Analysis and stability of Zymomonas mobilis ATCC 10988 plasmid pZMO3

PLASMID

23,59-66

(1990)

Analysis and Stability of Zymomonas mobilis ATCC 10988 Plasmid pZM03 ALEXANDRA SCORDAKI AND CONSTANTIN

DRAINAS’

Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of loannina, 451 10 loannina, Greece Received October 2, 1989; revised February 5, 1990 Plasmid pZM03 of Zymomonas mobilis strain ATCC 10988 was found to be nonhomologous either to chromosomal DNA or to any other plasmids of the strains ATCC 10988, NCIB 11163, and CP4. It contained single sites for the restriction endonucleases SphI, BglI, and HindIII, as well as at least four sites for Sau3A. Its origin of replication is located within the 1.54-kb Sau3A fragment as it was found that only the recombinant plasmid pDS3154, which contained this fragment, showed vectorial incompatibility with the native pZM03 plasmid. The stability of pZM03 may be controlled by partitioning sequences located in the 0.64-kb Sau3A fragment. Z. mobilis isolates, which had lost plasmid pZM03, were successfully isolated. 0 1990 Academic press, Inc.

Z. mobilis plasmids and production of plasmid-free strains will facilitate the genetic improvement of this important industrial microorganism. A variable number of native plasmids have been detected by many research groups in all the available 2. mobilis strains (Dally et al., 1982; Tonomura et al., 1982; Drainas et al., 1983; Skotnicki et al., 1984; Misawa et al., 1985). However, very little is known so far about their structural homologies, stability, and function. Strain ATCC 10988 harbors the largest number of native plasmids (Tonomura et al., 1982; Drainas et al., 1983). This strain contains at least six plasmid molecules, named pZMOl-6, which differ by molecular weights 1.6, 1.9, 2.7, 7.3, 16.7, and 31.6 kb, respectively (Drainas et al., 1983). However, a functional role has not yet been assigned for any of these stably replicating plasmids. Structural analysis and utilization of pZMO1 and pZM02 for the construction of recombinant plasmid vectors have been reported elsewhere (Scordaki and Drainas, 1987; Afendra and Drainas, 1987). The present report focuses on plasmid pZM03. Restriction fragments were subcloned in a known vector and sequences

During the last few years increasing scientific and industrial interest has been given for the ethanol producing anaerobe Zymomonas mobilis. However, its industrial application is still limited due to its small range of carbohydrate utilization and product formation (Swings and DeLey, 1977; Bringer et al., 1984; Rogers et al., 1984). Genetic improvement may overcome this limitation. Although plasmid vectors have been constructed for Z. mobilis, they show considerable instability in the bacterium, as they disappear (Tonomura et al., 1986) or loose their structural identity after several cell divisions (Byun et al., 1986; Liu et al., 1988). Incompatibility or genetic rearrangements with native plasmids of the Z. mobilis strains used may account for this instability. Reports on the stability of a recombinant vector used to transfer foreign genes in Z. mobilis have been based on very few cell divisions without testing the influence of the existing native plasmids on its stable inheritance (Misawa et al., 1986,1988; Misawa and Nakamura, 1989a). Therefore, studying of the structure, function, and stability of the native ’ To whom correspondence should be addressed.

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Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

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controlling replication and partitioning of pZM03 were identified. The use of the subcloned fragments for the isolation of strains cured for pZM03 is described. MATERIALS

