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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.

References Aboulmagd E, Oppermann-Sanio FB, Steinbüchel A (2000) Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Arch Microbiol 174:297– 306 Ajlani G, Meyer I, Vernotte C, Astier C (1989) Mutation in phenol-type herbicide resistance maps within the psbA gene in Synechocystis 6714. FEBS Lett 246:207–210 Alland D, Kramnik I, Weisbrod TR, Otsubo L, Cerny R, Miller LP, Jacobs WRJ, Bloom BR (1998) Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): the effect of isoniazid on gene expression in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 95:13227–13232 Allison G, Gough K, Rogers L, Smith A (1997) A suicide vector for allelic recombination involving the gene for glutamate 1-semialdehyde aminotransferase in the cyanobacterium Synechococcus PCC 7942. Mol Gen Genet 255:392–399

132 Anderson SL, McIntosh L (1991) Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol 173:2761– 2767 Andersson CR, Tsinoremas NF, Shelton J, Lebedeva NV, Yarrow J, Min H, Golden SS (2000) Application of bioluminescence to the study of circadian rhythms in cyanobacteria. Methods Enzymol 305:527–542 Angsuthanasombat C, Panyim S (1989) Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6. Appl Environ Microbiol 55:2428–2430 Astier C, Elmorjani K, Meyer I, Joset F, Herdman M (1984) Photosynthetic mutants of the cyanobacteria Synechocystis sp. strain PCC 6714 and PCC 6803: sodium p-hydroxymercuribenzoate as a selective agent. J Bacteriol 158:659–664 Atkins R, Rose T, Brown RS, Robb M (2001) The Microcystis cyanobacteria bloom in the Swan River–February 2000. Water Sci Technol 43:107–114 Avissar YJ, Margalit J, Spielman A (1994) Incorporation of body components of diverse microorganisms by larval mosquitoes. J Am Mosquito Control Assoc 10:45–50 Aviv H, Leder P (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acidcellulose. Proc Natl Acad Sci USA 69:1408–1412 Bartsevich VV, Shestakov SV (1995) The dspA gene product of the cyanobacterium Synechocystis sp. strain PCC 6803 influences sensitivity to chemically different growth inhibitors and has amino acid similarity to histidine protein kinases. Microbiology 141:2915–2920 Bauer CC, Haselkorn R (1995) Vectors for determining the differential expression of genes in heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. J Bacteriol 177:3332–3336 Berg H, Ziegler K, Piotukh K, Baier K, Lockau W, VolkmerEngert R (2000) Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin) Mechanism of the cyanophycin synthetase reaction studied with synthetic primers. Eur J Biochem 267:5561–5570 Bhalerao RP, Lind LK, Gustafsson P (1994) Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942. Plant Mol Biol 26:313–326 Bhaya D, Watanabe N, Ogawa T, Grossman AR (1999) The role of an alternative sigma factor in motility and pilus formation in the cyanobacterium Synechocystis sp. strain PCC6803. Proc Natl Acad Sci USA 96:3188–3193 Bhaya D, Bianco NR, Bryant D, Grossman A (2000a) Type IV pilus biogenesis and motility in the cyanobacterium Synechocystis sp. PCC6803. Mol Microbiol 37:941–951 Bhaya D, Vaulot D, Amin P, Takahashi AW, Grossman AR (2000b) Isolation of regulated genes of the cyanobacterium Synechocystis sp. strain PCC 6803 by differential display. J Bacteriol 182:5692–5699 Bhaya D, Takahashi A, Grossman AR (2001) Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC 6803. Proc Natl Acad Sci USA 98:7540–7545 Billi D, Wright DJ, Helm RF, Prickett T, Potts M, Crowe JH (2000) Engineering desiccation tolerance in Escherichia coli. Appl Environ Microbiol 66:1680–1684 Billi D, Friedmann EI, Helm RF, Potts M (2001) Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis. J Bacteriol 183:2298–2305 Black TA, Cai Y, Wolk CP (1993) Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol 9:77–84 Boerner RJ, Nguyen AP, Barry BA, Debus RJ (1992) Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex. Biochemistry 31:6660–6672 Boison G, Schmitz O, Schmitz B, Bothe H (1998) Unusual gene arrangement of the bi-directional hydrogenase and functional

analysis of its diaphorase subunit HoxU in respiration of the unicellular cyanobacterium Anacystis nidulans. Curr Microbiol 36:253–258 Bölter B, Soll J, Schulz A, Hinnah S, Wagner R (1998) Origin of a chloroplast protein importer. Proc Natl Acad Sci USA 95: 15831–15836 Borodin VB, Tsygankov AA, Rao KK, Hall DO (2000) Hydrogen production by Anabaena variabilis PK84 under simulated outdoor conditions. Biotechnol Bioeng 69:478–485 Borthakur D, Haselkorn R (1989) Tn5 mutagenesis of Anabaena sp. strain PCC 7120: isolation of a new mutant unable to grow without combined nitrogen. J Bacteriol 171:5759–5761 Boyd MR, Gustafson KR, McMahon JB, Shoemaker RH, O’Keefe BR, Mori T, Gulakowski RJ, Wu L, Rivera MI, Laurencot CM, Currens MJ, Cardellina JH, Buckheit RW, Nara PL, Pannell LK, Sowder RC, Henderson LE (1997) Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother 41:1521–1530 Brahamsha B (1996) A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus. Appl Environ Microbiol 62:1747–1751 Broedel SE, Wolf RE (1990) Genetic tagging, cloning, and DNA sequence of the Synechococcus sp. strain PCC 7942 gene (gnd) encoding 6-phosphogluconate dehydrogenase. J Bacteriol 172:4023–4031 Bruns BU, Briggs WR, Grossman AR (1989) Molecular characterization of phycobilisome regulatory mutants of Fremyella diplosiphon. J Bacteriol 171:901–908 Bryant DA, Marquardt J, Shen G, Nomura CT, Persson S, Zhao J, Li T, Huang X, Li S, Wang J, Wang J (2001) The complete genomic sequence of Synechococcus sp. strain PCC 7002: a progress report. In: Potts M, Slaughter S, Schroder M, Kennelly P (eds) Final program VIIth cyanobacterial workshop. A signal event, July 27–31, 200l, Pacific Grove, Calif. Virginia Polytechnic Institute and State University, Blacksburg, Va. p 26 Buikema WJ, Haselkorn R (1991) Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 173:1879–1885 Cai Y, Wolk CP (1990) Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol 172:3138–3145 Cai Y, Wolk CP (1997) Nitrogen deprivation of Anabaena sp. strain PCC 7120 elicits rapid activation of a gene cluster that is essential for uptake and utilization of nitrate. J Bacteriol 179:258–266 Cai YA, Murphy JT, Wedemayer GJ, Glazer AN (2001) Recombinant phycobiliproteins. Anal Biochem 290:186–204 Callahan SM, Buikema WJ (2001) The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120. Mol Microbiol 40:941–950 Casey ES, Grossman A (1994) In vivo and in vitro characterization of the light-regulated cpcB2A2 promoter of Fremyella diplosiphon. J Bacteriol 176:6362–6374 Cerniglia CE, Gibson DT, Van Baalen C (1979) Algal oxidation of aromatic hydrocarbons: formation of 1-naphthol from naphthalene by Agmenellum quadruplicatum, strain PR-6. Biochem Biophys Res Commun 88:50–58 Cerniglia CE, Van Baalen C, Gibson DT (1980a) Metabolism of naphthalene by the cyanobacterium Oscillatoria sp. strain JCM. J Gen Microbiol 116:485–494 Cerniglia CE, Gibson DT, Van Baalen C (1980b) Oxidation of naphthalene by cyanobacteria and microalgae. J Gen Microbiol 116:495–500 Chauvat F, De Vries L, Van der Ende A, Van Arkel GA (1986) A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803. Mol Gen Genet 204:185–191 Chiang GG, Schaefer MR, Grossman AR (1992) Transformation of the filamentous cyanobacterium Fremyella diplosiphon by conjugation or electroporation. Plant Physiol Biochem 30:315–325

133 Chitnis PR, Reilly PA, Nelson N (1989) Insertional inactivation of the gene encoding subunit II of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 264: 18381–18385 Chungjatupornchai W (1990) Expression of the mosquitocidalprotein genes of Bacillus thuringiensis subsp. israelensis and the herbicide-resistance gene bar in Synechocystis PCC 6803. Curr Microbiol 21:283–288. Ciferri O (1983) Spirulina, the edible microorganism. Microbiol Rev 47:551–578 Ciferri O, Tiboni O (1985) The biochemistry and industrial potential of Spirulina. Annu Rev Microbiol 39:503–526 Clément G, Giddey C, Menzi R (1967) Amino acid composition and nutritive value of the alga Spirulina maxima. J Sci Food Agric 18:497–501 Cobley JG, Zerweck E, Reyes R, Mody A, Seludo-Unson JR, Jaeger H, Weerasuriya S, Navankasattusas S (1993) Construction of shuttle plasmids which can be efficiently mobilized from Escherichia coli into the chromatically adapting cyanobacterium, Fremyella diplosiphon. Plasmid 30:90–105 Crameri A, Whitehorn EA, Tate E, Stemmer WP (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol 14:315–319 Currier TC, Haury JF, Wolk CP (1977) Isolation and preliminary characterization of auxotrophs of a filamentous cyanobacterium. J Bacteriol 129:1556–1562 Debus RJ, Barry BA, Babcock GT, McIntosh L (1988) Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA 85:427–430 Dolganov N, Grossman AR (1993) Insertional inactivation of genes to isolate mutants of Synechococcus sp. strain PCC 7942: isolation of filamentous strains. J Bacteriol 175:7644– 7651 Dolganov N, Grossman AR (1999) A polypeptide with similarity to phycocyanin α-subunit phycocyanobilin lyase involved in degradation of phycobilisomes. J Bacteriol 181:610–617 Dzelzkalns VA, Bogorad L (1988) Molecular analysis of a mutant defective in photosynthetic oxygen evolution and isolation of a complementing clone by a novel screening procedure. EMBO J 7:333–338 Elanskaya IV, Chesnavichene EA, Vernotte C, Astier C (1998) Resistance to nitrophenolic herbicides and metronidazole in the cyanobacterium Synechocystis sp. PCC 6803 as a result of inactivation of a nitroreductase protein encoded by drgA gene. FEBS Lett 428:188–192 Elhai J (1994) Genetic techniques appropriate for the biotechnological exploitation of cyanobacteria. J Appl Phycol 6:177– 186 Elhai J, Wolk CP (1988) A versatile class of positive selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68:119–138 Elhai J, Wolk CP (1990) Developmental regulation and spatial pattern of expression of the structural genes for nitrogenase in the cyanobacterium Anabaena. EMBO J 9:3379–3388 Elhai J, Thiel T, Pakrasi HB (1990) DNA transfer into cyanobacteria. Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual A12. Kluwer, Dordrecht, pp 1–23 Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP (1997) Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179: 1998–2005 Ellis BE (1977) Degradation of phenolic compounds by freshwater algae. Plant Sci Lett 8:213–216 Engebrecht J, Nealson K, Silverman M (1983) Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell 32:773–781 Ermakova-Gerdes S, Vermaas W (1999) Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding. J Biol Chem 274:30540–30549

Ermakova-Gerdes S, Yu Z, Vermaas W (2001) Targeted random mutagenesis to identify functionally important residues in the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803. J Bacteriol 183:145–154 Ernst A, Black T, Cai Y, Panoff J-M, Tiwari DN, Wolk CP (1992) Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism. J Bacteriol 174:6025–6032 Ferino F, Chauvat F (1989) A promoter-probe vector-host system for the cyanobacterium Synechocystis PCC6803. Gene 84: 257–266 Fernández-Piñas F, Wolk CP (1994) Expression of luxCD-E in Anabaena sp. can replace the use of exogenous aldehyde for in vivo localization of transcription by luxAB. Gene 150:169–174 Fernández-Piñas F, Leganes F, Wolk CP (2000) Bacterial lux genes as reporters in cyanobacteria. Methods Enzymol 305: 513–527 Finnegan J, Sherratt D (1982) Plasmid ColE1 conjugal mobility: the nature of bom, a region required in cis for transfer. Mol Gen Genet 185:344–351 Flores E, Wolk CP (1985) Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation. J Bacteriol 162:1339– 1341 Fogg GE, Stewart WDP, Fay P, Walsby AE (1973) The blue-green algae. Academic Press, London Foran DR, Brown WM (1988) Nucleotide sequence of the LuxA and LuxB genes of the bioluminescent marine bacterium Vibrio fisheri. Nucleic Acids Res 16:777 Fujita Y, Takahashi Y, Chuganji M, Matsubara H (1992) The nifHlike (frxC) gene is involved in the biosynthesis of chlorophyll in the filamentous cyanobacterium Plectonema boryanum. Plant Cell Physiol 33:81–92 Fulda S, Huang F, Nilsson F, Hagemann M, Norling B (2000) Proteomics of Synechocystis sp. strain PCC 6803. Identification of periplasmic proteins in cells grown at low and high salt concentrations. Eur J Biochem 267:5900–5907 Funk C, Vermaas W (1999) A cyanobacterial gene family coding for single-helix proteins resembling part of the light-harvesting proteins from higher plants. Biochemistry 38:9397–9404 Gao K (1998) Chinese studies on the edible blue-green alga Nostoc flagelliforme: a review. J Appl Phycol 10:37–49 Garczarek L, Hess WR, Holtzendorff J, van der Staay GWM, Partensky F (2000) Multiplication of antenna genes as a major adaptation to low light in a marine prokaryote. Proc Natl Acad Sci USA 97:4098–4101 Ghassemian M, Straus NA (1996) Fur regulates the expression of iron-stress genes in the cyanobacterium Synechococcus sp. strain PCC 7942. Microbiology 142:1469–1476 Gisby PF, Rao K, Hall DO (1987) Entrapment techniques for chloroplasts, cyanobacteria and hydrogenases. Methods Enzymol 135:440–454 Glazer AN (1988) Phycobiliproteins. Methods Enzymol 167: 291–303 Golden JW, Wiest DR (1988) Genome rearrangement and nitrogen fixation in Anabaena blocked by inactivation of xisA gene. Science 242:1421–1423 Golden SS (1988) Mutagenesis of cyanobacteria by classical and gene-transfer based methods. Methods Enzymol 167:714–727 Golden SS, Sherman LA (1983) A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2. J Bacteriol 155:966–972 Golden SS, Sherman LA (1984) Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2. J Bacteriol 158:36–42 Golden SS, Brusslan J, Haselkorn R (1986) Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J 5:2789–2798 Golden SS, Brusslan J, Haselkorn R (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymol 153: 215–231 Graham JE, Clark-Curtiss JE (1999) Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocyto-

134 sis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA 96:11554– 11559 Grigorieva G, Shestakov S (1982) Transformation in the cyanobacterium Synechocystis sp. 6803. FEMS Microbiol Lett 13: 367–370 Gupta R, Thomas P, Beddington RSP, Rigby PWJ (1998) Isolation of developmentally regulated genes by differential display screening of cDNA libraries. Nucleic Acids Res 26:4538– 4539 Haselkorn R (1995) Molecular genetics of nitrogen fixation in photosynthetic prokaryotes. In: Tikhonovich IA, Provorov NA, Romanov VI, Newton WE (eds) Nitrogen fixation: fundamentals and applications. Kluwer, Dordrecht, pp 29–36 Hein S, Tran H, Steinbüchel A (1998) Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria. Arch Microbiol 170:162–170 Herdman M, Carr NG (1972) The isolation