertion rate and the large insertion sizes, indicates that recombinant plasmids are enriched by transforming the ligation mixtures directly to A. calcoaceticus. It is not clear at present whether this is related to the chromosomal DNA from A. calcoaceticus in the recombinant plasmids or may just reflect a preference for large plasmid sizes. However, this property is of considerable advantage for direct cloning experiments. (d) Identification of the region necessary for stable, autonomous replication in ,4cinetobacter calcoaceticus A deletion analysis of the DNA from A. calcoaceficus in pWH 1266 was done to defme sequences necessary for stable replication and maintenance. This was carried out either by deleting parts ofpWH 1266 or by subcloning DNA fragments in pBR322. The restriction sites relevant for these approaches are indicated in Fig. 1 and the physical structures of the derived plasmids are described in Fig. 2. The plasmids pWH 1267through pWH 1274were initially cloned in E. coil, prepared and characterized by restriction mapping. They were then used to determine their efficiency to transform A. calcoacetlcus BD413 and quantitate their stability. The results of these experiments are presented in Table II. Only one deletion plasmid, pWHI274, shows the same transformation efficiency as pWH1266. This plasmid contains the 2.2-kb Accl-HinclI fragment from the cryptic plasmid inserted into the PmlI site ofpBR322. The size of pWH 1274 is about 6.5 kb, smaller than most of the broadhost-range plasmids. No transformants were found for pWHI267 containing a BamHI-BglIl deletion (from pWH 1266) and for pWH 1268 containing the deletion of the small Ndel fragment (from pWH 1266) (see Figs. 1 and 2). pWHI269 through pWHI273 yielded between 100 and 1000 transformants. These transformants, however, grew only to small colonies in comparison to those carrying pWHI266 or pWHI274. It is not obvious why these constructions gave rise to transformants while pWH 1268 carrying nearly the same A. calcoacetlcus DNA as pWH 1272 TABLE 11 Transformation efficiencies and stability of the derivatives of plasmid pWHI266 Plasmida pWHI266 pWH1267 pWHI268 pWHI269 pWHI270 pWHI271 pWHI272 pWHI273 pWHI274 pWHI296 pWHI297 Transformation efficiency (T/500 ng) b Growth': No 6×10 s 0 0 5 × 102 8 × 10' 7 × 102 " 1 x 10s 6 × 102 4 × l0 s 6 × l0 s 6×10 s 1×106 --No growth No growth No growth No growth 3 × l0 s 1 × 106 1 × 106 1× 106 StabilityC NA/No (¢ru/ml) 1.1 +0.11 -----5 × 10 -s + 1.5 x 10 - s 1.0 :t: 0.12 0.32 + 0.14 ° 1.0 +0.5 a See Figs. 1 and 2. For pWH1296 and pWH1297 see RESULTS AND DISCUSSION, section d. b The transformation efficiency is expressed as the number of transformants (T) obtained with 0.5/~g plasmid DNA per 2 x 108 cells of A. calcoaceticus BD413. A freshly prepared overnight culture of A. calcoaceticus was diluted 1000-fold in 20 ml LB and grown to an Asso of 3.0. 0.2 ml of the culture was removed, the appropriate amount of plasmid DNA was added, the culture was shaken at 30°C at 350 rpm for 1 h and plated out on LB-Ap plates. c To determine the stability of plasmids, the respective A. calcoaceticus strains were grown overnight in LB with 100/~g Ap/ml or 10 ~g Tc/ml, diluted 1000-fold in LB without an antibiotic and grown into the stationary phase. This was reached after roughly ten generations. The cultures were then titrated on LB plates (No) and LB plates with Ap (or Tc in the case ofpWHl296) (NA). The stability of the plasmid is defined