AND

METHODS

Bacterial strains and plasmids. Z. mobilis strains ATCC 10988, its derivative CU 1-Rif22 (Afendra and Drainas, 1987) CP4, and NCIB 11163 were used throughout this work. Escherichia coli strains RR1 (Bolivar et al., 1977), RR1 harboring plasmid pBR325 (Bolivar, 1979) and HBlOl harboring the conjugative plasmid pRK20 13 (Figurski and Helinski, 1979) were used in recombinant DNA and bacterial conjugation procedures (seebelow). Plasmid pDS2 12 is an EcoRI ligate of pBR325 with the native pZM02 Z. mobilis plasmid (Scordaki and Drainas, 1987) Growth. Z. mobilis strains were grown in a liquid complete medium without agitation at 30°C as described previously (Galani et al., 1985). E. coli strains were grown in Luria broth. For solid media 2% agar (Serva) was added. Chemicals. T4 DNA ligase, alkaline phosphatase, and restriction endonucleasesBanII, BglI, BglII, EcoRV, and PstI were from Bioellas. Agarose, sodium dodecyl sulfate, cesium chloride, phage X DNA, and restriction endonucleases BamHI, EcoRI, HincII, HindIII, HpaI, KpnI, NcoI, PvuII, SalI, Sau3A, SmaI, SphI, XbaI, and XhoI were from Bethesda Research Laboratories. Antibiotics were from Sigma. [‘*P]dATP was from Amersham. All other chemicals were from Serva. DNA isolation. Plasmid pZM03 was isolated from Z. mobilis strain ATCC 10988 extracts as previously described (Scordaki and Drainas, 1987). Chromosomal DNA was extracted from Z. mobilis cells and purified by CsCl density gradient ultracentrifugation (Byun et al., 1986). Miniscreen plasmid tests ’ Abbreviations used: Cm, chloramphenicol; Rif, rifampicin; Tc, tetracycline; R, resistant; SDS, sodium dodecyl sulfate.

were performed as described previously (Drainas et al., 1984). Large plasmid preparations from E. coli were made according to Maniatis et al. (1982). Recombinant DNA procedures. Enzyme digestions and ligations were performed by standard methods (Maniatis et al., 1982). Plasmid pBR325 was selected as the cloning vector because(a) it contains three antibiotic resistance markers, two of which (TcR and CmR) are expressed in Z. mobilis, and (b) it can be mobilized in Z. mobilis by helped conjugation and, therefore, it may be used for the construction of potential shuttle vectors for Z. mobilis (Afendra and Drainas, 1987). Recombinant plasmids (ligates of linearized pBR325 and various fragments of pZM03) were isolated by direct transformation of E. coli strain RR 1 (Cohen et al., 1972). Homogeneous fragments of pZM03 were isolated after complete digestion of the recombinant plasmids with the appropriate endonuclease, agarose gel electrophoresis, electroelution, and desalting. Sizesof restriction fragments were determined by comparison with X DNA Hind111or EcoRI digestson agarose,as well as on the polyacrylamide gel electrophoresis (Maniatis et al., 1982). DNA probes were labeled with 32Pby a BRL nick-translation kit according to the manufacturer’s instructions. For this purpose, 50 PC1(1.85 MBq) [32P]dATPwas used in the reaction mixture to give an incorporation of 45%. Southern blot hybridization. Nylon filters (Amersham, N Bond) were used for blotting. DNA was fixed on filters by uv irradiation according to the manufacturer’s instructions. All other steps were performed essentially as described by Maniatis et al. (1982) with minor modifications. Under high-stringency conditions filters were incubated with a radioactive probe at 68°C and washing was performed at 58°C in 0.1 X SSC(0.15 M NaCl, 0.0 15 M trisodium citrate, pH 7.0) + 0.005% SDS; under low-stringency conditions filters were incubated with the radioactive probe at 45°C and washing was performed at 35°C in 1X SSC + 0.05% SDS.

PLASMID pZM03 OF Zymomonas

Conjugation. E. coli donors transferred their plasmids into Z. mobilis recipients by filter matings (Afendra and Drainas, 1987). Donors were obtained by direct transformation of strain HB 101 harboring pRK20 13 with each of the recombinant constructs containing pZM03 sequences.Coexistanceof conjugative and mobilizable plasmid in donors was frequently tested by miniscreen plasmid analysis and back-transformation of E. coli RRl. Z. mobilis ATCC 10988derivative CU 1-Rif2 was used as recipient. Selection of Z. mobilis transconjugants was made on complete agar medium containing 100 pg ml-’ chloramphenicol (marker of mobilizable recombinant plasmids) and 12 pg ml-’ rifampicin as counterselection for E. coli. Resistant colonies were tested for harboring the mobilizable plasmid by in situ hybridization under high-stringency conditions, using a fragment carrying the chloramphenicol resistancemarker of pBR325 as a probe. Plasmid stability. Plasmid stability was estimated by measuring the inheritance of the antibiotic resistance plasmid marker by Z. mobifis transconjugants for up to several hundred cell divisions after growth under selective (with chloramphenicol) or nonselective (without chloramphenicol) conditions. The percentage of chloramphenicol resistant Z. mobilis colonies, which hybridized to the appropriate DNA probe by in situ colony hybridization per cell division, was determined.