and characterization of mutant strains of the blue-green alga Anacystis nidulans. J Gen Microbiol 70:213–220 Herdman M, Janvier M, Rippka R, Stanier RY (1979) Genome size of cyanobacteria. J Gen Microbiol 111:73–85 Herdman M, Coursin T, Rippka R, Houmard J, Tandeau de Marsac N (2000) A new appraisal of the prokaryotic origin of eukaryotic phytochromes. J Mol Evol 51:205–213 Hihara Y, Kamei A, Kanehisa M, Kaplan A, Ikeuchi M (2001) DNA microarray analysis of cyanobacterial gene expression during acclimation to high light. Plant Cell 13:793–806 Hirosawa M, Isono K, Hayes W, Borodovsky M (1997) Gene identification and classification in the Synechocystis genomic sequence by recursive gene mark analysis. DNA Seq 8:17–29 Hirschberg J, Chamovitz D (1994) Carotenoids in cyanobacteria. In: Bryant DA (ed), The molecular biology of cyanobacteria. Kluwer, Dordrecht, pp 559–579 Hitzfeld BC, Hoger SJ, Dietrich DR (2000) Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment. Environ Health Perspect 108:113–122 Holtman CK, Youderian PA, Golden SS (2001) Identification of genes necessary for circadian rhythm in Synechococcus elongatus PCC 7942. In: Potts M, Slaughter S, Schroder M, Kennelly P (eds) Final Program VIIth Cyanobacterial Workshop. A Signal Event, July 27–31, 200l, Pacific Grove, Calif. Virginia Polytechnic Institute and State University, Blacksburg, Va. p 71 Hondel CAMJJ van den, Verbeek S, van der Ende A, Weisbeek PJ, Borrias WE, van Arkel GA (1980) Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria. Proc Natl Acad Sci USA 77: 1570–1574 Hu N-T, Thiel T, Giddings TH, Wolk CP (1981) New Anabaena and Nostoc cyanophages from sewage settling ponds. Virology 114:236–246 Jung T, Dailey M (1989) A novel and inexpensive source of allophycocyanin for multicolor flow cytometry. J Immunol Methods 121:9–18 Kamei A, Yuasa T, Orikawa K, Geng XX, Ikeuchi M (2001) A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 183:1505–1510 Kaneko T, Tabata S (1997) Complete genome structure of the unicellular cyanobacterium Synechocystis sp. PCC6803. Plant Cell Physiol 38:1171–1176 Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S (1996a) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3:109–136

Kaneko T, Matsubayashi T, Sugita M, Sugiura M (1996b) Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome. Plant Mol Biol 31:19–201 Kaneko T, Nakamura Y, Sasamoto S, Wolk CP, Tabata S (2001a) Comparative genome analysis of cyanobacteria, Anabaena sp. PCC7120, Synechococcus elongatus BP-1, and Gloeobacter violaceus PCC 7421. In: Potts M, Slaughter S, Schroder M, Kennelly P (eds) Final Program VIIth Cyanobacterial Workshop. A Signal Event, July 27–31, 200l, Pacific Grove, Calif. Virginia Polytechnic Institute and State University, Blacksburg, Va. p 74 Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakazaki N, Shimpo S, Sugimoto M, Takazawa M, Yamada M, Yasuda M, Tabata S (2001b) Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8:205–213 Katayama M, Tsinoremas NF, Kondo T, Golden SS (1999) cpmA, a gene involved in an output pathway of the cyanobacterial circadian system. J Bacteriol 181:3516–3524 Kay RA (1991) Microalgae as food and supplement. Crit Rev Food Sci Nutr 30:555–573 Koksharova OA, Shestakov SV (1990) Mutants of the cyanobacterium Synechocystis 6803, resistant to inhibitors of photosynthesis (in Russian). Vest Mosk Univ Ser XVI Biol No 2:42–46 Koksharova O, Schubert M, Shestakov S, Cerff R (1998) Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 36:183–194 Kondo T, Ishiura M (1994) Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence. J Bacteriol 176:1881–1885 Kondo T, Strayer CA, Kulkarni RD, Taylor W, Ishiura M, Golden SS, Johnson CH (1993) Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc Natl Acad Sci USA 90:5672–5676 Kondo T, Tsinoremas NF, Golden SS, Johnson CH, Kutsuna S, Ishiura M (1994) Circadian clock mutants of cyanobacteria. Science 266:1233–1236 Koz’yakov SYa, Gromov BV, Khudyakov IYa (1972) A-1(L)cyanophage of the blue-green alga Anabaena variabilis (in Russian). Mikrobiologiya 41: 555–559 Kreps S, Ferino F, Mosrin C, Gerits J, Mergeay M, Thuriaux P (1990) Conjugative transfer and autonomous replication of promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803. Mol Gen Genet 221:129–133 Kufryk GI, Vermaas WFJ (2001) A novel protein involved in the functional assembly of the oxygen-evolving complex of photosystem II in Synechocystis sp. PCC 6803. Biochemistry 40:9247–9255 Kuhlemeier CJ, Borrias WE, van den Hondel CAMJJ, van Arkel GA (1981) Vectors for cloning in cyanobacteria: construction and characterization of two recombinant plasmids capable of transformation to Escherichia coli K-12 and Anacystis nidulans R2. Mol Gen Genet 184:249–254 Kunert A, Hagemann M, Erdmann N (2000) Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using light-emitting reporter systems Gfp and LuxAB. J Microbiol Methods 41:185–194 Kuritz T, Wolk CP (1995) Use of filamentous cyanobacteria for biodegradation of organic pollutants. Appl Environ Microbiol 61:234–238 Labarre J, Chauvat F, Thuriaux P (1989) Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803. J Bacteriol 171:3449–3457 Lang NJ, Simon RD, Wolk CP (1972) Correspondence of cyanophycin granules with structured granules in Anabaena cylindrica. Arch Mikrobiol 83:313–320