by the ratio NA/No. a The NA/No ratio after growth under selective pressure was 0.38 ± 0.14; thus no increased loss of plasmids occurs without selection. eoo~ piasmlds Pvull Hlncll Accl Nd Accl Ac ull pWH 1266 pWH 1267 pWH 1268 pWH 1269 pWH 1270 pWH 1271 pWH 1272 pWH 1273 pWH 1274 Fig.2. Physical maps of deletion derivatives from pWHI266. The A. calcoaceticus.derived portion of pWH1266 (see Fig. !) with some restriction sites is displayed on the top. The pBR322 derived sequences of the deleted plasmids are omitted, pWH1269, 1270, 1273 and 1274 contain the pBR322 portion in opposite orientation as compared to the other plasmids. The map is drawn to scale. did not. However, since the small colonies were not viable this effect was not studied further (see Table If). We concluded that the AccI-Hincll DNA fragment present in pWH1274 encodes the ability to efficiently transform A. calcoaceticus because deletions affecting this fragment resulted in plasmid instability. While the transformation efficiency was scored on LB-Ap plates, the stability of the plasmids was determined as described in the footnote to Table II. The transformants containing pWH 1269 through pWH 1272 failed to grow in overnight cultures in the presence of Ap while the transformant with pWH1273 grew only to a reduced density of 2.5 x l0 s cells per mi. This result indicates that the AccI fragment present in pWH 1273 leads to a greater stability in comparison to the other DNA segments. However, strains transformed with either pWH1266 or pWHI274 grew in overnight cultures to a higher density of about 1 x 109 cells per ml. This result is confirmed by growing the respective strains under non-selective conditions and scoring the pres- 49 ence of the Ap R phenotype afterwards. The results are also given in Table II. While both pWH 1266 and pWH 1274 are not segregated in the absence of selective pressure, pWH1273 is only maintained in about one in 105 cells after that growth period. This result confnmed that the 2.2-kb AccI-Hincll D N A is important and sufficient for stable replication in A. calcoaceticus, while plasmids with the neighboring 1.3-kb AccI D N A (see Figs. I and 2) can replicate, but segregate rapidly without selection. This indicates that the 0.7-kb Accl-HinclI D N A (see Fig. 1) contains an important feature for maintenance. Further characterization of the region necessary for maintenance in A. calcoaceticus was done by an insertion analysis using a lacZ indicator gene as shown in Fig. 3. It was designed to (i) identify positions where insertion of a D N A fragment interferes with replication and (ii) determine the transcriptional activity at the insertion sites. The replacement derivatives pWH 1293, pWH 1294, and pWHI295 showed a loss of transformation ability to Pvul~.~Accl Accl Bglll ~f t Accl t Hinc II f ,2m~7 ~ Pvull ~s,las,les,.m,eo t f f lae2~7 1290H ,'co[ ,.4[ ] - ,.,[ ---" ] - ] Fig. 3. Map ofthe insertion and substitution mutants ofpWHI26G.The black bar represents the 1337-bp replicon region, the nt sequence of which is shown in Fig, 4. Upward arrows indicate Sau3A sites. The order was not established for Sau3A fragments designated in two regions between arrows by their size in bp. The $au3A sites on the fight side are in the pBR322 vector portion, pWHI266 was partially digested with $au3A, ligated with the BamHl cleaved insertion sequence and transformed to E. coil RRI. Triangles indicate insertion sites of the BamHl insertion element