Autoradiographic localization of 13N after fixation of 13N-labeled nitrogen gas by a heterocyst-forming blue-green alga.

(13)N, generated by proton bombardment of (13)C powder, is rapidly and easily converted to (13)N-N(2), 0.01 atm pressure, ca. 10 mCi/ml, by automated Dumas combustion. (13)N fixed (as (13)N-N(2)) by algal filaments was localized by an autoradiographic technique which permits track autoradiography with isotopes having short half-lives. Our findings show directly that a minimum of about 25% of the N(2) fixation by intact, aerobically grown filaments of Anabaena cylindrica is carried out by the heterocysts. If all of the N(2) fixation takes place in the heterocysts, then the movement of nitrogen along the filaments can be characterized by a constant tau < ca. 5 s (cell(-2)).

Genetic analysis of cyanobacterial development.

Filamentous cyanobacteria, the only prokaryotes that form linear patterns of differentiated cells, can be genetically manipulated by the conjugative transfer of plasmids from Escherichia coli. It has become possible to determine the cellular localization of genetic transcription, including transcription of developmentally critical genes before morphological differentiation takes place, by using luciferase as a reporter. These techniques are facilitating developmental analysis.

Synthesis of nitrogenase by isolated heterocysts.

Heterocysts isolated from Anabaena variabilis incorporate [14C]leucine and [35S]methionine into trichloroacetic acid-precipitable material in the light. Analysis by polyacrylamide gel electrophoresis shows that the radioactivity is present in polypeptides of discrete sizes. However, the relative proportions of different proteins synthesized by isolated heterocysts differ from the relative proportions of those proteins incorporated by the heterocysts in intact filaments. The two components of nitrogenase are among the proteins synthesized by the isolated heterocysts.

Isolated heterocysts of Anabaena variabilis synthesize envelope polysaccharide.

Isolated heterocysts, incubated for 1 h at 30 degrees C [14C]mannose, synthesize [14C]arabinose and [14C]glucose, and incorporate the three 14C-labeled sugars into their envelopes with glycosidic linkages characteristic of their envelope polysaccharide. In extracts of metabolic intermediates with hot 80% methanol, [14C]mannose is associated with the nucleotide GDP and [14C]glucose and [14C]arabinose with UDP. Chloroform/methanolic extracts of the heterocysts contain phosphoglycolipids in which 14C-labeled mannose, arabinose, and glucose are present. The lipids have the same as dolichol phosphate mannose under varying chromatographic conditions, RF and like polyisoprenol monophosphate glycolipids are stable to treatment with mild alkali but labile to mild acid hydrolysis. If bacitracin is added to the incubation mixture, 14C-labeled nucleotide sugars accumulate, but incorporation of 14C into envelope polysaccharide is greatly diminished. This observation supports the interpretation that polyisoprenol phosphoglycolipids are intermediates in the biosynthesis of this polysaccharide.

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.

Expression of luxCD-E in Anabaena sp. can replace the use of exogenous aldehyde for in vivo localization of transcription by luxAB.

The genes luxCDABE from four luminescent bacteria suffice for light production in Escherichia coli [Meighen, Microbiol. Rev. 55 (1991) 123-142]. We have inserted these gene clusters between inverted polylinkers, and placed the resulting cassettes as reporters within derivatives of transposon Tn5. Anabaena sp. strain PCC 7120 was mutagenized with these transposons. The luminescence of all but the most highly self-luminescent resulting derivatives of Anabaena sp. was strongly dependent on exogenously added aldehyde. Thus, luminescence based on luxCDABE is multiplicatively limited by production of luciferase and aldehyde. No toxicity was observed over protracted periods of luminescence. By deletion, new cassettes were derived in which only the aldehyde biosynthetic genes, luxCD-E, remained intact. Transcription was localized at the single-cell level in strains of cyanobacteria bearing constitutively expressed Xenorhabdus luminescens luxCD-E on a plasmid and relatively weakly expressed, developmentally regulated luxAB from Vibrio spp. in the chromosome. The developmentally critical gene, hetR, was thereby shown to remain active in mature heterocysts.

Identification of the region of cyanobacterial plasmid pDU1 necessary for replication in Anabaena sp. strain M-131.

Shuttle vectors based on plasmid pDU1 from Nostoc sp. strain PCC7524 are able to replicate both in Escherichia coli and in strains of Anabaena and Nostoc spp. Derivatives partially deleted in the pDU1 portion were tested for their ability to replicate in Anabaena sp. strain M-131. Plasmid pRL6HE containing a 1.75-kb HindIII-ScaI fragment of pDU1 replicated stably in that cyanobacterium and also in Anabaena sp. strain PCC7120. Plasmid pRL6HC, containing an even smaller HpaI-ScaI fragment (1.3 kb) replicated in Anabaena sp. strain M-131 but not in Anabaena sp. strain PCC7120. Similarly, when the 1.75-kb fragment of pDU1 was transferred from pRL6HE to another vector (pRL40 delta), the resulting plasmids replicated in Anabaena sp. strain M-131 but not in Anabaena sp. strain PCC7120.

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers.

Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147-151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 x 366 to 2 x 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp. PCC 7120.

Fur (ferric uptake regulator) proteins are principally responsible for maintaining iron homeostasis in prokaryotes. Iron is usually a scarce resource. Its limitation reduces photosynthetic rates and cell growth in cyanobacteria in general and especially in cyanobacteria that are fixing dinitrogen, a process that requires the synthesis of numerous proteins with a high content of iron. This paper shows that in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120, levels of furA mRNA and FurA protein increase significantly in response to nitrogen deprivation, and that furA up-regulation takes place specifically in proheterocysts and mature heterocysts. Great differences in a Northern blot, probed with furA, of RNA from an ntcA mutant relative to wild-type Anabaena sp. were attributable to binding of NtcA, a global regulator of nitrogen metabolism, to the promoter of furA and to the promoter of the furA antisense transcript alr1690-alpha-furA.

Heterocyst formation.

Heterocysts are microaerobic, N2-fixing cells that form in a patterned array within O2-producing filamentous cyanobacteria. Structural features of heterocysts can be predicted from consideration of their physiology. This review focuses on the spacing mechanism that determines which cells will differentiate, and on the regulation of the progression of the differentiation process. Applicable genetic tools, developed primarily using Anabaena PCC 7120, but employed also with Nostoc spp., are reviewed. These tools include localization of transcription using fusions to lux, lac, and gfp, and mutagenesis with oriV-containing derivatives of transposon Tn5. Mature and developing heterocysts inhibit nearby vegetative cells from differentiating; genes patA, devA, hetC, and the hetMNI locus may hold keys to understanding intercellular interactions that influence heterocyst formation. Regulatory and other genes that are transcriptionally activated at different times after nitrogen stepdown have been identified, and should permit analysis of mechanisms that underlie the progression of heterocyst differentiation.

Role of bromine in the formation of the refractile inclusions of the vesicle cells of the Bonnemaisoniaceae (Rhodophyta).

Refractile inclusions characteristic of the vesicle cells of members of the Bonnemaisoniaceae fail to form if bromide is omitted from the culture medium. Electron microprobe analysis shows the localization of bromine in these cells. When grown in the presence of bromide, but not when grown in its absence, these organisms oxidize iodide to iodine; in Trailliella, this oxidation occurs at the vesicle cells.

Movement of carbon from vegetative cells to heterocysts in Anabaena cylindrica.

Radioactive carbon assimilated by vegetative cells of Anabaena cylindrica in the light passed via an intrafilamentous route into heterocysts in the dark. After several hours, label per heterocyst approximated label per vegetative cell. Much of the label entering heterocysts was not available for diffusional exchange back into vegetative cells.

Role for hetC in the transition to a nondividing state during heterocyst differentiation in Anabaena sp.

Nitrogen-deprived filaments of wild-type or hetC Anabaena sp. produce respectively, at semiregular intervals, heterocysts and weakly fluorescent cells. Unlike heterocysts, the latter cells can divide and elongate, producing a pattern of spaced series of small cells. Because a hetR::gfp fusion is expressed most strongly in the small cells, we propose that these small cells represent a very early stage of heterocyst differentiation. hetC::gfp is expressed most strongly in proheterocysts and heterocysts.

Use of filamentous cyanobacteria for biodegradation of organic pollutants.

Volume 61, no. 1, p. 234: the corresponding author footnote should read as follows: * Corresponding author. Present address: Center for Risk Management, Oak Ridge National Laboratory, Oak Ridge, TN 37830. Phone: (615) 241-6013. Fax: (615) 574-9887. [This corrects the article on p. 234 in vol. 61.].

Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing.

Transposon-generated mutant N10 of Anabaena sp. strain PCC 7120 has a Het- phenotype (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation reproduced a Het- phenotype, but reconstructions with other insertions at the position of the transposon produced strains that form multiple contiguous heterocysts. Sequence analysis around the site of insertion of the transposon showed that the insertion lies within the 5' end of an 861-bp open reading frame (ORF) (hetN). The product of translation of hetN (HetN) shows extensive similarity to NAD(P)H-dependent oxidoreductases that are involved in biosyntheses of fatty acids, poly-beta-hydroxybutyrate, nod factor, and polyketides. A second, 1,518-bp ORF (hetM) that ends 556 bp 5' from the start of hetN appears to encode a protein that has at least two functional domains: its amino terminus is similar to an acyl carrier protein, while its central portion is similar to domains of proteins that perform reductive reactions. A third, 711-bp ORF (hetI) encoded on the opposite strand ends 42 bp away from the 3' end of hetN. The protein encoded by hetI, HetI, is similar to Sfp from Bacillus subtilis and EntD from Escherichia coli, proteins that are required for the biosynthesis or export of cyclic peptides. Clones from a lambda-EMBL3 library that contain the wild-type DNA for hetN do not complement the hetN::Tn5-1063 mutation in N10. The presence of hetN, as the only ORF, on a replicating plasmid suppresses heterocyst formation in wild-type cells, whereas the additional presence of hetI alleviates this effect.

Genetic tools for cyanobacteria.

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.