mobilis

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FIG. 1. Restriction map of pZM03 and recombinant plasmids containing pZM03 sequences. I, pZM03; 2, pDS3 154;3, pDS364; 4, pDS329; 5, pDS3 17; 6, pDS3270; 7, pBR325. B, BarnHI; Bg, Egll; H, HindIII; E, EcoRV; S, Sau3A; Sa, SalI; Sp, SphI.

present report (Misawa and Nakamura, 1989b), of a plasmid (pZM2, 2749 bp) probably equivalent to pZM03. According to this sequence, 11 Suu3A sites should exist on pZM03. The pattern of the partial Suu3A digest of pZM03, which in our hands was reproduced repeatedly, is shown in Fig. 2B (lane 2). The above sequence contained the SphI and the BglI sites at the same location as shown in Fig. 1. Four Sau3A fragments with sizes0.17,0.29, 0.64, and 1.54 kb, which represent the total length of pZM03, were subcloned in the BamHI site of pBR325 (Fig. 1). The recombinant plasmids produced were RESULTS AND DISCUSSION named pDS317, pDS329, pDS364, and pDS3154, respectively. Each of the above Subcloning and Hybridization Analysis of fragments was not homologous with any of pZMO3 Sequences the other three (see below). Therefore, seRestriction endonuclease analysis of quence overlaps due to the partial Suu3A hypZM03 purified from Z. mobilis ATCC drolysis among the four subcloned fragments 10988 extracts contained 1 SphI, 1 Bgll and were ruled out. Further restriction analysis of at least 4 Sau3A sites (Fig. 1). The latter were the subcloned fragments, recovered from E. deduced from the analysis of a partial Sau3A coli extracts, revealed a single Hind111 site digest of purified pZM03. It is possible, how- within the 1.54-kb Suu3A fragment (Fig. l), ever, that other Suu3A sites may also exist on which was unidentifiable on pZM03 when pZM03. This possibility is supported by the isolated from the Z. mobilis extracts. This renucleotide sequenceanalysis, which appeared sult, along with the partial hydrolysis of in the literature after the submission of the Suu3A, could be due to either the modification

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SCORDAKI AND DRAINAS 2

3 4

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6 7 6

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A

6

FIG. 2. Hybridization pattern of a ‘*P-labeled pZM03 probe to plasmid and chromosomal DNA of Z. mobilis. Lane 1, pDS3 I%/EcoRV + SalI; lane 2, pZM03jSau3A; lane 3, pZM06jPstI; lane 4, pZMOS/ EglII; lane 5, pZMO1 + pZM02/BglII; lane 6, pDS212/EcoRI; lanes 7-9, plasmid DNA of strains CP4, NCIB I 1163,and ATCC 10988,respectively; lanes 10-12, chromosomal DNA ofstrains CP4, NCIB I1 163, and ATCC 10988, respectively; lane 13, X DNA/EcoRI. Lanes 1-6, restriction digest with the marked endonuclease.Hybridization was performed under low stringency conditions. Probe, 1.54-kbSau3A fragment of pZM03 extracted from pDS3 154 by a double EcoRV + Sal1 digestion. Hybridization observed on lane 6 was due to pBR325 sequencescarried by the probe. pDS2 I2 was used as a source of purified pZM02. (A) Autoradiograph; (B) agarose.gel electrophoresis.

