Genetic tools for cyanobacteria - Springer Link

Appl Microbiol Biotechnol (2002) 58:123–137 DOI 10.1007/s00253-001-0864-9

MINI-REVIEW

O. A. Koksharova · C. P. Wolk

Genetic tools for cyanobacteria

Received: 17 August 2001 / Received revision: 5 October 2001 / Accepted: 5 October 2001 / Published online: 1 December 2001 © Springer-Verlag 2001

Abstract Cyanobacteria are oxygenic photosynthetic bacteria that have been used increasingly to study diverse biological processes, including photosynthesis and its regulation; cell differentiation and N2 fixation; metabolism of nitrogen, carbon, and hydrogen; resistance to environmental stresses; and molecular evolution. Many vectors and other genetic tools have been developed for unicellular and filamentous strains of cyanobacteria. Transformation, electroporation, and conjugation are used for gene transfer. Diverse methods of mutagenesis allow the isolation of many sought-for kinds of mutants, including site-directed mutants of specific genes. Reporter genes permit measurement of the level of transcription of particular genes, and assays of transcription within individual colonies or within individual cells in a filament. Complete genomic sequences have been obtained for the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803 and the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120. Genomic sequence projects are under way for Nostoc punctiforme strain PCC 73102 (ATCC 29133) and strains of the unicellular genera, Synechococcus, Prochlorococcus, and Gloeobacter. Genomic sequence data provide the opportunity for global monitoring of changes in genetic expression at transcriptional and translational levels in response to variations in environmental conditions. The availability of genomic sequences accelerates the identification, study, modification and comparison of cyanobacterial genes, and facilitates analysis of evolutionary relationships, including the relationship of chloroplasts to ancient cyanobacteria. The many available genetic tools enhance the opportunities for possible biotechnological applications of cyanobacteria. O.A. Koksharova · C.P. Wolk (✉) MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA e-mail: [email protected] Tel.: +1-517-3532049, Fax: +1-517-3539168 Permanent address: O.A. Koksharova, N. Vavilov Institute of General Genetics, Moscow 117809, Russia

Introduction Cyanobacteria, ancient relatives of chloroplasts, are outer membrane-bearing, chlorophyll a-containing, photosynthetic bacteria that carry out photosynthesis much as do plants. Cyanobacteria are believed to have been responsible for introducing oxygen into the atmosphere of primitive Earth. A small fraction of the cells of certain cyanobacteria may differentiate into heterocysts, in which dinitrogen (N2) fixation can take place in an oxygen-containing milieu. Cyanobacteria are capable of growth, and in some cases differentiation, when provided with little more than sunlight, air, and water. Their potentialities are being enhanced by the availability of genetic tools and genomic sequences. During the past half century, cyanobacteria have been used increasingly to study, among other topics, photosynthesis and its genetic control; photoregulation of genetic expression; cell differentiation and N2 fixation; metabolism of nitrogen, carbon, and hydrogen; resistance to environmental stresses; and molecular evolution. The availability of powerful genetic techniques allows the biotechnological application of cyanobacteria to produce specific products (including photosynthetic pigments and molecular hydrogen), to biodegrade organic pollutants in surface waters, to control mosquitoes, and for other purposes. Genetic analysis of cyanobacteria has been thoroughly and excellently reviewed by Thiel (1994). Elhai et al. (1990) and Elhai (1994) have reviewed ways in which to approach application of genetic techniques to analysis of a cyanobacterium of one’s choice. Between their discussions and ours, there have been new developments, but none as far-reaching as the availability of genomic sequences of unicellular and differentiating, filamentous cyanobacteria.

Means of gene transfer Transformation and electroporation The single most important requirement for genetic manipulation is genetic transfer. Gene transfer can be per-

124 Table 1 Cyanobacteria to which DNA has been mobilized from Escherichia coli and/or transformed by electroporation Sectiona and genus

Section I Synechococcus

Synechocystis

Strains transformed by: conjugation(C), electroporation(E)b

Referenceb

Comments

R2(1C, 2E), PCC 6301(5C), and 7942(5C); marine strains WH7803(6C), WH8102(6C), WH8103(6C), NKBG15041c(3,4,8C), NKBG042902(2,3C), NKBG15031C(3C), PCC 7335(4C), NKBG15031a(4C), NKBG040606B(3C), NKBG040607(3C); S. elongatus(7C,7E)

1Wolk et al. 1984; 2Matsunaga et al. 1990; 3,4Sode et al. 1992a, b; 5Marraccini et al. 1993

Conjugation is substantially more efficient than transformation (Tsinoremas et al. 1994)

PCC 6714(1,3C) and 6803(3C); marine strain 7a(2C)

and Mermet-Bouvier et al. 1993; 6Brahamsha 1996 (C); 7Mühlenhoff and Chauvat 1996 (C,E); 8Yu et al. 2000 (C)

1Kreps et al. 1990 (C); 2Sode et al. 1992a, b; 3Marraccini et al. 1993

and Mermet-Bouvier et al. 1993 Section II Chroococcidiopsis

Strains 029(C,E), 057(C) and 123(C,E)

Billi et al. 2001

Section III Plectonema

UTEX 596(1C) and IAM-M101(2E)

1Tuli et al. 1990 and Vachhani et al. 1993; 2Fujita et al. 1992

Pseudanabaena

NKBG040605C (C)

Sode et al. 1992a

PCC 7120(1C), PCC 7118 (1C), ATCC 29413 (PCC 7937)(3C), M131(1C,2E), 90(4E)

1Wolk et al. 1984; 2Thiel and Poo 1989; 3Murry and Wolk 1991; 4Rouhiainen et al. 2000

Nostoc

PCC 6310(1C), 7107(1C), 7121(2C,2E), 73102 (ATCC 29133) (1C)

1Flores 2Moser

Fremyella/Calothrix

PCC 7601(1,2C,1E)

