PLASMID 10, 196-198(1983)

SHORT COMMUNICATIONS

A Restriction Map of Plasmid pDC1 from the Filamentous

Cyanobacterium Nostoc sp. MAC PCC 8009’

GRANTLAMBERT'ANDNOELCARR

Deparrmenr of Biochemistry, University ~1’ Liverpool. Liverpool L69 3BX, United Kingdom

Received May 27, 1983

A plasmid designated pDC I from the cyanobacterium Nostoc sp. MAC PCC 8009 was incubated with 16 different restriction enzymes, of which 8 cleaved pDC I. Plasmid pDC 1 has a single site for C/al, two sites for each of Bgfi. EcoRI. EcoRV, and MluI, three sites for HpaI, and four for HindIlL A restriction map of pDCl for these 7 enzymes was constructed.

Plasmids in filamentous cyanobacteria, first observed by Simon (4) are of interest in terms of their encoded functions and because of their potential for use as cloning vectors in these organisms. Recently a physical map of plasmid pDU1 from Nostoc PCC 7524 has been re- ported (3). As it is yet uncertain as to which strains or plasmids will be suitable for devel- oping a cloning system, there is a need to characterize several plasmids from different filamentous cyanobacteria. Nostoc sp. MAC PCC 8009 is a cyanobacterium capable of chromatic adaptation and it contains at least two prominent plasmid DNAs (2). Interest- ingly, plasmid involvement in chromatic ad- aptation in another cyanobacterium Fre- myella has recently been demonstrated (I). As a necessary preliminary to cloning the replicon and investigating its function(s), we com- municate here a physical map of a 5.3-MD cryptic plasmid, designated pDC 1, from Nos- tot sp. MAC PCC 8009.

METHODS AND MAPPING STRATEGY

Plasmid DNA isolation from 41 cultures was based on van den Hondel et al. (5). Plas- mid DNAs were separated on 5-20% sucrose

’ This communication represents a brief summary of the data used to construct the map; complete documen- tation is on file at the editorial office of Plusmid.

* To whom correspondence should be addressed.

gradients at 75,OOOg for 16 h at 15°C. The restriction endonucleases BamH 1, BglI, BglII, EcoRI, EcoRV, HindIII, HpaI, HpaII, PvuII, SalI, and XhoI were obtained from Boehringer Mannheim; M/u1 from Amersham Interna- tional; PstI from NBL Enzymes; ClaI and HaeIII from Bethesda Research Laboratories, and SphI from New England Biolabs. Incu- bations were performed in a volume of 11 ~1 at 37°C (except for &I, incubation at 30°C) for 2 h with a twofold excess of enzyme. The incubation buffer was 10 mM Tris, 10 mM MgS04, and 1 mM dithiothreitol, pH 7.4, in the case of BgfII, HaeIII, HpaI, and HpaII, with 50 mM NaCl also present in the case of BarnHI, BgfI, C’laI, HindIII, WI, and fiuI1; in 100 mM NaCl, 50 mM Tris, and 10 mM MgSO,, pH 7.4, in the case of EcoRI, Eco- RI’, SalI, SphI, and XhoI; 10 mrvr Tris, 7 mM MgClz, 100 mrvt NaCl, and 7 mrvt 2-mercap- toethanol, pH 7.5 for A4luI. In the case of double digests, enzymes were added sequen- tially, with appropriate adjustment of the buffer for the second enzyme, where necessary. Incubations were terminated by the addition of 10 ~1 of 4 M urea, 50% sucrose, 50 mM EDTA, and 0.1% bromophenol blue, pH 7.0, prior to restriction analysis.

Primary digests of pDC1 on horizontal 1% agarose gels run for 16 h at 45 V (see (2) for other conditions). revealed a single site for C/a& generating a linear 5.3-MD molecule,

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All rtghrs of reproductmn in an) form rewved.

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two sites for each of BglI, EcoRI, EcoRV, and MU, three sites for HpaI, and four for HindIII. With the exception of HpaII, which generated at least six fragments which were not characterised further, all other enzymes tested failed to cleave pDC 1. The construction of the map was facilitated by the four enzymes which cleave pDC 1 twice. These were first un- ambiguously positioned relative to each other by all combinations of double digests between BgA, EcoRI, EcoRV, and MluI. The single ClaI site (assigned position 0, see Fig. 1) was then added to the map by double digests with BgfI and EcoRI, which were sufficient to place it, then confirmed by the results of the double digests ClaIIEcoRV and ClaIjA4luI. The three HpaI sites were positioned by means of double digests with each of the five enzymes which cleaved pDC 1 one or two times (see Fig. 1).

In the case of the four Hind111 fragments, here designated A, B, C, and D in order of de- creasing size, a partial digest indicated the fragment order -B-A-C-D-. The HindI sites were then placed on the map by double digests using Hind111 with CZaI, BglI, EcoRI, EcoRV, MfuI, and, finally, HpaI. The results of these digests were internally consistent, enabled un- ambiguous placing of the Hind111 sites and yielded the same fragment order as deduced above.

The limit of detection in the gel system we have used is about 0.2 MD. The number of such tiny fragments is probably very small, if they exist, since in all areas of the map dis- crepancies were minor (less than 0.15 MD) between sizes obtained using different enzymes and combinations thereof. The complete re- striction map for pDC1 is given in Fig. 1. At

FIG. 1. ClaI, BglI, EcoRI, EcoRV, MluI, HpaI, and Hind111 restriction map of p&nid plX 1, oi+nbrecj with respect to the single CluI site.

198 SHORT COMMUNICATION

present the cloning possibilities for this repli-

con reside primarily in its single ClaI site. Li- gation into the single C&z1 site possessed by pBR 322 and many of its derivatives is a fea- sible cloning route. Regarding the paucity of restriction sites on pDC 1, a similar phenom- enon has been noted in the case of pDUl from Nostoc PCC 7524 (3). Moreover, a re- sistance of chromosomal DNA from the cy- anobacterium Fremyella diplosiphon PCC 7601 to cleavage by many restriction endo- nucleases has recently been documented (6). We have evidence, to be presented elsewhere, of extensive modification of chromosomal DNA from Nostoc sp. MAC PCC 8009. Thus, additional restriction sites on pDC1 may be revealed when this replicon is transferred to a more amenable host.

REFERENCES

1. BOCORAD, L., GENDEL, S. M., AND KOLLER, K.-P., In “Photosynthetic Prokaryotes Cell Differentiation and Function” (G. C. Papageorgiou and L. Packer, eds.), Elsevier, New York, 1983.

2. LAMBERT, G. R., AND CARR, N. G., Arch. Microbial. 133, 122-125 (1982).

3. REASTON, J., VAN DEN HONDEL, C. A. M. J. J., VAN ARKEL, G. A., AND STEWART, W. D. P., Plasntid 7, 101-104 (1982).

4. SIMON, R. D., 1. Bacferiol. 136, 414-418 (1978).

5. VAN DEN HONDEL, C. A. M. J. J., KEEGSTRA, W., BORRIAS, W. E., AND VAN ARKEL, G. A., Plasmid 2, 323-333 (1979).

6. VAN DEN HONDEL, C. A. M. J. J., VAN LEEN. R. W., VAN ARKEL, G. A., DUYVESTEYN, M.. AND DE WAARD, A., FEMS Microbial. Lelt. 16, 7-12 (1983).