Identification of Nostoc punctiforme akinete-expressedgenes using differential displayClaudia Argueta, Kamile Yuksek, Ronak Patel andMichael L. Summers*California State University Northridge, Department ofBiology, 18111 Nordhoff St. Northridge, CA 91330-8303,USA.SummaryAkinetes are spore-like resting cells formed by certainfilamentous cyanobacteria that have increased resis-tance to environmental stress. They can be found atlow frequencies in dense cultures experiencing lowlight or phosphate limitation, but also form at highfrequencies in a zwf mutant strain of Nostoc puncti-forme following dark incubation in the presence offructose. The wild-type strain is capable of facultativeheterotrophic growth under these conditions anddoes not form akinetes. To identify genes associatedwith akinete development, differential display wasused to amplify and compare cDNA from a wild-typeand zwf mutant strain of N. punctiforme following aswitch to dark heterotrophic conditions. Screening ofcandidate genes by reverse transcriptase real-timequantitative PCR and subsequent testing for akinete-specific expression using GFP transcriptionalreporter plasmids lead to the identification of threenovel akinete-expressed genes. The genes identifiedfrom the screening encoded for proteins homologousto an aminopeptidase (aapN), a zinc protease (hap)and an ATP-binding cassette (ABC)-type transporter(aet). Expression of hap was also increased in devel-oping hormogonia, a transient type of differentiatedfilament capable of gliding motility. Transcriptionalstart sites for akinete-expressed genes were deter-mined using random amplification of cDNA ends(RACE), and promoter regions were compared withorthologues in other filamentous cyanobacteria toidentify putative regulatory sequences.IntroductionNostoc punctiforme is a filamentous cyanobacteriumcapable of differentiating its normal vegetative cells intonitrogen-fixing heterocysts, motile hormogonia or spore-like akinetes (Meeks et al., 2002). Like others compris-ing the subset of strains capable of akinete formationin the order Nostocales and Stigonematales, akineteinduction by N. punctiforme usually occurs in culturesexposed to light limitation or phosphate starvation(Campbell et al., 1996; Wong and Meeks, 2002),although low intracellular ATP levels may not be thedirect signal for their formation (Argueta and Summers,2005). The size and position of akinetes relative to het-erocysts vary between strains, with those of N. puncti-forme appearing only slightly larger than vegetativecells, forming first midway between heterocysts withinthe filament. Subsequent akinetes then form adjacent tothe first and spread out towards the heterocysts. In fila-ments grown with nitrate or ammonia that lack hetero-cysts, akinetes appear randomly in the filaments (Meekset al., 2002). As akinetes mature, they become morerounded than vegetative cells, and separate from thefilament as cell–cell contacts diminish.Akinetes contain a thickened envelope composed ofpolysaccharides and glycolipids (Cardemil and Wolk,1981; Soriente et al., 1993), and usually contain refractilegranules of storage material for carbon in the form ofglycogen, and nitrogen in the form of cyanophycin(copolymers of arginine and aspartate). The importance ofcyanophycin for akinete germination has been demon-strated in a Nostoc ellipsosporum arginine biosynthesismutant incapable of accumulating this polymer (Leganeset al., 1998). These characteristics, and others yet to bediscovered, are likely to be required for functional aki-netes capable of surviving environmental extremes thatkill normal vegetative cells, and their subsequentgermination. Although not heat-resistant like theendospores of Gram-positive bacteria, cyanobacterial aki-netes have been reported to survive 5–7 years of desic-cation (Yamamoto, 1975; Sili et al., 1994), months of cold(4°C) dark conditions (Sutherland et al., 1979), and havebeen isolated from sediments as old as 64 years (Living-stone and Jaworski, 1980). Little is known about themolecular basis for such resistance to environmentalextremes.The little that is known about regulation of akinete dif-ferentiation has been gleaned through effects of genesoriginally identified for their role in heterocystdifferentiation. Overproduction of DevR, a response regu-lator component of a two-component regulatory systemAccepted 30 May, 2006. *For correspondence. E-mail [email protected]; Tel. (+1) 818 677 7146; Fax (+1) 818 677 2034.Molecular Microbiology (2006) 61(3), 748–757 doi:10.1111/j.1365-2958.2006.05263.xFirst published online 15 June 2006© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltdinvolved in heterocyst polysaccharide synthesis, causes-increased akinete induction in N. punctiforme, possiblydue to cross-talk (Campbell et al., 1996). A second tran-scriptional regulatory protein, HetR, was shown to beessential for both heterocyst and akinete formation inN. ellipsosporum (Leganes et al., 1994); however, a hetRmutant strain of N. punctiforme was able to form cold-resistant akinete-like cells that lacked obvious granulationtypical of akinetes (Wong and Meeks, 2002). In addition,the hepA gene, shown to be essential for formation of apolysaccharide layer in heterocysts (Wolk et al., 1994),was also required for normal akinete envelopes of Ana-baena variabilis (Leganes, 1994). These results support acommon envelope and possible storage materialsbetween heterocysts and akinetes, but gives no clearevidence for a common regulatory pathway controllingtheir differentiation.Prior to this report, the only study focusing specificallyon identification of genes involved in akinete formation orfunction utilized a proteomic approach (Zhou and Wolk,2002). A highly expressed protein from Anabaena cylin-drica akinetes was identified, and the orthologous genefrom Anabaena sp. strain PCC 7120 (A. PCC 7120) wasused to design primers for amplification of avaK genefrom the genome of A. variabilis.AnA. variabilis strainbearing the avaK promoter region fused to a GFP tran-scriptional reporter exhibited high levels of expression inakinetes as compared with heterocysts or vegetativecells, confirming this gene as an akinete-marker gene(Zhou and Wolk, 2002). The orthologue of avaK inN. punctiforme is similarly regulated (Argueta et al.,2004), but its function remains unknown.Although the germination of akinetes has receivedmuch attention (Chauvat et al., 1982; Sutherland et al.,1985; Sili