1Chiang 2Cobley

Section V Fischerella muscicola

UTEX 1829 (C)

Flores and Wolk 1985

Section IV Anabaena

a Rippka et al. 1979 b Superscript numbers

and Wolk 1985; et al. 1993

et al. 1992; et al. 1993

Desiccation-tolerant

For PCC 7121, electroporation was more efficient than conjugation (Moser et al. 1993) Electroporation mutagenic (Bruns et al. 1989)

Results only presumptive

in column 2 refer to references in column 3, of the same row, that have identical superscript numbers

formed by transformation, conjugation, and transduction. As discovered by Shestakov and his colleagues (Shestakov and Khyen 1970; Grigorieva and Shestakov 1982), a few cyanobacteria are naturally transformable by exogenous DNA. As a result, Synechocystis sp. strain PCC 6803 (hereinafter, Synechocystis), which is capable of “lightactivated” growth in the dark with glucose (Anderson and McIntosh 1991), and to a lesser extent Synechococcus, have played a major role in analysis of cyanobacterial photosynthesis. Circadian rhythms have been studied extensively in Synechococcus. A unicellular cyanobacterium, Agmenellum quadruplicatum strain PR-6, also known as Synechococcus sp. strain PCC 7002, also

proved transformable (Stevens and Porter 1980). The most elegant example of abundant transformation was reported by Dzelzkalns and Bogorad (1988), who demonstrated transformation by individual slices of a lowmelting-point electrophoresis gel of HindIII-restricted wild-type DNA that were melted and dotted onto a lawn of a mutant. In conjunction with the known sequence of PCC 6803 DNA, this technique allows identification of a specific restriction fragment that complements a mutation (Vermaas 1998; Kufryk and Vermaas 2001). Why transformation is possible with several unicellular strains but not more generally is unclear. Extracellular nucleases, frequently encountered in heterocyst-forming, filamen-

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tous strains (Wolk and Kraus 1982), may be part of the reason. Molecular details of natural transformation in cyanobacteria are only now being elucidated. Yoshihara et al. (2001) have shown that a product of Synechocystis ORF slr0197 is required for the uptake of DNA. Electroporation, an artificial form of transformation by exogenous DNA, has been used to transform diverse cyanobacteria (Table 1, “Comments”), but may be weakly mutagenic (Bruns et al. 1989; Mühlenhoff and Chauvat 1996). Despite intensive study of various cyanophages, including lysogenic strains (Koz’yakov et al. 1972; Rimon and Oppenheim 1975; Sherman and Brown 1978; Hu et al. 1981; Mendzhul et al. 1985; Sarma and Kaur 1997), no viral vector has been found capable of transducing any cyanobacterium. Conjugation To date, the most general means for transforming cyanobacteria has proven to be conjugation, mediated by the broad-host-range, P-incompatibility-group plasmid RP4 and its close relatives. Although there is no evidence that this plasmid can itself replicate in cyanobacteria, it has been shown to mobilize plasmids to a diversity of cyanobacteria (Table 1), including marine strains. DNA to be transferred can be made mobilizable by provision of an “origin-of-transfer” (oriT) fragment of RP4. An RP4-encoded enzyme nicks its own doublestranded DNA at oriT, providing single-stranded DNA for transfer. RP4 can also mobilize DNA that bears the oriT region of broad-host-range plasmid RSF1010. Alternatively, one can include the oriT region (also known as bom, basis of mobilization) from plasmid pBR322 (a region that has been lost from pUC19 and closely related plasmids), provided that one includes, in the donor cell, the mobilization genes of colicinogenic plasmids ColD, ColK, or ColE1 (Finnegan and Sherratt 1982).

Effects of restriction and how to elude them Once the barriers of the outer membrane, cell wall and plasmalemma have been breached, a formidable barrier to stable transformation often remains: degradation of transferred DNA by restriction endonucleases native to the recipient strain. Although some strains – perhaps Synechococcus sp. strain PCC 7942 and Nostoc PCC 73102 – may be devoid of such endonucleases, others have them in abundance. Nostoc PCC 7524, for example, has four that are isoschizomers of Bsp1286I (G[g/a/t] GC[c/t/a]C), AvaI (CyCGrG), Sau96I (GGnCC), and AsuII (TTCGAA), and a fifth that targets rCATGy (Reaston et al. 1982). For Anabaena sp. strain PCC 7120 (hereinafter, Anabaena 7120), it has been shown that: “for low numbers of sites, the efficiency of conjugal transfer decreased as an exponential function of the number of unprotected sites” (Elhai et al. 1997; see also Cobley et al. 1993 and Moser et al. 1993). Thiel and Poo

(1989) showed that a single unmethylated AvaII site reduced transfer by electroporation by a factor of 100. Therefore, transfer is likely to be enhanced, perhaps greatly, if it is possible to identify the restriction endonucleases of a strain to which genetic transfer is sought, and if the DNA to be transferred can be appropriately methylated. The result may make the difference between observing and not observing transfer.

Plasmid vectors Plasmid vectors either can replicate in one’s host of choice, or cannot. Each type of behavior has experimental uses. Replicating vectors can potentially express an exogenous gene in a new host. Such vectors may have either broad or narrow host range. Derivatives of plasmid RSF1010, for example, are capable of replicating in Synechocystis (Marraccini et al. 1993; Mermet-Bouvier et al. 1993) as well as in Anabaena 7120 (Thiel 1994), Synechococcus (including a thermophilic strain) and Pseudanabaena (Sode et al. 1992a; Mühlenhoff and Chauvat 1996), and warrant testing for replication in a strain new to genetic analysis. In contrast, plasmids based on the 6.3-kb plasmid pDU1 from Nostoc 7524 can replicate in several Section-IV strains (sensu Rippka et al. 1979; Table 1) and a Section-II strain (Billi et al. 2001), but have not been reported to replicate in Section-I strains. Plasmid vectors that can be transferred to, but cannot replicate in, a new host are ideal for transporting DNA that must either transpose (e.g., a transposon, used for mutagenesis) or integrate (e.g., by homologous recombination) in order to be stably maintained. Thiel (1994) has described a variety of each for cyanobacteria.