and brackets the deleted Sau3A fragments.The insertion element is a 4.7-kb DNA containing a promoterless lacZ reading frame followedby the fd terminator and a KmRgene flanked by BamHI, Sail and Pstl sites (Kokotek and Lotz, 1989). The arrows within the triangles and brackets mark the direction of lacZ transcription. The double arrows in pWH1289 indicates a double insertion of the lacZ fragment. The numbers at the triangles and brackets are the respective pWH plasmid designations. The resulting plasmids were prepared and transformed to A. calcoaceticusBD413. Compared with pWHI266 all of these plasmids yielded at least 100-fold fewer transformants (data not shown). This result is not due to modifications of the on"sequence, as is most clearly demonstrated by pWHI288 having the insertion in the pBR322 portion. Furthermore, pWHI289, pWHI281 and pWHI291 were used to construct pWHI297, pWHI298 and pWHI299, respectively, by Pstl deletion of the insertion sequence. In all three cases this resulted in the same transformation efficiencies as found for pWHI266. This result indicates that the insertion sequence itselfis indeed responsible for the 100-foldreduced transformation efficiency. A. calcoaceticus, indicating that the minimal repficon was affected in these plasmids. Taken together these results locate the ori for A. calcoaceticus in pWHI266 to about 1350 bp from the insertion in pWHI281/1283 to that in pWHI282/1284. This conclusion was confirmed by stability tests of pWHI297 (pWHI289 after deletion of the insertion sequence) and pWHI296 (EcoRl deletion of pWHI281 removing nearly the entire insertion sequence and the pBR322 b/a region to the E c o R l site). Strains with either plasmid were grown for 8 h without selection and the plasmids turned out to be stable with no detectable loss within this time period (see Table II). Thus, the 1350-bp segment marked in Fig. 3 contains all the sequences required for replication and maintenance in A. calcoaceticus. (e) Transcriptional activity of pWH1266 in Acinetobacter calcoaceticus and E . coU The expression of pGal from the lacZ gene in the insertion sequence was measured to determine promoter activity in pWH 1266. The pGal activities are given in Table III. A strong promoter directs transcription into the lacZ gene in pWHI285. Since this part of the DNA is not required for replication, this activity serves as a positive control, indicating that A. calcoaceticus promoters can be detected by this method. It is interesting to note that this strong promoter is also active in E. coil RRI iacZAMI5, suggesting that this A. calcoaceticus promoter may be also functional in E. coll. This promoter activity is terminated in pWH 1287, indicating the presence of a transcriptional terminator between these insertions. The results obtained with pWHI281 and pWHI282 show that no strong promoter TABLE III pGal activities in A. calcoacet/cusand £. coli Plasmidsa pGal activityu A. calcoaceticus E. coli (BD413) pWHI281 pWHI282 pWH1285 pWH!287 pWHI290 pWHI291 pWH1293 pWHI294 pWHI295 45 + 28 !.6 + 0.3 11890 +476 8 + ! 