135 Lindblad P (1999) Cyanobacterial H2-metabolism: knowledge and potential/strategies for a photobiotechnological production of H2. Biotechnol Appl 16:141–144 Liu Y, Golden SS, Kondo T, Ishiura M, Johnson CH (1995) Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria. J Bacteriol 177:2080–2086 Marin K, Zuther E, Kerstan T, Kunert A, Hagemann M (1998) The ggpS gene from Synechocystis sp. strain PCC 6803 encoding glucosyl-glycerol-phosphate synthase is involved in osmolyte synthesis. J Bacteriol 180:4843–4849 Marraccini P, Bulteau S, Cassier-Chauvat C, Mermet-Bouvier P, Chauvat F (1993) A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. Plant Mol Biol 23:905–909 Marraccini P, Cassier-Chauvat C, Bulteau S, Chavez S, Chauvat F (1994) Light-regulated promoters from Synechocystis PCC6803 share a consensus motif involved in photoregulation. Mol Microbiol 12:1005–1012 Martinez MR (1988) Nostoc commune Vauch. a nitrogen-fixing blue-green alga, as source of food in the Philippines. Philippine Naturalist 71:295–307 Martinez-Férez IM, Vioque A (1992) Nucleotide sequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803 and characterization of a new mutation which confers resistance to the herbicide norflurazon. Plant Mol Biol 18:981–983 Matsunaga T, Takeyama H, Nakamura N (1990) Characterization of cryptic plasmids from marine cyanobacteria and construction of a hybrid plasmid potentially capable of transformation of marine cyanobacterium, Synechococcus sp. and its transformation. Appl Biochem Biotechnol 24/25:151–160 Matveyev AV, Young KT, Meng A, Elhai J (2001) DNA methyltransferases of the cyanobacterium Anabaena PCC 7120. Nucleic Acids Res 29:1491–1506 Megharaj M, Venkateswarlu K, Rao AS (1987) Metabolism of monocrotophos and quinalphos by algae isolated from soil. Bull Environ Contam Toxicol 39:251–256 Mendzhul MI, Nesterova NV, Goryushin VA, Lysenko TG (1985) Cyanophages – viruses of cyanobacteria (in Russian). Naukova Dumka, Kiev Mermet-Bouvier P, Cassier-Chauvat C, Marraccini P, Chauvat F (1993) Transfer and replication of RSF1010-derived plasmids in several cyanobacteria of the genera Synechocystis and Synechococcus. Curr Microbiol 27:323–327 Mikheeva LE, Koksharova OA, Shestakov SV (1994) Mutant of the cyanobacterium Anabaena variabilis ATCC 29413 producing molecular hydrogen (in Russian). Vest Mosk Univ Ser XVI Biol No 2:54–57 Mikheeva LE, Schmitz O, Shestakov SV, Bothe H (1995) Mutants of the cyanobacterium Anabaena variabilis altered in hydrogenase activities. Z Naturforsch Teil C 50:505–510 Miyake M, Takase K, Narato M, Khatipov E, Schnackenberg J, Shirai M, Kurane R, Asada Y (2000) Polyhydroxybutyrate production from carbon dioxide by cyanobacteria. Appl Biochem Biotechnol 84:991–1002 Montesinos ML, Herrero A, Flores E (1997) Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids. J Bacteriol 179:853–862 Moser DP, Zarka D, Kallas T. (1993) Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121. Arch Microbiol 160:229–237 Mrázek J, Bhaya D, Grossman AR, Karlin S (2001) Highly expressed and alien genes of the Synechocystis genome. Nucleic Acids Res 29:1590–1601 Mühlenhoff U, Chauvat F (1996) Gene transfer and manipulation in the thermophilic cyanobacterium Synechococcus elongatus. Mol Gen Genet 252:93–100 Murphy RC, Stevens SE (1992) Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity. Appl Environ Microbiol 58:1650– 1655

Murry MA, Wolk CP (1991) Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp strain M-131. Mol Gen Genet 227:113–119 Narro ML, Cerniglia CE, Van Baalen C, Gibson DT (1992) Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6. Appl Environ Microbiol 58:1351–1359 Narusaka Y, Narusaka M, Kobayashi H, Satoh K (1998) The herbicide-resistant species of the cyanobacterial D1 protein obtained by thorough and random in vitro mutagenesis. Plant Cell Physiol 39:620–626 Nicholson AW (1997) Escherichia coli ribonucleases: paradigm for understanding cellular RNA metabolism and regulation. In: D’Alessio G, Riordan JF (eds) Ribonucleases structures and functions. Academic Press, San Diego, Calif. pp 1–49 Nyhus KJ, Thiel T, Pakrasi HB (1993) Targeted interruption of the psaA and psaB genes encoding the reaction-centre proteins of photosystem I in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Mol Microbiol 9:979–988 Ochoa de Alda JA, Houmard J (2000) Genomic survey of cAMP and cGMP signalling components in the cyanobacterium Synechocystis PCC 6803. Microbiology 146:3183–3194 Ohkawa H, Price G, Badger M, Ogawa T (2000) Mutation of ndh genes leads to