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Journal of Bacteriology, May 2002, p. 2529-2532, Vol. 184, No. 9
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.9.2529-2532.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of an Akinete Marker Gene in Anabaena variabilis

Ruanbao Zhou^, and C. Peter Wolk^*

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

Received 16 November 2001/ Accepted 3 February 2002

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Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium. Because no akinete marker gene has been identified, akinete formation has been little studied genetically. We report the first identification of an akinete marker gene.

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The vegetative cells of many filamentous cyanobacteria, including Anabaena spp., can differentiate into encysted spores called akinetes as well as usually spaced, N₂-fixing cells called heterocysts ("other cysts"). Akinetes provide the capacity for germination after long-term exposure to stresses such as cold, desiccation, and phosphate limitation (1, 8, 13, 17). Available evidence supports the hypothesis that in Anabaena cylindrica and certain other taxa, heterocysts induce adjacent cells to become akinetes, generating a juxtaposition pattern, and thus present a prokaryotic example of an embryogenetic-type induction (10, 19). Although akinete and heterocyst differentiation may be related biochemically and perhaps evolutionarily (12, 21), much less is known about the differentiation of akinetes (1, 8) than about that of heterocysts (21, 22). Just as N₂ fixation as a marker has greatly aided genetic analysis of heterocyst differentiation, genetic analysis of akinete differentiation would be greatly facilitated by the identification of a marker gene for developing or mature akinetes. We provide the first report of a gene that is expressed primarily in akinetes.

A protein characteristic of akinetes Anabaena cylindrica strain ATCC 29414 was grown for 1 month in medium AA/8 (11). Heterocysts and akinetes were isolated (20) and broken by cavitation in the presence of fine glass beads (25% vol/vol in water) and phenylmethylsulfonyl fluoride (1 mM) (18). Vegetative-cell lysate was the supernatant solution obtained by passing a cell suspension (20% wt/vol in 50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) twice through a French press (American Instrument Co., Div. Travenol Laboratories, Inc., Silver Spring, Md.) at 600 lb/in² and sedimenting the eluate for 10 min at 3,200 rpm in a GLC-2B centrifuge (Dupont Sorvall, Newtown, Conn.). Total protein from each type of cell was treated with 10% trichloroacetic acid on ice for 2 h, sedimented at 16,000 x g for 30 min, washed twice with 80% methanol and twice with 80% acetone, boiled for 3 min in 300 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer per 2 liters of starting suspension culture, and clarified by centrifugation (16,000 x g, 20 min). The supernatant solution was subjected to SDS-PAGE, and proteins were stained with Coomassie brilliant blue R-250. In initial experiments, proteins with masses of 20, 43, and 66 kDa were observed in extracts of akinetes but not in extracts of vegetative cells or heterocysts (Fig. 1). After highly sensitive staining with silver (9), only the position of 43 kDa showed no band in extracts of vegetative cells or heterocysts (data not shown). Faint bands may have resulted from breakage of a small number of akinetes. The 43-kDa band is seen in a silver-stained gel as a negative band against a brownish background (Fig. 2, lane 3). Because protein AcAK43, purified from the 43-kDa band, like glycoproteins (15), is stained positively by a combination of periodic acid and silver nitrate (Fig. 2, lane 4), it may be a glycoprotein.

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FIG. 1. SDS-12% (A) and -15% (B) PAGE gels of total cell proteins of A. cylindrica stained by Coomassie brilliant blue R-250. Vertically arrayed numbers are sizes of standards in kilodaltons. The arrows indicate potential akinete-specific proteins. (A) Total cell protein extracted from akinetes (lane 1, 35 µg of protein) and vegetative cells (lane 2, 35 µg of protein). (B) Total cell protein extracted from vegetative cells (lane 1, 50 µg of protein), akinetes (lane 2, 30 µg of protein), and heterocysts (lane 3, 20 µg of protein).

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FIG. 2. SDS-12% PAGE gel, stained by silver nitrate (lanes 1 to 3) or by a combination of periodic acid and silver (lane 4). Numbers 14 to 97 refer to sizes of standards in kilodaltons. The arrows indicate candidate akinete-specific proteins. Five micrograms of total cell protein extracted from vegetative cells (lane 1), heterocysts (lane 2), and akinetes (lane 3) and 0.2 µg of purified 43-kDa, candidate akinete-specific protein (lane 4) were loaded.

Tryptic peptides and homology searching A 43-kDa band excised from SDS-PAGE gels was electroeluted into 1 ml of buffer (25 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 0.1% SDS). After dialysis overnight against redistilled H₂O at 4°C, the solution of eluted protein was lyophilized. The lyophilized powder, mixed with 1x SDS-PAGE loading buffer, was subjected to SDS-PAGE, and a protein band with a molecular mass of 43 kDa was collected for protein microsequencing. Two peptides were isolated from a tryptic digest of that band. Their sequences, LTTDYDYEEQVR and TVASGEASFR, were determined by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer at the Harvard Microchemistry Facility. Fragments similar in sequence were identified, by BLAST search (2), within the predicted product (AnAK) of gene all4050 (which we designate anaK) of the Kazusa database of PCC 7120 DNA (URL: http://www.kazusa.or.jp/cyano/Anabaena). Three proteins similar to AnAK, the products of the genes all4051, all5215, and alr5332, were found in the database of PCC 7120 sequences, and four more were found in that of Nostoc punctiforme sequences (URL: http://www.jgi.doe.gov/JGI_microbial/html/nostoc_homepage.html). Protein se-quences were aligned with Corpet's software (5) (Fig. 3). No protein similar to AnAK was predicted by the genome of Synechocystis sp. strain PCC 6803, a cyanobacterium that does not form akinetes.