1.7 + 0.4 100 + 61 n.d. n.d. 14876 + 1018 n.d. n.d. n.d. 15 + 16 + 830 + !.2 1.4 25 See Fig. 3. b pGai activity is given in units as defined by Miller (1972). A. calcoaceticusBD413without a plasmid gave 2.9 :!:0.4 units, n.d. ffi not determined; -- = no transformunts in A. calcoaceticus. a 50 GATCGTAGAAATATCTATGATTATCTTGAAGAACGCAACCCTATAGCAGCTATTGAAATTGATGATTTAATTGAAGAAAAGACAGATTTA 9O GTTGTTGATAATI;GACTGATGGGGCGCACAGGCAGACAGAAAGATACTAGGGAGTTAGTGATACATCCGCATTATGTGGTTGTATATGAC 180 ATCACTGATATAATACGGATACTCAGAGTGCTACACACATCGCAGGAGTGGTCATGACTTACTCATGTACTTTGGATTATTTAGTGTTAT 270 AAAAT¢I;:TGATTTATAAAiTTTTTTTTGTTA.a_a_a_a_a.GATAAm'~TT~TTG G G ~ A C C GTAATTTATGGGGTACAGAT 360 CTTCGATACTGACATATCGGCAATCGAAAGCATTAAGGTTTGACGACCGCTAATGATTTCACCACAGGGGCTTAATGTACCTGTCTTA.qA 45O ACTCAATCTGT£CATGTGTGGGTGATGAGG 54O TTCTAAGGTTTTAACTCGCTTTGTCAAGCA TAGACCCCAAAAATTTAGCCAATGTCTGTA GGACGCAATGAGTAGTCATTTAATTGGCGG 630 TACAGTGACGCTAGCACACATCGGAAAAACGCTATTACTAGGGGAACTGAACAGAGTAGC TGATGTCATCCCTGATGCGAAAGCGACCGA 720 TTATGAGCGTGTTCAGGCGG TGCTAT£AATCGTAATCATAACAGTGGCAGCTTGATACAG TCCCTCCCTC 810 CCGACGGTACATCGAATGGGAATACTTTAG GGTGATI'TTTAAGAATCGCT CTAGGGTGAGTATTTCCCATTCAGCTCTGC ATTAAACAAT 9OO TGGTACTTTAATCAAAAGCACTACTA.qACATATGTTTTTAAAT[~AT ATTGATATAGAGATAATATTAGTAAGAATA A 990 TGAATATAGATAAATCATTGTTAAATAAAGATTAATTATTAAAATGAATGTATACTTATATATAAATCAATGATFIFAAAATATTTGATA 1080 AGAAAACTTTTCAAA&~ATATAATTGAGATTGTGTCATTTCGGTCAATTCTTAATATGTTCCACGCAAGTTTTAGCTATGGTGCTAAA CAGAAATTTGCTGAAAAAGAACTTTTCACTGAACTGGTTAAAATGTAAGCAGCCTGAGAGCCGCCAJ~TlrTTAAAAACA.qACCGCCTT1170 )260 AATCATCTTCAAAAAATACC TCTAAAACCTCACCATTTGCGTTTTAAGACCCATATTTCATCCTGCCCTTATGTTCCCATGCTGATAGCT GCAAAATGTTAAAATCC 1337 ATA.qAGTGTCTGTAATCGCTTCCTATGACGTTCTAGGCTGTTGATAACTTTTGGAACAAC Fig. 4. Nucleotide sequence of the or/region of pWHI266. The 1337-bp fragment is sufficient for replication and stable maintenance of plasmids in A. calcoaceticus.It correspondsto the one indicated by the black bar in Fig. 3; the Sau3A site immediatelyfollowingthe 3'-end is not shown.The A + T-rich regions are underlined,a palindromicelementis indicated by the heavyconvergentarrows and elevenrepeats with at least 7 nt ofthe consensus sequence AAAAAATATare printed in bold letters. The Bglll site is at nt 357 and the Accl site at nt 950. Sequencingwas cloneby the dideoxy-chain-termination method (Sanger et al., 1977)on both strands. transcribes out of the sequence required for replication; however, pWH 1281 shows a weak activity which is clearly above background. In pWHI290 this activity is reduced to background levels. The significance of this promoter activity for replication is not clear. We conclude that fusions with a promoterless lacZ gene can detect promoter activity in A. calcoaceticus; however, a promoter activity likely to be associated with the replication sequence was not found. Bramhill and Kornberg, 1988; and references cited there). This notion is supported by the reduced stability of pWH 1273 (see Fig. 2 and Table II) which lacks D N A from the Accl site at nt 950 in Fig. 4 including four of the nine AAAAAATAT repeats. (f) Specificity of the replicons on plasmid pWHI266 In plasmid pWH 1296, the ColE 1 on' was deleted without decreasing the number of transformants obtained in A. calcoaceticus (Table If). This indicates that the Cole I or/ had no activity in A. calcoaceticus which is confirmed by the observation that pBR322 yields no transformants in A. calcoaceticus. Attempts to transform pWHI296 into E. coil RRI yielded no transformants. Therefore, it was concluded that the replicon of the cryptic plasmid pWH1277 showed no activity in E. coll. We thank Mrs. B. Weins for helpful discussions, M. Will and I. Schiessl for excellent technical assistance and Mrs. R. Oster for typing the manuscript. This work was supported by the Fonds der chemischen Industrie. V.K. was supported by a DAAD fellowship. (g) Nucleotide