inhibition of CO2 uptake rather than HCO3– uptake in Synechocystis sp. strain PCC 6803. J Bacteriol 182: 2591–2596 Pakrasi HB, Williams JGK, Arntzen CJ (1988) Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II. EMBO J 7:325–332 Papen H, Kentemich T, Schmülling T, Bothe H (1986) Hydrogenase activities in cyanobacteria. Biochimie 68:121–132 Partensky F, Hess WR, Vaulot D (1999) Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol Mol Biol Rev 63:106–127 Patterson MLG, Baldwin CL, Bolis CM, Caplan FR, Karuso H, Larsen LK, Levine IA, Moore RE, Nelson CS, Tschappat KD, Tuang GD, Furusawa E, Furusawa S, Norton TR, Raybourne RB (1991) Antineoplastic activity of cultured blue-green algae (Cyanophyta). J Phycol 27:530–536 Pecker I, Ohad N, Hirschberg J (1987) The chloroplast-encoded type of herbicide resistance is a recessive trait in cyanobacteria. In: Biggins J (ed) Progress in photosynthesis research, vol III. Nijhoff, Dordrecht, pp 811–814 Pieulle L, Guedeney G, Cassier-Chauvat C, Jeanjean R, Chauvat F, Peltier G (2000) The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of Synechocystis PCC6803. FEBS Lett 487:272–276 Polukhina LE, Koksharova OA, Maisuryan NA, Shestakov SV (1985) Mutants of the cyanobacterium Anabaena variabilis, resistant to methionine sulfoximine and metronidazole (in Russian). Vest Mosk Univ Ser XVI Biol No 2:59–65 Poncelet M, Cassier-Chauvat C, Leschelle X, Bottin H, Chauvat F (1998) Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC 6803 by plasmid shuffling. Mol Microbiol 28:813–821 Prentki P, Binda A, Epstein A (1991) Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon. Gene 103:17–23 Reaston J, Duyvesteyn MGC, de Waard A (1982) Nostoc PCC7542, a cyanobacterium which contains five sequencespecific deoxyribonucleases. Gene 20:103–110 Reumann S, Davila-Aponte J, Keegstra K (1999) The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog. Proc Natl Acad Sci USA 96:784–789 Richmond CS, Glasner JD, Mau R, Jin H, Blattner FR (1999) Genome-wide expression profiling in Escherichia coli K-12. Nucleic Acids Res 27:3821–3835 Richter R, Hejazi M, Kraft R, Ziegler K, Lockau W (1999) Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin).

136 Molecular cloning of the gene of Synechocystis sp. PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme. Eur J Biochem 263:163–169 Rimon A, Oppenheim AB (1975) Heat induction of the blue-green alga Plectonema boryanum lysogenic for the cyanophage SP1cts1. Virology 64:454–463 Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61 Rocap G, Stilwagon S, Lamerdin JE, Chisholm SW, Larimer FW (2001) Comparative genomics of Prochlorococcus. In: Potts M, Slaughter S, Schroder M, Kennelly P (eds) Final Program VIIth Cyanobacterial Workshop. A Signal Event, July 27–31, 2001, Pacific Grove, Calif. Virginia Polytechnic Institute and State University, Blacksburg, Va. p 40 Rosenberg C, Christian J, Bricker TM, Putnam-Evans C (1999) Site-directed mutagenesis of glutamate residues in the large extrinsic loop of the photosystem II protein CP 43 affects oxygen-evolving activity and PS II assembly. Biochemistry 38: 15994–16000 Rouhiainen L, Paulin L, Suomalainen S, Hyytiäinen H, Buikema W, Haselkorn R, Sivonen K (2000) Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90. Mol Microbiol 37:156–167 Rujan T, Martin W (2001) How many genes in Arabidopsis come from cyanobacteria? An estimate from 386 protein phylogenies. Trends Genet 17:113–120 Sarma TA, Kaur B (1997) Characterization of host-range mutants of cyanophage N-1. Acta Virol 41:245–250 Sazuka T, Ohara O (1997) Towards a proteome project of cyanobacterium Synechocystis sp. strain PCC6803: linking 130 protein spots with their respective genes. Electrophoresis 18: 1252–1258 Sazuka T, Yamaguchi M, Ohara O (1999) Cyano2Dbase updated: linkage of 234 protein spots to corresponding genes through N-terminal microsequencing. Electrophoresis 20:2160–2171 Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470 Schmetterer G, Wolk CP, Elhai J (1986) Expression of luciferases from Vibrio harveyi and Vibrio fischeri in filamentous cyanobacteria. J Bacteriol 167:411–414 Schmitz O, Boison G, Hilscher R, Hundeshagen B, Zimmer W, Lottspeich F, Bothe H (1995) Molecular biological analysis of a bidirectional hydrogenase from cyanobacteria. Eur J Biochem 233:266–276 Schwartz SH, Black TA, Jäger K, Panoff J-M, Wolk CP (1998) Regulation of an osmoticum-responsive gene in Anabaena sp. strain PCC 7120. J Bacteriol 180:6332–6337 Shen G, Boussiba S, Vermaas WFJ (1993) Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function. Plant Cell 5:1853–1863 Sherman LA, Brown RM (1978) Cyanophages and viruses of eukaryotic algae. In: Fraenkel-Conrat H, Wagner RR (eds) Comprehensive virology, vol 12. Plenum Press, New York, pp 145–234 Shestakov SV, Khyen NT (1970) Evidence for genetic transformation in the blue-green alga Anacystis nidulans. Mol Gen Genet 107:372–375 Simon RD (1971) Cyanophycin granules from the blue-green alga Anabaena cylindrica: a reserve material consisting of copolymers of aspartic acid and arginine. Proc Natl Acad Sci USA 68:265–267 Simon RD (1973a) The effect of chloramphenicol on the production of cyanophycin granule polypeptide in the blue-green alga Anabaena cylindrica. Arch Mikrobiol 92:115–123 Simon RD (1973b) Measurement of the cyanophycin granule polypeptide contained in the blue-green alga Anabaena cylindrica. J Bacteriol 114:1213–1216 Simon RD (1976) The biosynthesis of multi-L-arginyl-poly(Laspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. Biochim Biophys Acta 422:407–418