of the Hind111and at least of some Sau3A sites in the Z. mobilis cells or the existence of some inhibitor of restriction endonuclease activity in our plasmid preparations. However, subcloning of the whole pZM03 plasmid in the Hind111site of pBR325 (Fig. 1) was achieved after prolonged treatment (3 h) of a low concentration of pZM03 DNA with Hind111 (1 unit/O.1 pg DNA), although linearized pZM03 plasmid was undetectable by agarose gel electrophoresis. The latter recombinant plasmid was named pDS3270. The Hind111 site is also contained in the sequencepublished by Misawa and Nakamura (1989b). The HindIII-linearized pZM03 and the subcloned Sau3A fragments were isolated from the recombinant plasmids by single restriction with Hind111 and double restriction with EcoRV + SalI, respectively. These fragments were all used as 32P-labeledprobes to study their structural relation with other natural plasmids, as well as chromosomal DNA of the Z. mobilis strains ATCC 10988, NCIB

11163, and CP4. The hybridization pattern of the 1.54-kb fragment is shown in Fig. 2. No detectable homologies between the above fragment and any of the DNA molecules tested were observed following hybridization even under low-stringency conditions. The hybridization signals appearing in Fig. 2A concern only self-hybridizing sequences. Specifically, in lane 1, the strong signal corresponds to selfhybridization of the 1.54-kb fragment. The weak bands are not visible on the agarosegel electrophoresis (Fig. 2B) and are probably due to some nonspecific hydrolysis of pDS3154. In lane 2, the probe hybridized only with bands which contain the 1.54-kb fragment. In lane 6, the signal is due to pBR325 sequencesalso carried by the probe, while there is not any detectable hybridization at the area where the linear pZM02 migrates.In lane 9, two strongly hybridizing bands indicate that pZM03 probably exists in two forms (most likely CCC and OC) in the Z. mobilis cytosol. This was also supported by the identical restriction pattern

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PLASMID pZM03 OF Zymomonas mobilis TABLE 1 FREQUENCY OF HELPED CONJUGAL TRANSFER OF RECOMBINANT PLASMIDS CONTAINING pZM03 SEQUENCESIN Z. mobilis

Plasmid

Ligation site

Insert size W)

pDS3 17 pDS329 pDS364 pDS3 154 pDS3270 pBR325

Sau3AIBamHI SadA/BamHI Sau3AfBamHI Sau3A/BamHI Hind111 -

0.17 0.29 0.64 0.54 2.70 -

f <10-g 4.7 x lo-’ 2.3 x 1O-6 5.2 x lo-’ 1.8 x 1O-6 <1o-8

Note. f; frequency = transconjugants/recipients. Standard error of six repeats < kO.5 X IO-‘. Donor, E. coli HB 101 carrying helper plasmid pRK20 13 and the appropriate recombinant plasmid. Recipient, Z. mobilis CU lRif2.

of plasmid DNA isolated from both bands. Finally, in lane 12, the hybridizing band corresponds to the OC form of pZM03 (lane 9), indicating that chromosomal DNA preparation of strain ATCC 10988 was contaminated with pZM03 molecules. The results were identical for all pZM03 probes and, therefore, hybridization of other probes was omitted from Fig. 2. None of the probes hybridized to eachother under high- or under low-stringency conditions. These results strongly support that pZM03 is a DNA molecule with unique sequences among the Z. mobilis strains used. Transfer and Stability of the Recombinant Plasmids in Z. mobilis Attempts were made to understand the mechanism which controls stability of plasmid pZM03, based on the stable expression in Z. mobilis of the recombinant E. coli plasmids carrying the native Z. mobilis pZM03 sequences. The recombinant plasmid pDS2 12, which contains the whole pZM02 plasmid (Afendra and Drainas, 1987), has been transferred in Z. mobilis from E. coli donors by helped conjugation with higher frequencies, and it also was stably expressedin the Z. mo-

bilis host, as compared to the parental vector pBR325. Similarly, the recombinant plasmids pDS329, pDS364, pDS3154, and pDS3270 were transferred with higher frequencies (- IOO-fold), as compared to the transfer of pDS3 17 and pBR325 (Table 1). The stability of the recombinant plasmids in the 2. mobilis hosts was studied following the resistance of the hosts on chloramphenicol, as well as detecting the presenceof plasmids by in situ hybridization. It was found that plasmids pDS3 17 and pDS329 and the parental pBR325 were very unstable under both selective and nonselective conditions, as they disappeared after only a few cell divisions (Fig. 3). In particular, it was not possible to maintain by sequential subculturing Z. mobilis transconjugants carrying pDS317 or the parental pBR325. Plasmids pDS3154 and pDS3270 were extremely stable under selective conditions, as they showed strong hybridiza-

