[89] Cyanobacterial genetic tools: Current status

808

MOLECULAR GENETICS

[89]

aqueous hybridization buffer at 50, 55, 60 or 65 °, or at 42 ° in presence of formamide at a final concentration of 10, 30, or 50% (v/v). Hybridization in the presence of formamide is preferred because it solves the evaporation problem encountered with aqueous buffer at temperatures up to 60°. It should be noted, however, that the rate of hybridization is 2 times slower in the presence of 50% formamide than in an aqueous solution.16 Filters are prehybridized at the desired temperature and concentration of formamide in a 6× S S C - l x Denhardt's solution 16 containing 15/xg denatured herring sperm DNA solution per milliliter. The denatured probe is then added to the hybridization mix (routinely 1 x 106 cpm/filter) and hybridized to the filter for 20 hr. Filters are then washed 2 times for 10 min in I × SSC, 0. I% SDS at room temperature and 2 times for 15 min in 0.1 × SSC, 0.1% SDS at 42 °. To reduce nonspecific hybridization and give optimal signal-to-noise ratios, an additional high-stringency wash in 0.1 × SSC, 0.1% SDS at 50-65 ° for 5-20 min may be necessary. After the final wash, filters are air dried and autoradiographed. The rate of hybridization can be increased by the addition of dextran sulfate (final concentration of 10%) to the hybridization buffer. Nevertheless, it should be noted that the use of this polymer may lead to increased background. Up to now, hybridizations of the Anabaena PCC 7120 nifD and nifH probes to DNA from all the nitrogen-fixing unicellular and filamentous cyanobacteria tested have been performed in stringent conditions (65 ° in aqueous buffer). 3,4A1,12 Fragments hybridizing to nifK probe 4 and nifS probe (unpublished observations) are observed under less stringent conditions (58-60 ° in aqueous buffer).

[89] C y a n o b a c t e r i a l G e n e t i c Tools: C u r r e n t Status By

JEAN HOUMARD

and NICOLE TANDEAU DE MARSAC

In recent years, cyanobacteria have become increasingly popular, and the development of cyanobacterial genetics has arisen with the new possibilities offered by molecular genetics.l The following tables are a compilation of the information concerning the genetic tools available for various cyanobacteria. The literature dealing with cyanobacterial taxonomy is somewhat confused by different names and numbers appended to the same strain. Table z N. T a n d e a u de M a r s a c a n d J. H o u m a r d , in " T h e C y a n o b a c t e r i a " (P. Fay and C. Van Baalen, eds.), p. 251. Elsevier, A m s t e r d a m , 1987.

METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988by AcademicPress, Inc. All rights of reproduction in any form reserved.

[89]

CYANOBACTERIAL GENETIC TOOLS

809

I lists alternative strain designations and numbers in the different culture collections for the cyanobacteria mentioned in the other tables. Whenever possible, we have referred to specific strains by their number in the Pasteur Culture Collection (PCC). Nearly all cyanobacteria harbor extrachromosomal DNA elements, but, unfortunately, no function has yet been shown to be encoded by any of these plasmids. Table II presents the plasmid content of nearly a hundred cyanobacterial strains in which both the number and the size of these plasmids vary greatly. On the other hand, it is important to avoid DNA restriction when introducing DNA into heterologous strains. Most of the strains examined so far do contain sequence-specific endonucleases. They are listed in Table III together with their specific recognition sequences. Since none of the endogenous plasmids contain selectable genetic markers, a large number of cloning vectors has been developed for the strains which are able to receive DNA either by transformation or by conjugation. The available cyanobacterial cloning vectors are listed in Table IV. Finally, Table V is a compilation of cyanobacterial genes which were cloned up to November I, 1987. Since several multigene families have been recognized in different cyanobacteria, it appears that the nomenclature of cyanobacterial genes will rapidly become confusing. Consequently, we propose the following rules: Gene designations proposed for Escherichia coh"z and/or Bacillus subtilis 3 must be used whenever possible. For functions specifically related to photosynthesis, gene designations employed for photosynthetic bacteria or plants will be used. The designations already used for bacterial genes must be avoided if gene products either have not been identified or are functionally different from the ones which were previously published. Genes involved in the formation of multimolecular complexes (structure, assembly, and/or regulation) or which are part of a given metabolic pathway can be designated by difference capital letters appended to a unique three-lowercase-letter root. The following are given as examples:

apcA to apcZ: genes related to allophycocyanin atpA to atpZ: genes related to the ATPase complex cpcA to cpcZ: genes related to phycocyanin cpeA to cpeZ: genes related to phycoerythrin metA to metZ: genes related to the methionine biosynthetic pathway nifA to nifZ: genes involved in nitrogen fixation 2 B. J. Bachmann, Microbiol. Rev. 47, 180 (1983). 3 p. j. Piggot and J. A. Hoch, Microbiol. Rev. 49, 158 (1985).