Markers Host cells that contain the transferred DNA are usually identified by transferring a gene whose product has the potential to inactivate an antimetabolite, such as an antibiotic. When cultured in the presence of the antimetabolite, only those cells to which the gene has been transferred should grow. First, however, one must identify the concentration range above which that antimetabolite prevents growth of the new host. Because cyanobacteria are, structurally, Gram-negative bacteria, they have most often been used with antibiotics and antibiotic-resistance genes that are known to be effective for that group of bacteria. These antibiotics include neomycin and kanamycin and the corresponding neomycin phosphotransferase (npt)-encoding genes from transposons Tn5 and Tn903, and streptomycin and spectinomycin and the corresponding aminoglycoside adenyltransferase (aadA)encoding genes from Tn7 (Wolk et al. 1993) and the omega interposon (Prentki et al. 1991). Bleomycin, although expensive, is sufficiently active to be costeffective with Anabaena 7120. Chloramphenicol and chloramphenicol acetyl transferase (cat) have proven

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useful for unicellular cyanobacteria (Golden and Sherman 1983; Chauvat et al. 1986) and Anabaena sp. strain 90 (Rouhiainen et al. 2000). Antibiotics such as tetracycline and rifampicin, which are light-sensitive, are of much reduced utility for cyanobacteria, unless one screens out the wavelengths of light to which the antibiotics are sensitive. Because the ampicillin-degrading enzyme β-lactamase is secreted, cells of a conjugal donor strain that bears a β-lactamase-encoding gene may protect cells of a potential recipient strain even if the gene has not been transferred. Therefore, a plasmid that encodes β-lactamase may be of great use for isolation of transformants, in the narrow sense of that term, or to confirm conjugal transfer, but of much less use for initial selection of exconjugants. Conveniently usable antibiotic-resistance cassettes were described by Elhai and Wolk (1988). The only antibiotic heretofore used for selection in cyanobacteria that is normally used only with Gram-positive bacteria, erythromycin, offers fine selection in cyanobacteria (Shestakov and Khyen 1970; Grigorieva and Shestakov 1982; Wolk et al. 1984; Shen et al. 1993), suggesting that additional determinants normally used specifically for Gram-positives [e.g., thiostrepton, hygromycin (see Ohkawa et al. 2000), puromycin, and apramycin] may be effective with cyanobacteria. Antimetabolites that target cyanobacterial photosynthesis, including atrazine or 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU), which block electron transfer from photosystem II; metronidazole, which interacts with electron transfer from photosystem I; and nitrophenolic herbicides (Golden and Sherman 1984; Polukhina et al. 1985; Ajlani et al. 1989; Koksharova and Shestakov 1990; Bartsevich and Shestakov 1995; Elanskaya et al. 1998; Narusaka et al. 1998) have been used to select resistant mutants, but the genes that confer resistance have been little tested as markers for gene transfer. In the presence of the wild type gene, atrazine resistance is recessive (Pecker et al. 1987). Gabaculin, which inhibits the synthesis of δ-aminolevulinic acid, and thus of chlorophyll, and a gene that confers resistance to gabaculin, can be used for selection (Allison et al. 1997). Similarly, “bleaching herbicides” (Hirschberg and Chamovitz 1994), such as norflurazon (Martinez-Férez and Vioque 1992), which inhibit synthesis of carotenoids, in combination with a mutant target gene that is functional but resistant to the herbicide, can be used for selection. It sometimes helps to include a screenable gene such as one that encodes luciferase (Engebrecht et al. 1983; Foran and Brown 1988) or green fluorescent protein (GFP) (Crameri et al. 1996), because luminescence or fluorescence provides an easy and sensitive test of gene transfer to a presumptive recipient (Schmetterer et al. 1986; Murry and Wolk 1991; Billi et al. 2001).

Means of mutagenesis Mutations are central to genetics, both to identify unknown genes that are involved in a particular process, and to elucidate the function of known genes. Spontane-

ous mutants can be isolated (Astier et al. 1984; Montesinos et al. 1997), or mutations can be generated physically [e.g., with UV light (Singh and Tiwari 1969; Herdman and Carr 1972; Wolk et al. 1988; Vega-Palas et al. 1990)], chemically [e.g., with methyl sulfonates or N-methyl-N′-nitro-N-nitrosoguanidine (Herdman and Carr 1972; Currier et al. 1977; Vega-Palas et al. 1990; Buikema and Haselkorn 1991)] or biologically (e.g., with transposons or insertional mutagenesis). Because cyanobacteria often have on the order of 10 copies of their chromosome per cell (Herdman et al. 1979) and – for filamentous strains – many linked cells, and transposon mutants can be selected, random mutagenesis with transposons has often facilitated the search for mutants (Borthakur and Haselkorn 1989; Ernst et al. 1992; Wolk et al. 1991). Transposons have the additional advantage that they “tag” the genomic site of the mutation, facilitating recovery of the wild-type form of the mutated locus, whereas untagged mutations are normally cloned by complementation of a mutation with a library of chromosomal fragments, e.g., the ca. 20,000 cosmid-clone library of Buikema and Haselkorn (1991). Mutagenesis with the β-lactamase-encoding transposon, Tn901, played a major role in the spread of cyanobacterial transformation technology westward from Russia: van den Hondel et al. (1980) introduced Tn901 into a native plasmid of Synechococcus, and transformed the resulting derivative into Synechococcus. Kuhlemeier et al. (1981) and Tandeau de Marsac et al. (1982) thereupon used transposition of Tn901 from the plasmid to the chromosome as a means of mutagenizing chromosomal loci. Later, Borthakur and Haselkorn (1989) showed that transposon Tn5 can transpose in Anabaena. However, use of that transposon became much more effective with the introduction of variants, e.g., Tn5–1058 and its progeny that had (1) a much stronger promoter driving the antibiotic-resistance operon, (2) enhanced transposition, and (3) an Escherichia coli origin of replication within the transposon. The latter variation allows the cloning of sequences contiguous with the transposon, by cutting genomic DNA with a restriction endonuclease that does not cut within the transposon, recircularizing in vitro, and transforming E. coli with the resulting ligation mixture (e.g., Wolk et al. 1991; Ernst et al. 1992; Black et al. 1993; Cai and Wolk 1997). More recently, transposon Tn5–692 (and derivatives thereof) has increased the transposition rate ca. 100-fold, providing large numbers of transposon mutants of Anabaena variabilis strain ATCC 29413 (PCC 7937) and Synechococcus 7942 (C.P. Wolk and O.A. Koksharova, unpublished), but at the price that repeated transposition may sometimes be observed. In the interim, Synechocystis and Synechococcus were also mutagenized by reintroduction of fragments of their own DNA coupled to a selective marker (“random cassette mutagenesis”; Labarre et al. 1989; Dolganov and Grossman 1993; Tsinoremas et al. 1994; see also Broedel and Wolf 1990), thus also tagging the sites of mutation. Particular genes can be mutagenized by homologous recombination, either to observe the effect of inactivat-