Simon RD (1987) Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies. In: Fay P, Van Baalen C (eds) The cyanobacteria. Elsevier, Amsterdam, pp 199–225 Singh AK, Sherman LA (2000) Identification of iron-responsive, differential gene expression in the cyanobacterium Synechocystis sp. strain PCC 6803 with a customized amplification library. J Bacteriol 182:3536–3543 Singh RN, Tiwari DN (1969) Induction by ultraviolet irradiation of mutation in the blue-green alga Nostoc linckia (Roth) Born. et Flah. Nature 221:62–64 Sode K, Tatara M, Takeyama H, Burgess JG, Matsunaga T (1992a) Conjugative gene transfer in marine cyanobacteria: Synechococcus sp. Synechocystis sp. and Pseudanabaena sp. Appl Microbiol Biotechnol 37:369–373 Sode K, Tatara M, Ogawa S, Matsunaga T (1992b) Maintenance of broad host range vector pKT230 in marine unicellular cyanobacteria. FEMS Microbiol Lett 99:73–78 Soltes-Rak E, Kushner DJ, Williams DD, Coleman JR (1993) Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942. Appl Environ Microbiol 59:2404–2410 Soltes-Rak E, Kushner DJ, Williams DD, Coleman JR (1995) Factors regulating cryIVB gene expression in the cyanobacterium Synechococcus PCC 7942. Mol Gen Genet 246:301–308 Sorkhoh N, Al-Hasan R, Radwan S, Höpner T (1992) Self-cleaning of the Gulf. Nature 359:109 Sorkhoh NA, Al-Hasan RH, Khanafer M, Radwan SS (1995) Establishment of oil-degrading bacteria associated with cyanobacteria in oil-polluted soil. J Appl Bacteriol 78:194–199 Stevens SE, Porter RD (1980) Transformation in Agmenellum quadruplicatum. Proc Natl Acad Sci USA 77:6052–6056 Suzuki I, Los DA, Kanesaki Yu, Mikami K, Murata N (2000) The pathway for perception and transduction of low-temperature signals in Synechocystis. EMBO J 19:1327–1334 Suzuki I, Kanesaki Y, Mikami K, Kanehisa M, Murata N (2001) Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis. Mol Microbiol 40:235–244 Sveshnikov DA, Sveshnikova NV, Rao KK, Hall DO (1997) Hydrogen metabolism of mutant forms of Anabaena variabilis in continuous cultures and under nutritional stress. FEMS Microbiol Lett 147:297–301 Takenaka H, Yamaguchi Y, Sakaki S, Watarai K, Tanaka N, Hori M, Seki H, Tsuchida M, Yamada A, Nishimori T, Morinaga T (1998) Safety evaluation of Nostoc flagelliforme (nostocales [sic], Cyanophyceae) as a potential food. Food Chem Toxicol 36:1073–1077 Tamagnini P, Costa J-L, Almeida L, Olivera M-J, Salema R, Lindblad P (2000) Diversity of cyanobacterial hydrogenases, a molecular approach. Curr Microbiol 40:356–361 Tandeau de Marsac N, Borrias WE, Kuhlemeier CJ, Castets AM, van Arkel GA, van den Hondel CAMJJ (1982) A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant. Gene 20:111–119 Tandeau de Marsac N, de la Torre F, Szulmajster J (1987) Expression of the larvicidal gene of Bacillus sphaericus 1593M in the cyanobacterium Anacystis nidulans R2. Mol Gen Genet 209:396–398 Taroncher-Oldenburg G, Stephanopoulos G (2000) Targeted, PCR-based gene disruption in cyanobacteria: inactivation of the polyhydroxyalkanoic acid synthase genes in Synechocystis sp. PCC 6803. Appl Microbiol Biotechnol 54:677–680 Taroncher-Oldenburg G, Nishina K, Stephanopoulos G (2000) Identification and analysis of the polyhydroxyalkanoate-specific β-ketothiolase and acetoacetyl coenzyme A reductase genes in the cyanobacterium Synechocystis sp. strain PCC6803. Appl Environ Microbiol 66:4440–4448 Thiel T (1994) Genetic analysis of cyanobacteria. In: Bryant DA (ed) The molecular biology of cyanobacteria. Kluwer, Dordrecht, pp 581–611 Thiel T, Poo H (1989) Transformation of a filamentous cyanobacterium by electroporation. J Bacteriol 171:5743–5746