L

y.. 20

, ‘I\. 40

60

80

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FIG. 3. Stability of recombinant plasmids in Z. mobilis hosts. The recombinant plasmids were transferred in Z. mobilis hosts by helped conjugation from E. coli donors. Transconjugants carrying pDS364, pDS3 154, and pDS3270 were grown under selective conditions for over 300 cell divisions and then transferred under nonselective conditions to study their stability. pDS329 was transferred, under nonselective conditions, a few cell divisions after isolation. Stability was calculated as the percentageof Cma colonies which hybridized to the appropriate probe per cell division. (m) pDS329; (0) pDS364; (A) pDS3 154; (A) pDS3 154in pZM03-free hosts;(0) pDS3270, or pDS3270 in pZM03-free hosts.

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tion with pZM03 probes and high resistance to chloramphenicol (up to 200 pg ml-‘) for over 300 cell divisions. However, under nonselective conditions they disappeared after 40 and 65 cell divisions, respectively (Fig. 3). On the contrary, plasmid pDS364 conferred a low resistance to chloramphenicol (up to 100 pg mll’) and a low hybridization profile with pZM03 probes, probably due to its maintenance in a low copy number in the Z. mobilis cells. However, pDS364 showed 100%stability for over 300 cell divisions under both selective or nonselective conditions (Fig. 3). Total plasmid DNA, isolated from the above stable transconjugants of Z. mobifis, were prepared and used for transformation of E. coli RR 1. The recombinant plasmids were reisolated from the E. coli transformants and they were subjected to restriction analysis. It was found that all plasmids retained their original size and restriction pattern after 100 cell divisions. However, after 300 cell divisions only plasmids pDS3154 and pDS3270 retained their original size and restriction pattern, whereas pDS364 showed an altered structure (Fig. 4, lanes 1 and 2, other results not shown, for clarity), possibly due to intracellular genetic modifications. Curing of Plasmid pZMO3 To investigate further the function of the 1.54- and 0.64-kb fragments in relation with the stability of pZM03, the vectorial incompatibility of the recombinant plasmids pDS3 154, pDS3270, and pDS364 against the native pZM03 was examined. Vectorial incompatibility of plasmids is known to be based upon stable inheritence of one against the other when both are sharing the same origin of replication (Novick, 1987). Plasmid preparations from Z. mobifis transconjugants carrying the recombinant plasmids pDS364, pDS3 154, or pDS3270, growing under selective conditions for over 300 cell divisions, were subjected to hybridization analysis using pZM03 fragments as probes, under highstringency conditions. In preparations con-

A

B

FIG. 4. Hybridization pattern of ‘*P-labeled pZM03 probe to plasmid preparations from Z. mobilis transconjugants. Lane 1, CU lRif2/pDS364, lane 2, pDS364; lanes 3 and 4, CUl-Rif2/pDS3 154; lane 5, pDS3154; lane 6, CUl-Rif2. Trasconjugants were grown under selective conditions and extracted for plasmids after 100 cell divisions (lane 4) or 300 cell divisions (lanes I and 3). The hybridization was made with the 2.7-kb Hi&III-linearized pZM03 as a probe under high-stringent conditions. Lanes 1,3,4, and 6, plasmid miniscreens from Z. mobilis transconjugants. Lanes 2 and 5, plasmids purified on CsCl density gradient from E. coli extracts. (A) Autoradiograph; (B) agarosegel electrophoresis.