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ing that gene or to observe the effect of small changes in the sequence of the gene. Williams and Szalay (1983) were the first to demonstrate homologous recombination in cyanobacteria, and Golden and Wiest (1988) first achieved homologous recombination in Anabaena. Single-crossover homologous recombination within a gene inactivates the gene only if the fragment recombined contains neither the start nor end of the transcriptional unit. To obtain mutation with a more extensive fragment requires double reciprocal recombination (Williams and Szalay 1983; Golden et al. 1986, 1987; Golden1988), an approach that has permitted the characterization of the function of many cyanobacterial genes (e.g., Pakrasi et al. 1988; Chitnis et al. 1989; Nyhus et al. 1993; Bhalerao et al. 1994; Ghassemian and Straus 1996; Boison et al. 1998; Koksharova et al. 1998; Marin et al. 1998; Yoshihara et al. 2001). Because the phenotype of a transposon mutant can result from the combined effects of a spontaneous mutation elsewhere in the genome and antibiotic resistance conferred by the transposon, it is standard practice to reconstruct transposon mutants by homologous recombination to test whether the phenotype is, in fact, due only to insertion of the transposon. Depending on the strain of cyanobacterium studied, double recombination may be frequently or infrequently obtained. The combined presence of the sacB gene and high concentrations of sucrose added to solid medium is lethal. Use of sacB as a means of selecting for a second recombination proved very helpful for Anabaena 7120 (Cai and Wolk 1990; Black et al. 1993). By introducing a mutant allele by single-crossover recombination, and then using sacB to select for one of two resulting copies of the introduced allele, it is possible to replace the wildtype with the mutant allele without the introduction of an antibiotic-resistance determinant (Andersson et al. 2000). Such an approach can be very useful if, for example, one wants to derive a strain that will be extensively used (e.g., if one were to want to inactivate the gene for a restriction endonuclease) without precluding the use of a particular antibiotic-resistance determinant for later constructions. Entire genes have been deleted, and recombinant forms of the original gene then introduced to determine, for example, the effects of single or multiple amino acid changes on the function of the encoded protein (e.g., Debus et al. 1988; Vermaas et al. 1990; Boerner et al. 1992; and recently, Ermakova-Gerdes and Vermaas 1999; Rosenberg et al. 1999; Tichy and Vermaas 1999; Ermakova-Gerdes et al. 2001). Analysis of pseudorevertants, in which a suppressor mutation maps to a locus that differs from that of the primary mutation, can identify genes whose products interact with the protein affected by the primary mutation (Tichy and Vermaas 2000; Kufryk and Vermaas 2001). A PCR-based method was described recently for the efficient construction of targeted gene disruptions and gene fusions in Synechocystis (Taroncher-Oldenburg and Stephanopoulos 2000). Vermaas (1996, 1998) has summarized approaches to mutagenizing Synechocystis.

Despite protracted opportunity for segregation, it often appears impossible to inactivate all of the multiple copies of a particular gene in cyanobacterial cells. Two approaches have been taken to determine whether such a gene is indeed essential. Both approaches involve driving the gene in question by a regulated promoter. In one variation (Poncelet et al. 1998; Pieulle et al. 2000), a copy of the gene promoted by a temperature-regulated, coliphage lambda-derived promoter is placed into the cell in a replicating plasmid. At a temperature appropriate to maintain the lambda-derived promoter active, the copy of the gene driven by the wild-type promoter is insertionally inactivated and the mutation segregated. The temperature is then changed so that the sole remaining copy of the gene is no longer transcribed, and the effect of transcriptional inactivation is assessed. In a second procedure (Callahan and Buikema 2001), the natural promoter of the gene is replaced by the copper-regulated, petE promoter; so long as copper is present, the gene is maintained active. Copper is then removed, and the effect of transcriptional inactivation of the gene becomes evident.