137 Thiel T, Lyons EM, Erker JC, Ernst A (1995) A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium. Proc Natl Acad Sci USA 92:9358–9362 Thiery I, Nicolas L, Rippka R, Tandeau de Marsac N (1991) Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae. Appl Environ Microbiol 57:1354–1359 Thor JJ van, Gruters OW, Matthijs HC, Hellingwerf KJ (1999) Localization and function of ferredoxin:NADP(+) reductase bound to the phycobilisomes of Synechocystis. EMBO J 18: 4128–4136 Tichy M, Vermaas W (1999) In vivo role of catalase-peroxidase in Synechocystis sp. strain PCC 6803. J Bacteriol 181:1875–1882 Tichy M, Vermaas W (2000) Combinatorial mutagenesis and pseudorevertant analysis to characterize regions in loop E of the CP47 protein in Synechocystis sp. PCC 6803. Eur J Biochem 267:6296–6301 Tiwari DN (1978) The heterocysts of the blue-green alga Nostochopsis lobatus: effects of cultural conditions. New Phytol 81:853–856 Tsinoremas NF, Kutach AK, Strayer CA, Golden SS (1994) Efficient gene transfer in Synechococcus sp. strains PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination. J Bacteriol 176:6764–6768 Tuli R, Vachhani AK, Iyer RK (1990) Plasmids in Plectonema boryanum and a mobilisable vector system for the cyanobacterium. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: achievements and objectives. Chapman Hall, NewYork, p 593 Vachhani AK, Iyer RK, Tuli R (1993) A mobilizable shuttle vector for the cyanobacterium Plectonema boryanum. J Gen Microbiol 139:569–573 Vega-Palas MA, Madueño F, Herrero A, Flores E (1990) Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol 172:643–647 Vermaas WFJ (1996) Molecular genetics of the cyanobacterium Synechocystis sp. PCC 6803: principles and possible biotechnology applications. J Appl Phycol 8:263–273 Vermaas W (1998) Gene modifications and mutation mapping to study the function of photosystem II. Methods Enzymol 297: 293–310 Vermaas W, Charité J, Shen GZ (1990) Glu-69 of the D2 protein in photosystem II is a potential ligand to Mn involved in photosynthetic oxygen evolution. Biochemistry 29:5325–5332 Vinnemeier J, Hagemann M (1999) Identification of salt-regulated genes in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 by subtractive RNA hybridization. Arch Microbiol 172:377–386 Wilkinson CR, Fay P (1979) Nitrogen fixation in coral reef sponges with symbiotic cyanobacteria. Nature 279:527–529 Williams JG, Szalay AA (1983) Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2. Gene 24:37–51 Wolk CP (1996) Heterocyst formation. Annu Rev Genet 30:59–78 Wolk CP, Kraus J (1982) Two approaches to obtaining low, extracellular deoxyribonuclease activity in cultures of heterocystforming cyanobacteria. Arch Microbiol 131:302–307 Wolk CP, Vonshak A, Kehoe P, Elhai J (1984) Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci USA 81:1561–1565 Wolk CP, Cai Y, Cardemil L, Flores E, Hohn B, Murry M, Schmetterer G, Schrautemeier B, Wilson R (1988) Isolation

and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen. J Bacteriol 170:1239–1244 Wolk CP, Cai Y, Panoff J-M (1991) Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium. Proc Natl Acad Sci USA 88: 5355–5359 Wolk CP, Elhai J, Kuritz T, Holland D (1993) Amplified expression of a transcriptional pattern formed during development of Anabaena. Mol Microbiol 7:441–445 Wu J, Pond W (1981) Amino acid composition and microbial contamination of Spirulina maxima, a blue-green alga, grown on the effluent of different fermented animal wastes. Bull Environ Contam Toxicol 27:151–159 Wu X, Vennison SJ, Liu H, Ben-Dov E, Zaritsky A, Boussiba S (1997) Mosquito larvicidal activity of transgenic Anabaena strain PCC 7120 expressing combinations of genes from Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 63:4971–4975 Xu X, Wolk CP (2001) Role for hetC in the transition to a nondividing state during heterocyst differentiation in Anabaena sp. J Bacteriol 183:393–396 Xu X, Kong R, Hu Y (1993) High larvicidal activity of intact recombinant cyanobacterium Anabaena sp. PCC 7120 expressing gene 51 and gene 42 of Bacillus sphaericus sp. 2297. FEMS Microbiol Lett 107:247–250 Xu X, Yan G, Kong R, Liu X, Yu L (2000) Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control. Curr Microbiol 41:352–356 Yoon H-S, Golden JW (1998) Heterocyst pattern formation controlled by a diffusible peptide. Science 282:935–938 Yoshihara S, Suzuki F, Fujita H, Geng X, Ikeuchi M (2000) Novel putative photoreceptor and regulatory genes required for the positive phototactic movement of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 41:1299–1304 Yoshihara S, Geng XX, Okamoto S, Yura K, Murata T, Go M, Ohmori M, Ikeuchi M (2001) Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 42:63–73 Yu R, Yamada A, Watanabe K, Yazawa K, Takeyama H, Matsunaga T, Kurane R (2000) Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococcus sp. Lipids 35:1061–1064 Yura K, Toh H, Go M (1999) Putative mechanism of natural transformation as deduced from genome data. DNA Res 6:75–82 Zhao N, Hashida H, Takahashi N, Misumi Y, Sakaki Y (1995) High-density cDNA filter analysis: a novel approach for largescale quantitative analysis of gene expression. Gene 156: 207–213. Zhu J, Kong R, Wolk CP (1998) Regulation of hepA of Anabaena sp. strain PCC 7120 by elements 5′ from the gene and by hepK. J Bacteriol 180:4233–4242 Zhulin IB (2000) A novel phototaxis receptor hidden in the cyanobacterial genome. J Mol Microbiol Biotechnol 2:491–493 Ziegler K, Diener A, Herpin C, Richter R, Deutzmann R, Lockau W (1998) Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). Eur J Biochem 254:154–159