810

MOLECULAR GENETICS

[89]

psaA to psaZ: genes related to p h o t o s y s t e m I psbA to psbZ: genes related to p h o t o s y s t e m II Arabic numerals following a gene designation are used to identify the various copies within a multigene family (psbA1, psbA2, psbA3 . . . . ~). Allele numbers o f a given locus must be indicated by a hyphen preceding arabic numerals (atpA-1, cpcB2-1, psbD1-3 . . . . ). Acknowledgments We are deeply grateful to Prof. A. de Waard for compilation of the cyanobacterial restriction enzymes presented in Table III. Our thanks also go to colleagues who provided us with information in advance of publication and to Dr. A. Pugsleyfor critical reading of the manuscript.

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lsoschizomer

Recognition sequence ~

PuuII AvaI BamHl EspI FnuDII

CAGCTG C ~ PyCGPuG GGATCC GCTNAGC CGCG

1 2 1

Synechococcus sp. PCC 7202

Gspl Aqul CelI CelIl Scel

Synechococcus sp. PCC 7418

Ahal

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ccCGG

3

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AcyI DraI

GPu ,~ CGPyC TTT ~ AAA n.d. C ~ CNNGG CCGG CCTNAGG GGTNACC CAGCTG GC ~ TNAGC n.d.

Organism Section I Gloeothece sp. PCC 6909 Synechococcus sp. PCC 7002 Synechococcus sp. PCC 7003

Synechococcus sp. PCC 7942 Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6711 Synechocystis sp. PCC 6906 Section III Microcoleus sp. UTEX 2220

Pseudanabaena sp. PCC 6901 Pseudanabaena sp. PCC 7409 Section IV Anabaena sp. PCC 6309

Anabaena sp. PCC 7108 Anabaena sp. PCC 7122 Anabaena sp. PCC 7937 Anabaena sp. CCAP 1403/1

Anabaena sp. CCAP 1403/9

Anabaena sp. CCAP 1403/11

Anabaena sp. CCAP 1403/12

Endonuclease

Mstl MstlI Pspl Psel Asu! AsulI AsulI1 AstWl Acyl AcylI AorI AvrlI AcaI AcalI AcallI AcalV AocI

MspI MstlI BstEII PoulI

Saul AsuI Asul

Acyl AcyI

AvaI AsulI BamHI MstI Haelll MstlI

Ref. b

1

4 5 6

1 7

TGCGCA CC ~ TNAGG GGNCC GGNCC

8 9 10 1

G ~ GNCC TT ~ CGAA GPuCGPyC GPuCGPyC GPu ~ CGPyC n.d. C ,~ PyCGPuG CCTAGG TTCGAA GGATCC TGCGCA GGCC CC ~ TNAGG

11 12

AoclI

SduI

G C GAGCTC T A

AosI AoslI AoslIl AspHI AspHII

Mstl AcyI SaclI AsulI MstI

TGC ~, GCA GPu ~, CGPyC CCGCGG TTCGAA TGCGCA

12, 13 14 13, 15, 16 16 1

1

17 18 18

(continued)

TABLE II1 (continued)

Organism Anabaena sp. CCAP 1403/13f

Endonuclease

Isoschizomer

Recognition sequence"

Ref. h

Avail

G $ GAcc

Calothrix sp. PCC 7101

Aflll Afllll AinI Ainll AspTAil AspTAilI AspTAilll Ttel

Pstl BamHl Pstl BamHl Haelll Haelll

C ~ TTAAG A ~ CPuPyGT CTGCAG GGATCC CTGCAG GGATCC GGCC GGCC

20

Calothrix sp. PCC 7601

FdiI

Avail

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21

Calothrix sp. CCAP 1410/5 Cylindrospermum sp. UTEX 1828

Fdill Cscl Clcl Clcll

Mstl Sacll Pstl Mstl

TGC $ GCA CCGCGG CTGCAG TGCGCA

22 1

CliI

Avail

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23

ClitI Clilll Nsp(6302)l Nsp(6310)l Nsp(6310)II Asp(6411)I

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13

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Asull

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15

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15

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CPyCGPuG GGTNACC C ~ PyCGPuG

25

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25, 26

AvallI Asp(7119)I

Aval

ATGCAT CPyCGPuG

27 15

Asp(7119)11

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Anabaena sp. CCAP 1446/1a Anabaena sp. TAi

Cylindrospermum sp. UTEX 2014 Nostoc sp. PCC 6302 Nostoc sp. PCC 6310 Nostoc sp. PCC 6411