Reporters Reporter genes enable very sensitive measurement of the level of transcription of a particular gene (e.g., Wolk et al. 1993; van Thor et al. 1999; Kunert et al. 2000; reviews by Wolk 1996, and Fernández-Piñas et al. 2000), permitting assay of transcription within particular colonies on a Petri plate (Wolk et al. 1991; Kondo and Ishiura 1994; Kondo et al. 1994; Cai and Wolk 1997; Schwartz et al. 1998; Zhu et al. 1998) or within particular types of cells in a filament. For example, in studies of the mechanisms that underlie the positions at which heterocysts form, it is crucial to know whether particular genes are transcribed in all cells, or only within differentiating or mature heterocysts, or only in vegetative cells of heterocyst-bearing filaments. Such determinations have proven possible by use of luxAB, encoding luciferase (Elhai and Wolk 1990; Black et al. 1993; Wolk et al. 1993; Fernández-Piñas et al. 2000, for review); gfp, encoding GFP (Haselkorn 1995; Yoon and Golden 1998; Callahan and Buikema 2001; Xu and Wolk 2001); and lacZ, encoding β-galactosidase (Thiel et al. 1995). Fernández-Piñas and Wolk (1994) demonstrated the use of luxC, luxD and luxE to synthesize the substrate for luciferase, thereby preventing the toxicity experienced upon exogenous addition of that substrate. As noted above, expression of a reporter gene in an axenic recipient convincingly demonstrates gene transfer (Murry and Wolk 1991; Billi et al. 2001). Very weak expression of a promoter can be amplified, permitting localization of expression, as follows. The promoter is coupled transcriptionally to the gene that encodes coliphage T7 RNA polymerase and a promoter recognized by that polymerase is placed upstream from the luxAB genes (Wolk et al. 1993). By use of luxAB to report on circadian clock-

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controlled expression within individual colonies of Synechococcus 7942, Kondo and associates (Kondo et al. 1993; Liu et al. 1995; Katayama et al. 1999) identified and isolated colonies exhibiting mutations in clock genes. When placed in a transposon, luxA and luxB have been used to identify transpositions downstream from promoters that are regulated by a change in particular environmental variables, such as ambient nitrogen supply, temperature, or salt concentration (Wolk et al. 1991; Cai and Wolk 1997; Schwartz et al. 1998). Second-site mutations that result in non- or constitutively-luminescent phenotypes identify regulatory loci (e.g., Schwartz et al. 1998; Zhu et al. 1998). Promoter probe vectors constructed for Synechocystis (Kunert et al. 2000) allow the transcriptional fusion of promoter sequences with luxAB and gfp reporter genes and their stable integration into a neutral site of the Synechocystis chromosome. Each reporter has advantages and disadvantages. Bacterial luxAB has high sensitivity and low background, but requires oxygen and expensive instrumentation. Clear images are difficult to obtain at high cell density. Use of LacZ as a reporter has a long history for quantitative studies. When used with a fluorescent β-galactoside substrate for localization of genetic expression, cells must be permeabilized by fixation with glutaraldehyde (Thiel et al. 1995). GFP requires oxygen for formation and stabilization of the fluor, hours may be required for maturation of the fluor, and photobleaching can prove a problem. As with lacZ, high cell densities are not problematic, but for both of these reporters, the background caused by the natural fluorescence of cyanobacterial cells limits the sensitivity of the assay. All three kinds of reporter genes work well; the researcher needs only to choose which is best for particular experiments. The cat gene, which encodes chloramphenicol acetyl transferase, has also been used as a reporter in cyanobacteria (Ferino and Chauvat 1989; Marraccini et al. 1993, 1994; Bauer and Haselkorn 1995), as has uidA, which encodes β-glucuronidase (GUS; Casey and Grossman 1994; Dolganov and Grossman 1999).

Genomic sequences A genomic sequence is an, as yet, only slightly comprehensible encyclopedia, created by nature through billions of years of evolution. As the language of such sequences becomes better understood, much more should be discerned about the proteins, metabolic regulation, evolution and ecological relationships of cyanobacteria. Genomic sequences have been finalized for the unicell, Synechocystis (3.57 Mb plus sequenced plasmids) and the filamentous, heterocyst-forming cyanobacterium, Anabaena 7120 (6.41 Mb plus sequenced plasmids; Kaneko et al. 1996a, b, 2001b; Kaneko and Tabata 1997; http://www.kazusa.or.jp/cyano/), and are being finalized for two ecotypes (MED4, a high-light-adapted ecotype from the Mediterranean Sea, and MIT9313, a low-lightadapted ecotype from the Gulf Stream) of the unicellular

cyanobacterium Prochlorococcus (http://www.jgi.doe.gov/ JGI_microbial/html/prochlorococcus/ prochlo_pickastrain. html). Extensive sequence data are available for Nostoc 73102, a strain that forms heterocysts, akinetes, hormogonia, and symbiotic associations with the hornwort Anthoceros and the cycad Macrozamia (http://www.jgi.doe. gov/JGI_microbial/html/nostoc/nostoc_homepage.html). In addition, sequencing projects are under way for Gloeobacter and several strains of Synechococcus (Bryant et al. 2001; Holtman et al. 2001; Kaneko et al. 2001a). Most of these strains can be manipulated genetically and have been the subject of many of the studies cited above. The available genomic sequence data are extremely useful for finding, studying, modifying, and comparing cyanobacterial genes. Cyanobacterial genomic data have already been used to identify regulatory and structural genes (e.g., Hein et al. 1998; Vinnemeier and Hagemann 1999; Ochoa de Alda and Houmard 2000; Zhulin 2000), to investigate molecular mechanisms of natural genetic transformation (Yura et al. 1999; Yoshihara et al. 2001), and to analyze evolutionary events (Bölter et al. 1998; Reumann et al. 1999; Herdman et al. 2000; Rujan and Martin 2001). For sequencing projects, it is helpful to have large clones – called bridging clones – that span the content of a number of sequencing clones, so as to test the distance between the ends of the bridges predicted by assembling contigs of individual sequences. The bacterial artificial chromosome (BAC) vector used for construction of bridging clones for the Anabaena 7120 sequencing project was constructed so that clones that it bears can be mobilized to, and selected in, that host, for complementation of unmarked mutations (Kaneko et al. 2001a). In addition, broad-host-range, RSF1010-based vectors (see above) have been designed with a polylinker identical to that of the BAC vector, to facilitate subcloning from the BAC to the RSF1010-based vector. Whereas the BAC vector would replicate in the cyanobacterium only upon recombination with the genome, the RSF1010-based vector can replicate autonomously (C.P. Wolk, unpublished). Sequence analysis of Prochlorococcus, the smallest and most abundant photosynthetic organism in the ocean and presumably on Earth (Partensky et al. 1999), may illuminate the evolution of cyanobacteria and chloroplasts. Strains of Prochlorococcus have a genome size of approximately 2 Mbp, lack the phycobilisome antennae that are characteristic of cyanobacteria, and possess an unorthodox pigment composition of divinyl derivatives of chlorophyll a and b, α-carotene, zeaxanthin and a pigment related to chlorophyll c. The low-light-adapted Prochlorococcus strain SS120 contains a type of phycoerythrin (reviewed by Partensky et al. 1999). Strain SS120 possesses seven transcribed genes that encode different chlorophyll a/b-binding proteins, suggesting that possession of multiple genes for an antenna protein may enhance survival at very low levels of light (Garczarek et al. 2000). Because Prochlorococcus contributes 30–80% of the total photosynthesis in the oligo-