taining pDS3 154, the hybridization signal corresponding to the pZM03 band was reduced after 100 cell divisions (Fig. 4A, lane 4) and was undetectable after 300 cell divisions (Fig. 4A, lane 3). However, this was not the case for either pDS364 (Fig. 4A, lane 1) or pDS3270 (not shown). To verify these results, curing of pZM03 in the presence of the recombinant plasmids was also tested on the single colony level. Transconjugants carrying pDS364, pDS3 154, or pDS3270 were cultured on selective medium for over 300 cell divisions, transferred under nonselective conditions for N 100 cell divisions, serially diluted, and plated on solid nonselective media. Cloramphenicol sensitivity and in situ hybridization testsshowedthat over 90% of the colonies which carried pDS3 154 and only 8% of the colonies which carried pDS3270 had lost pZM03. On the contrary, none of the colonies which carried pDS364 had lost pZM03. The results strongly support that, under the selective conditions used, plasmid pZM03 can be cured in the presence of pDS3154 and to a significantly lower degree in the presence of

PLASMID pZM03 OF Zymomonas mobilis

pDS3270. In the absenceof pZM03, the stability of pDS3154 under nonselective conditions increases significantly (Fig. 3). On the contrary, pDS3270 transferred in pZM03-free recipients showed the same stability pattern. After serial subculturing (over 100 cell divisions) of pZM03-free 2. mobilis transconjugants carrying pDS3 154 under nonselective conditions it was possible to isolate colonies sensitive to Cm, which did not hybridize at all with any pZM03 probe. Six 2. mobilis isolates, which had lost plasmid pZM03, were tested. These were subsequently named CU lRif2-pZM03- l-6 and had the same growth kinetics and requirements as the parental CU I-Rif2 strain (results not shown for clarity). CONCLUSIONS

The above results indicate that the 0.64-kb fragment carries partitioning-like sequences (Austin, 1988), as it confers high stability on pDS364, as well as compatibility of pDS364 with the native pZM03. The origin of replication of pZM03 is most likely located in the 1.54-kb Suu3A fragment, as indicated by the vectorial incompatibility of plasmid pDS3 154 against the native pZM03. In support of this hypothesis is the fact that the stability of the recombinant pDS3 154 was increased significantly in 2. mobilis hosts, which had lost the native pZM03. Surprisingly, the recombinant plasmid pDS3270, which carries the whole pZM03 molecule, did not follow the same stability and curing pattern when compared to pDS3 154. It is possible that the Hind111site may have some topological relation with sequences, which control initiation and/or rate of pZM03 replication. Sequenceslocated in the 0.64-kb Sau3A and 1.54-kb Sau3A fragments of pZM03 may be valuable tools for the construction of powerful vectors for 2. mobilis. Moreover, the successful isolation of derivatives, which have lost the native pZM03, consistsof a major steptoward the increase in the stability of recombinant plasmids in 2. mobilis.

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ACKNOWLEDGMENTS This work was supported by the Greek Government (Ministry of Industry, Energy and Technology, PROPE 1986)and the Commission of the European Communities (Biotechnology Action Programme, BAP-0153-GR).

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(1984). Zymomonas ethanol fermentations. Microbial. Sci. 1, 133-136. SCORDAKI,A., AND DRAINAS,C. (1987). Analysis of natural plasmids of Zymomonas mobilis ATCC 10988.J. Gen. Microbial. 133, 2547-2556. SKOTNICKI,M. L., GOODMAN,A. E., WARR, R. G., AND ROGERS,P. L. ( 1984).Isolation and characterization of Zymomonas mobilis plasmids. Microbios 40, 53-6 1. SWINGS,J., AND DELEY,J. (1977). The biology of Zymomonas Bacterial. Rev. 41, 1-46. TONOMURA,K., KUROSE,N., KONISHI, S., AND KAWASAKI,H. ( 1982).Occurrenceof plasmids in Zymomonus mobilis. Agric. Biol. Chem. 46, 2851-2853. TONOMURA, K., OKAMOTO T., YASUI, M., AND YANASE,

H. (1986). Shuttle vectors for Zymomonas mobilis. Agric. Biol. Chem. 50, 805-808. Communicated by S. Dusko Ehrlich