Nostoc sp. PCC 6705

Afll

Mstl Aval

NspBll Nostoc sp. PCC 6719

Nostoc sp. PCC 6720 Nostoc sp. PCC 7118

Nostoc sp. PCC 7119

Nostoc sp. PCC 7120

Nostoc sp. PCC 7413

Nostoc sp. PCC 7524

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Avail

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NspHI

NspCl

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NspHll

A vail

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NspCl

NspHl

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Sdul

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19

I 1

1, 13 13, 24 15

15

1

15

15

28

(continued)

822

TABLE Ill (continued)

Organism

Endonuclease

Isoschizomer

Recognition sequence"

Ref)

NspCIII NspCIV NspCV NspMACI NliI

Aval AsuI Asull Bglll Aval

CPyCGPuG GGNCC TTCGAA A J, GATCT CPyCGPuG

NlilI

Avail

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NmuI

Aval

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NmuII

Avail

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Avail

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13, 23

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Nsp23-9Bl

Avail

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1, 13

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Nsp78-12Bl NspUM-3I NspUM-3II NspUM-311I NspUM-31V

Asul Mstl AsuI

GGNCC TGCGCA GGNCC n.d. n.d.

1, 13 13, 23

FspI Fspll MlaI

MstI Asull Asull

TGCGCA TTCGAA TT $ CGAA

30

Nostoc sp. PCC 8009 Nostoc linckia

Nostoc sp. M-131-G

Section V Fischerella sp. PCC 73103 Fischerella sp. PCC 7414

29 15

15

31

a Arrows indicate the cleavage site in the recognition sequence, n.d., Not determined. b References to Table III: (1) F. Call6ja and A. de Waard, personal communication; (2) R. H. Lau and W. F. Doolittle, FEBS Lett. 121, 200 (1980); (3) P. R. Whitehead and N. L. Brown, Arch. Microbiol. 141, 70 (1985); (4) P. R. Whitehead and N. L. Brown, FEBS Lett. 143, 296 (1982); (5) M. L. Gallagher and W. F. Burke, Jr., FEMS Microbiol. Lett. 26, 317 (1985); (6) F. Call6ja, N. Tandeau de Marsac, T. Coursin, H. van Ormondt, and A. de Waard, Nucleic Acids Res. 13, 6745 (1985); (7) F. Call6ja, B. M. M. Dekker, T. Coursin, and A. de Waard, FEBS Lett. 178, 69 (1984); (8) T. R. Gingeras, J. P. Milazzo, and R. J. Roberts, Nucleic Acids Res. 5, 4105 (1978); (9) 1. Schildkraut, unpublished observations; (10) B. Mulligan and M. Szekeres, unpublished observations; (11) S. G. Hughes, T. Bruce, and K. Murray, Biochem. J, 185, 59 (1980); (12) A. de Waard and M. Duyvesteyn, Arch. Microbiol. 128, 242 (1980); (13) C. P. Wolk and J. Kraus, Arch. Microbiol. 131, 302 (1982); (14) A. de Waard, J. Korsuize, C. P. van Beveren, and J. Maat, FEBS Left. 96, 106 (1978); (15) M. G. C. Duyvesteyn, J. Korsuize, A. de Waard, A. Vonshak, and C. P. Wolk, Arch. Microbiol. 134, 276 (1983); (16) E. C. Rosenvold and A. Honigman, Gene 2, 273 (1977); (17) A. de Waard, C. P. van Beveren, M. Duyvesteyn, and H. van Ormondt, FEBS Lett, 101, 71 (1979); (18) A. de Waard, personal communication; (19) P. R. Whitehead and N. L. Brown, J. Gen. Microbiol. 131,951 (1985); (20) B. Siegelman, unpublished observations; (21) C. A. M. J. J. van den Hondel, R. W. van Leen, G. A. van Arkel, M. Duyvesteyn, and A. de Waard, FEMS Microbiol. Lett. 16, 7 (1983); (22) M. G. C. Duyvesleyn, J. Korsuize, and A. de Waard, Plant Mol, Biol. 1, 75 (1981); (23) C. Karreman and A. de Waard, personal communication; (24) E. Flores and C. P. Wolk, personal communication; (25) S. G. Hughes and K, Murray, Biochem, J. 185, 65 (1980); (26) J. G. Sutcliffe and G. M. Church, Nucleic Acids Res. 5, 2313(1978); (27) G. Roizes, P.-C. Nardeux, and R. Monier, FEBS Lett. 104, 39 (1979); (28) J. Reaston, M. G. C. Duyvesteyn, and A. de Waard, Gene 20, 103 (1982); (29) R. H. Lau, L. P. Visentin, S. M. Martin, J. D. Hofman, and W. F. Doolittle, FEBS Lett. 179, 129 (1985); (30) M. Szekeres (New England Biolabs), personal communication (1985); (31) M. Duyvesteyn and A. de Waard, FEBS Lett. 111, 423 (1980). 823

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