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trophic oceans, it plays a significant role in the global carbon cycle and in the climate of our planet. The availability of the complete genomic sequence of two of these microbes should accelerate understanding of the regulation of these globally important processes and of the ecological diversity of the two strains (Rocap et al. 2001). The availability of genomic sequences has drastically changed certain strategies for genetic analysis of the sequenced cyanobacteria. For example, one need only sequence outward from a transposon to identify its site of insertion; one need only sequence a fragment of a protein to identify the gene that encodes it; and once a DNA-binding site is identified, all such sites can be determined. All genes that encode particular types of protein (e.g., a sigma factor or a protein-histidine kinase) can be identified, and their functions determined by systematically mutating them. For example, Ochoa de Alda and Houmard (2000) identified Synechocystis genes presumptively encoding cyclic nucleotide-binding proteins; by systematically inactivating genes for protein-histidine kinases and performing extensive random mutagenesis of the Synechocystis genome by cassette mutagenesis, Suzuki et al. (2000, 2001) could identify Synechocystis genes involved in sensing and responding to low temperatures; Ohkawa et al. (2000) demonstrated that Synechocystis has multiple NAD(P)H dehydrogenase complexes with specific roles; and Funk and Vermaas (1999) found five genes that are predicted to encode small Cab-like proteins, a new group of cyanobacterial proteins that may be involved in transient binding of pigments. That Anabaena 7120 possesses nine DNA methyltransferases was deduced by an analysis of genomic sequences and enzymatic activities (Matveyev et al. 2001). Of the nine, four are associated with type-II restriction enzymes, four function outside of a restriction/modification system, and one could not be classified. The availability of genomic sequence information led to the development of methods for its analysis (Hirosawa et al. 1997). For example, horizontally transferred genes and highly expressed genes can be presumptively identified in silico (Mrázek et al. 2001). High-density microarrays of clones that cover much of the genome of Synechocystis are available from the company TaKaRa Shuzo (in the US, from Panvera, Madison, Wis.), and permit assessment of the transcriptional responses of most of the organism’s genes to environmental variation, e.g., an increase in light intensity (Hihara et al. 2001). Microarrays are planned also for Anabaena 7120. The complete genomic sequence of a small number of cyanobacteria is a valuable resource for study of other cyanobacteria. For example, peptides from a presumptively akinete-specific protein isolated from Anabaena cylindrica showed similarity to peptides in a protein predicted from Anabaena 7120, facilitating the isolation of a corresponding gene from Anabaena variabilis ATCC 29413 (R. Zhou and C.P. Wolk, unpublished). Although cyanobacteria lack flagella, some can glide when in contact with a solid surface and a few are

known to swim in liquid. Once a conceptual linkage was made between pili genes and motility in Synechocystis (Bhaya et al. 1999), other pili-related genes identified by making use of the genomic sequence were mutagenized, confirming and extending the relationship of motility to thick pili (Bhaya et al. 2000a; Yoshihara et al. 2001). Investigation of putative Ser/Thr kinases identified one that was involved in motility (Kamei et al. 2001). Positive phototaxis in Synechocystis can be mediated by a phytochrome-like photoreceptor and a CheA/CheY-type signal transduction system (Yoshihara et al. 2000). Analysis of mutants affected in phototaxis suggested that the Che-like polypeptides can control both pilus biosynthesis and orientation of movement with respect to light (Bhaya et al. 2001). Sazuka and coauthors (Sazuka and Ohara 1997; Sazuka et al. 1999) linked 234 protein spots on twodimensional gel electrophoretograms to corresponding genes. The resulting analysis showed characteristics of cyanobacterial signal sequences and modification of cyanobacterial proteins. Proteome analysis was subsequently used to identify periplasmic proteins of Synechocystis cells, including a group whose production responded strongly to salt-stress conditions; all proved to have one or other of two types of signal peptides (Fulda et al. 2000). Genomic sequence data provide the opportunity to monitor global changes in genetic expression at transcriptional and translational levels in response to various environmental conditions. Techniques used to analyze global gene expression in eukaryotes (Schena et al. 1995; Zhao et al. 1995; Gupta et al. 1998) are less easily applicable to prokaryotes because one cannot use poly(A) tails (Aviv and Leder 1972) to separate bacterial mRNAs from rRNA. In analyses of hybridization with cDNA derived from total RNA in prokaryotes, the great contribution from rRNA increases the background and thereby decreases the sensitivity of such analyses. Despite the success of Richmond et al. (1999) in work with E. coli without reduction of rRNA, the greater sensitivity that might be achieved by removal of rRNA may be important in cyanobacteria. The challenge is how to reduce the abundance of rRNA without simultaneously impoverishing the population of mRNAs. Because of its seeming simplicity, a method of choice for selectively reducing the abundance of rRNA may be a procedure developed by Affymetrix: primers specific to 23 S and 16 S rRNA are hybridized to those rRNA species, and then used with reverse transcriptase to generate first-strand rRNA “cDNA,” resulting in an rRNA/cDNA hybrid. RNAse H, which specifically degrades the RNA in RNA-DNA hybrids (Nicholson 1997), is used to degrade the RNA strand of the duplex. Finally, the sample is treated with RNAse-free DNAse I to destroy the remaining rRNA cDNA strand. None of these steps should affect the mRNA. An alternative approach, based on work of Graham and Clark-Curtiss (1999), is to capture cDNA reverse-transcribed from total RNA by binding to an amount of biotinylated genom-