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FIG. 3. Sequence alignment of homologues of AnAK. AnAK, AnAKb, AnAKc, and AnAKd were translated from Anabaena sp. strain PCC 7120; NoAK, NoAKb, NoAKc, and NoAKd were translated from the genome of N. punctiforme ATCC 29133; AvAK was translated from A. variabilis; and AcAK43 was from A. cylindrica.

Liquid cultures of Anabaena variabilis strain ATCC 29413 and derivatives of it were grown at 30°C in the light on a rotary shaker in medium AA/8 supplemented with appropriate antibiotics (4) and with or without nitrate. avaK' was amplified from genomic DNA of A. variabilis by use of the primers 5'-GGAATTCCATATGATTAAGAGGCATTTTATATATTTGAGG-3' and 5'-CGGGATCCTTAGCGTTCTTCAATGGGAAGACCAGGAGCATT-3', based on anaK. The PCR product was cloned into the EcoRV site of pBluescript SK⁺ (Stratagene, La Jolla, Calif.), producing pRL2476, and was sequenced. The sequence of the native gene, avaK, was subsequently determined from multiple, independently obtained PCR products using primers based on sequences 5' (upstream) and 3' (downstream) from anaK. avaK' differs from avaK only in two C-terminal primer-determined base pairs, but not in predicted amino acid sequence. avaK showed 93% nucleotide sequence identity to anaK, and AvAK showed 96.5% amino acid identity to AnAK, confirming homology.

Localization of expression of avaK Because A. cylindrica is not genetically manipulable, we sought to test in which cells of A. variabilis there is expression of avaK. A 1,055-bp SpeI-XhoI avaK'-containing fragment from pRL2476 was ligated between the SpeI and XhoI sites of pRL278 (4), which bears a neomycin-resistance determinant, producing pRL2721. gfp encodes green fluorescent protein (GFP) (6), and the omega interposon (16) bears a streptomycin- and spectinomycin-resistance determinant. A cassette bracketed by SmaI and PvuII sites and bearing promoterless gfp plus, for selection, the omega interposon was transferred from pRL2379 (23) to the unique SmaI site 25 bp downstream from the stop codon of avaK' in pRL2721. In the product, pRL2722a, avaK' is transcriptionally fused to gfp. To enhance transfer to A. variabilis, a unique AvrII site was destroyed by deletion from it to a nearby SpeI site, both in the vector portion of pRL2722a, producing pRL2726.

pRL2726 was transferred to A. variabilis by conjugation (7, 14) with selection on 30 µg of kanamycin sulfate ml^-1 (14). Homologous recombination between pRL2726 and chromosomal DNA was confirmed by both Southern blotting and diagnostic PCR (data not shown). Single recombinant A. variabilis::pRL2726 (also known as SR2726) bore a copy of avaK and a copy of avaK', one fused transcriptionally to gfp. Fluorescence was visualized from SR2726 grown for 12 ± 2 days in AA/8 medium containing 5 µg of neomycin sulfate ml^-1 plus 1 µg of spectinomycin ml^-1. An Axiophot microscope (Carl Zeiss) with a x100 oil immersion objective was used to obtain bright-field images and images of fluorescence, the latter using bandpass filters providing actinic illumination of 470 ± 20 nm and emission at 510 ± 11.5 nm. Images were captured with an MDS100 digital video camera (Eastman Kodak Co., Rochester, N.Y.) and processed with Adobe Photoshop 5.5.

Akinetes and heterocysts of A. variabilis both form a conspicuous envelope, but they are distinguishable as follows. Whereas the pole of the heterocyst is perforated by a cytoplasmic channel which is surrounded by a thick layer of glycolipid, no such structure is found in the akinete. Often, the akinete is also more strongly pigmented and has a more granular interior. Although very slight fluorescence at wavelengths characteristic of GFP emission was visualized with the wild-type strain (Fig. 4F), GFP-based fluorescence originated primarily from akinetes (Fig. 4A to E). Therefore, expression of avaK is primarily, perhaps completely, akinete specific. When pRL2726 was fused to Anabaena sp. strain PCC 7120, which is not known to form akinetes, no GFP-based fluorescence was observed (data not shown). The inability of PCC 7120 to form akinetes despite the presence of anaK is attributable to a spontaneous mutation ("many cyanobacteria which formed akinetes when first isolated can no longer do so" [8]).

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FIG. 4. (A to E) Fluorescence from an avaK43::gfp fusion in A. variabilis::pRL2726. (F) Wild-type A. variabilis. Heterocysts (h) and akinetes (a) are indicated, and vegetative cells are also seen.

Because of known similarities in the chemical composition of the envelopes of heterocysts and akinetes (21) and because germination of akinetes gives rise to filaments of vegetative cells, where might proteins unique to akinetes be present? One possibility is additional envelope layers (3); another might be proteins related specifically to the response of akinetes to the stresses to which they confer resistance. Unfortunately, the sequence of AvAK provides no obvious clues to its function.

Nucleotide sequence accession number The sequence of the native gene, avaK, was submitted to GenBank under accession no. AY072915.

 
This work was supported by the U.S. Department of Energy under grant DOE-FG02-91ER20021 and by NSF grant MCB 9723193.

 
* Corresponding author. Mailing address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-2049. Fax: (517) 353-9168. E-mail: wolk{at}msu.edu.

Present address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Permanent address: College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, People's Republic of China.

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Journal of Bacteriology, May 2002, p. 2529-2532, Vol. 184, No. 9
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.9.2529-2532.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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