sequence of the ori active in Acinetobacter caleoaceticus The nt sequence of the 1.35-kb fragment is shown in Fig. 4. No ORFs were detected within the segment necessary for stable maintenance in A. calcoaceticus. This indicates that the ori region in plasmid pWH 1277 is not dependent on a plasmid specific replication protein as found for most replicons (Gallic and Kado, 1988). However, other features of a plasmid replicon were found in the sequence. Two A+T-rich regions (88%) were observed at nt 256-312 and 835-1017. Eleven repeats defmed by at least ? nt out of the 9-bp consensus sequence AAAAAATAT are found. Eight ofthese are clustered between nt 835 and 1189 of the sequence shown in Fig. 4. A palindromic sequence at nt 310-337 was also found. Repeat sequences were postulated to be essential for many replicons (Novick, 1987; ACKNOWLEDGEMENTS REFERENCES Birnboim, H.C. and Doly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7 (1979) 1513-1523. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S.: Construction and characterization of new cloning vehicles, II. A multipurpose cloning system. Gene 2 (1977) 95-113. Bramhill, D. and Kornberg, A.: A model for initiation at origins of DNA replication. Cell 54 (1988) 915-918. Cleton-Jansen, A.-M., Goosan, N., Wenzel, T.J. and Van de Putte, P.: Cloning of the gene encoding quinoprotein glucose dehydrogenase fromAcinetobactercalcoaceticus:evidencefor the presenceof a second enzyme. J. Bacteriol. 170 (1988) 2121-2125. Cohen, S.N., Chang, A,C.Y. and Hsu, L.: Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherlchla coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69 0972) 2110-2114. Gallic, D.R. and Kado, CJ.. Minimal region necessary for autonomous replication of pTAR. J. Bacteriol. 170 0988) 3170-3176. Goosen, N., Vermans,D.A.M. and Van de Putte, P.: Cloningofthe genes involved in synthesis of enzyme pyrrolo-quinollne-quinon from Acinetobacter calcoaceticus. J. Bacteriol. 169 (1987) 303-307. Hillen, W., Klein, R.D. and Wells, R.D.: Preparation of milligram 51 amounts of 21 deoxyribonucleic acid restriction fragments. Biochemistry 20 (1981) 3748-3756. Hillen, W., Klock, G., KalTenberger, J., Wray Jr., L.V. and Reznikoff, W.S.: Purification of the Tet repressor and tet operator from the transposon TnlO and characterization of their interaction. J. Biol. Chem. 257 (1982) 6605-6613. Juni, E.: Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J. Baeteriol. 112 (1972) 917-931. Juni, E.: Genetics and physiology ofAcinetobacter. Annu. Rev. Microbiol. 32 (1978) 349-371. Kokotek, W. and Lotz, W.: Construction ofa lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene 84 (1989) 467--471. Legler, F.: (.~er das Vorkommen, die Pathogenitit und die AntibiotikaEmpflndlichkeit des Ac.~omobacaer mucosus. Zbl. Bakt., !. Abt. Orig. 215 (1970) 466-474. Miller, J.H.: Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, pp. 352-355. Novik, R.P.: Piasmid incompatibility. Microbiol. Rev. 51 (1987) 381-395. Sanger, F., Nieklen, S. and Coulson, A.R.: DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Singer, J.T., Van Tuiji, JJ. and Finnerty, W.R.: Transformation and mobilization of cloning vectors in Acre"eto/mcter ca/coacetfcus spp. J. Bacteriol. 165 (1985) 301-305. 本文献由“学霸图书馆-文献云下载”收集自网络,仅供学习交流使用。 学霸图书馆(www.xuebalib.com)是一个“整合众多图书馆数据库资源, 提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。 图书馆致力于便利、促进学习与科研,提供最强文献下载服务。 图书馆导航: 图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具