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ic DNA sufficient to bind most message-derived cDNA but only a small fraction of rRNA-derived cDNA. Yet a third possibility, one that has recently been applied to analysis of Synechocystis (Singh and Sherman 2000), involves PCR amplification of all non-rRNA genes (generating a so-called customized amplification library, or CAL), saturation of biotin-labeled cDNA with the CAL, capture of the combination on streptavidin beads, washing of the beads, and elution and labeling of the remaining bound CAL, culminating in its use to probe a DNA array (Alland et al. 1998). Differential display, a technique based on reverse transcriptase (RT)-mediated PCR, permits rapid screening for genes that are expressed under specific conditions, and has been used to identify genes regulated by light intensity in Synechocystis (Bhaya et al. 2000b).

Biotechnology Cyanobacteria play an important role in diverse ecological systems. They are common in aqueous environments, including marine, brackish and fresh waters; in soil and rocks, and especially on moist surfaces; and in some habitats that lack eukaryotic life, such as some hot springs and highly alkaline lakes. They form symbioses with algae, bryophytes, ferns, cycads and an angiosperm, and with invertebrates (corals) (Fogg et al. 1973; Wilkinson and Fay 1979). In association with fungi, they form lichens that help to transform rocks to soil in which other forms of life can grow. It is thought that cyanobacteria were the first colonizers of land, providing a physical and chemical substrate for the later growth of eukaryotic plants. How a cyanobacterium survives extreme desiccation is now open to genetic analysis, thanks to the development of a genetic system for Chroococcidiopsis, which dominates microbial communities in the most extremely arid hot and cold deserts (Billi et al. 2001). Transfer of just the sucrose-6-phosphate synthase gene (spsA) from Synechocystis to desiccation-sensitive E. coli resulted in a 104-fold increase in survival compared to wild-type cells following freeze-drying, air drying, or desiccation over phosphorus pentoxide (Billi et al. 2000). If care is not taken in the disposal of phosphate- and nitrate-containing industrial, agricultural, and human wastes, these wastes can eutrophicate lakes and ponds, resulting in massive growth (“blooms”) of cyanobacteria (Atkins et al. 2001). The surface of the water becomes turbid and light cannot penetrate to lower levels. A portion of the cyanobacteria then dies, producing unpleasant odors; bacteria that decompose the cyanobacteria use up available oxygen; and fish then die for lack of oxygen. Some bloom-forming cyanobacteria produce toxins that may render water unpotable (Hitzfeld et al. 2000). Certain of these toxins and other natural products of cyanobacteria have potential for medicinal uses (Patterson et al. 1991; Boyd et al. 1997). Naturally occurring aromatic hydrocarbons (Cerniglia et al. 1979, 1980a, b; Ellis 1977; Narro et al. 1992) and

xenobiotics (Megharaj et al. 1987; Kuritz and Wolk 1995) can both be degraded by cyanobacteria, and cyanobacteria can be genetically engineered to enhance their biodegradative ability (Kuritz and Wolk 1995). Microbial mats rich in cyanobacteria facilitate the remediation of oil-polluted waters and desert in the region of the Arabian Gulf by utilizing crude oil and individual n-alkanes as sources of carbon and energy (Sorkhoh et al. 1992, 1995). Cyanobacteria can also perform syntheses that are of biotechnological significance. Like many genera of eubacteria, they synthesize polyhydroxyalkanoates (PHAs), a thermoplastic class of biodegradable polyesters that includes polyhydroxybutyrate (PHB). PHAs are carbon- and energy-storage compounds that are deposited in the cytoplasm as inclusions. Miyake and colleagues (Miyake et al. 2000) isolated a Tn5 insertion mutant of Synechococcus sp. MA19 with enhanced accumulation of PHB. By using the genomic sequence of Synechocystis, Hein et al. (1998) identified and characterized a gene encoding PHB synthase. Two related genes, a PHAspecific β-ketothiolase and an acetoacetyl-CoA reductase, have been identified and characterized (TaroncherOldenburg et al. 2000). Another such synthesis is that of eicosapentaenoic acid (20:5n-3, EPA), a polyunsaturated fatty acid that is an essential nutrient for marine fish larvae and is important for human health. Yu et al. (2000) introduced the EPAbiosynthetic gene cluster from an EPA-producing bacterium, Shewanella sp. SCRC-2738 into a marine Synechococcus sp., strain NKBG15041c, by conjugation. Transgenic cyanobacteria produced amounts of EPA and its precursor, 20:4n-3, that depended upon the culture conditions used. A cyanobacterial polymer called cyanophycin is a copolymer of arginine and aspartic acid, multi-L-arginylpoly(aspartic acid), discovered and structurally analyzed by Simon (1971, 1987), which comprises the so-called structured granules within the cells (Lang et al. 1972). Simon (1973a, b, 1976) presented evidence that the polymer is synthesized non-ribosomally and can be degraded to serve as a cellular nitrogen reserve, and extensively purified an enzyme involved in its biosynthesis. The cyanophycin synthetase from Anabaena variabilis ATCC 29413 was isolated, microsequenced, and the partial amino acid sequence used to identify the corresponding gene in the Synechocystis PCC 6803 database (Ziegler et al. 1998). It, in turn, permitted isolation, and thence sequencing, of the corresponding gene from Anabaena variabilis ATCC 29413, overexpression of the corresponding protein, and analysis of the mechanism of synthesis of cyanophycin (Berg et al. 2000). The one gene evidently sufficed for cyanophycin synthesis in E. coli. The cyanophycinase gene of PCC 6803, expressed in E. coli and purified, hydrolyzed cyanophycin granule polypeptide to an asp-arg dipeptide (Richter et al. 1999). On the basis of the sequence of those genes, the corresponding genes from Synechocystis PCC 6308 were then cloned, leading to heterologous production of cyanophycin exceeding 26% of cell dry mass (Aboulmagd et al. 2000).

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Use of Spirulina as a source of protein and vitamins for humans or animals has been reviewed by Ciferri (1983) and Kay (1991). Spirulina platensis and Spirulina maxima are thought to have been consumed since ancient times as a food in a part of Africa that is now in the Republic of Chad, and in Mexico, respectively (Ciferri and Tiboni 1985). These species have an unusually high protein content for photosynthetic organisms – up to 70% of the dry weight. Nostoc flagelliforme is considered a delicacy in China (Gao 1998; see also Takenaka et al. 1998). Other cyanobacteria are eaten in India and the Philippines (Tiwari 1978; Martinez 1988). The amino acid composition of S. maxima (Clément et al. 1967), which can grow on animal wastes (Wu and Pond 1981), is also among the best, for human nutrition, of a photosynthetic organism. Like other microalgae, Spirulina is used as a source of natural colorants in food, and as a dietary supplement (Kay 1991). C-phycocyanin and allophycocyanin are phycobiliproteins, pigmented components of the photosystem-II antenna structure, the phycobilisome (Glazer 1988). Phycobiliproteins, coupled to monoclonal and polyclonal antibodies to form fluorescent antibody reagents, are valuable as fluorescent tags in cell sorting, studies of cell surface antigens, and screening of high-density arrays. Spirulina is a convenient and inexpensive source of allophycocyanin and C-phycocyanin (Jung and Dailey 1989). As an alternative approach, genetic engineering of Anabaena 7120 has permitted the in vivo production of stable phycobiliprotein constructs bearing affinity purification tags, usable as fluorescent labels without further chemical manipulation (Cai et al. 2001). Cyanobacteria, because they inhabit the same ecological niches as mosquito larvae, and are eaten by them (Thiery et al. 1991; Avissar et al. 1994), are attractive candidates for mosquito control. Transgenic mosquitocidal cyanobacteria, filamentous as well as unicellular, have been engineered (Tandeau de Marsac et al. 1987; Angsuthanasombat and Panyim 1989; Chungjatupornchai 1990; Murphy and Stevens 1992; Soltes-Rak et al. 1993, 1995; Xu et al. 1993, 2000; Wu et al. 1997). A high level of toxicity was observed with recombinant clones of Anabaena 7120 bearing two δ-endotoxin genes (cryIVA and cryIVD) and gene p20 of Bacillus thuringiensis subsp. israelensis (Wu et al. 1997). Outdoor tests indicated that genetically altered Anabaena 7120 could keep containers with natural water from being inhabited by Culex larvae for over 2 months (Xu et al. 2000). Although one might expect laboratory strains to have low competitive ability compared with indigenous species, that same characteristic may help to prevent unwanted spread of a genetically modified microorganism. Notably, a lyophilized (and presumably nonproliferative) preparation of the recombinant cells retained the same high mosquitocidal activity as the original culture. Liquid suspension cultures or immobilized cells of cyanobacteria offer opportunities for photoproduction of molecular hydrogen (Gisby et al. 1987; Lindblad 1999).

Cyanobacteria possess several enzymes directly involved in hydrogen metabolism: (1) nitrogenase(s), catalyzing the production of H2 as a side product of reduction of N2 to NH3; (2) an uptake hydrogenase, catalyzing the consumption of H2 produced by the nitrogenase; and (3) a bidirectional hydrogenase, which has the capacity both to take up and to produce H2 (Papen et al. 1986; Schmitz et al. 1995; Tamagnini et al. 2000). To maximize H2 production, mutants of A. variabilis strain ATCC 29413 defective in H2-utilization were isolated by PolukhinaMikheeva and Koksharova (Mikheeva et al. 1994). Two mutants altered in hydrogen metabolism were characterized (Mikheeva et al. 1995; Sveshnikov et al. 1997) and one of them, PK 84, has been used for hydrogen production in an automated helical tubular photobioreactor (Borodin et al. 2000). H2-production aside, no biotechnological use has yet been made of the capacity of heterocyst-forming cyanobacteria while growing in air to support reactions that require microoxic conditions.

Summary and outlook Cyanobacteria, structurally Gram-negative prokaryotes and ancient relatives of chloroplasts, can assist analysis of photosynthesis and its regulation more easily than can studies with higher plants. Many genetic tools have been developed for unicellular and filamentous strains of cyanobacteria during the past three decades. Gene transfer systems are available, as are many cloning vectors, transposons, methods of mutagenesis, reporter genes, and genomic sequences. These tools provide abundant opportunity for identifying novel genes; for investigating the structure, regulation and evolution of genes; for understanding the ecological roles of cyanobacteria; and for possible practical applications, only a few of which we have discussed. Acknowledgements We thank Karen Bird, Franck Chauvat, Arthur Grossman, Theresa Thiel, Wim Vermaas and Galyna Kufryk for helpful suggestions concerning the manuscript. This work was supported by the US Department of Energy under grant DOE-FG02–91ER20021.

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