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Microbiology (2012), 158, 380–389 DOI 10.1099/mic.0.054783-0 The putative phosphatase All1758 is necessary for normal growth, cell size and synthesis of the minor heterocyst-specific glycolipid in the cyanobacterium Anabaena sp. strain PCC 7120 Sasa K. Tom and Sean M. Callahan Correspondence Sean M. Callahan [email protected] Received 16 September 2011 Revised 30 October 2011 Accepted 1 November 2011 Department of Microbiology, University of Hawaii, Honolulu, HI 96822, USA The filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocysts arranged in a periodic pattern when deprived of a fixed source of nitrogen. In a genetic screen for mutations that prevent diazotrophic growth, open reading frame all1758, which encodes a putative serine/threonine phosphatase, was identified. Mutation of all1758 resulted in a number of seemingly disparate phenotypes that included a delay in the morphological differentiation of heterocysts, reduced cell size, and lethality under certain conditions. The mutant was incapable of fixing nitrogen under either oxic or anoxic conditions, and lacked the minor heterocyst-specific glycolipid. Pattern formation, as indicated by the timing and pattern of expression from the promoters of hetR and patS fused transcriptionally to the gene for green fluorescent protein (GFP), was unaffected by mutation of all1758, suggesting that its role in the formation of heterocysts is limited to morphological differentiation. Transcription of all1758 was constitutive with respect to both cell type and conditions of growth, but required a functional copy of all1758. The reduced cell size of the all1758 mutant and the location of all1758 between the cell division genes ftsX and ftsY may be indicative of a role for all1758 in cell division. Taken together, these results suggest that the protein encoded by all1758 may represent a link between cell growth, division and regulation of the morphological differentiation of heterocysts. INTRODUCTION Heterocysts are terminally differentiated, non-dividing cells that allow the simultaneous execution of two incompatible processes, fixation of nitrogen and oxygen-evolving photosynthesis, in some filamentous cyanobacteria. They maintain a micro-oxic environment where oxygen-labile nitrogenase can function. The envelope of heterocysts includes a layer of distinct glycolipid that reduces the diffusion of molecular oxygen into cells and an outer layer of heterocyst-specific exopolysaccharides that protect the integrity of the glycolipid layer, and increased respiration reduces the level of molecular oxygen that does enter heterocysts (Murry & Wolk, 1989). Fixed nitrogen from heterocysts supports the growth of vegetative cells in the filament, which in turn supply heterocysts with fixed carbon (Meeks et al., 1978; Wolk, 1968). In the cyanobacterium Anabaena sp. PCC 7120, under conditions that require fixation of nitrogen for growth, heterocysts are arranged in a periodic pattern at intervals of approximately 10 cells along unbranched filaments (Wolk et al., 1994). Abbreviations: HGL, heterocyst-specific glycolipid; ORF, open reading frame; pNPP, 4-nitrophenol phosphate. 380 Activation of the heterocyst differentiation signalling pathway in response to nitrogen limitation leads to an accumulation of the metabolite 2-oxoglutarate in cells (Laurent et al., 2005). Binding of 2-oxoglutarate stimulates the DNA-binding activity of the global transcriptional regulator of nitrogen and carbon metabolism in cyanobacteria, NtcA (Vázquez-Bermúdez et al., 2002). NtcA indirectly leads to upregulation of the master regulator of heterocyst differentiation, hetR, which feeds back on the regulation of transcription of ntcA (Muro-Pastor et al., 2002) and, together with NtcA, directly or indirectly activates genes involved in the patterning and morphogenesis of heterocysts (Olmedo-Verd et al., 2005). Induction of differentiation, pattern formation and morphogenesis involves the transcription of hundreds of genes specific for the formation of heterocysts. The reversibility of protein phosphorylation mediated by kinases and phosphatases is an essential part of many signalling cascades and regulatory networks. In Anabaena sp., genes with homology to signalling components have been reported to influence nitrogen metabolism and the early regulatory stages of heterocyst development. NrrA, which encodes a response regulator of the OmpR family, acts early in the differentiation process by directly Downloaded from www.microbiologyresearch.org by 054783 G 2012 SGM IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 Printed in Great Britain all1758 and heterocyst development upregulating hetR expression (Ehira & Ohmori, 2006). The PP2C-type protein phosphatases PrpJ1 and PrpJ2 are also involved in the initiation of heterocyst development through mutual regulation of both ntcA and hetR (Jang et al., 2009). The protein kinases Pkn41 and Pkn42, which contain serine/threonine kinase and histidine kinase domains, respectively, are cotranscribed specifically under iron-deficient conditions and regulated by NtcA (Cheng et al., 2006). The nitrogen regulatory protein PII encoded by glnB is differentially modified in the two cell types. It goes from a phosphorylated to a non-phosphorylated state during transition from vegetative cell to heterocyst (Laurent et al., 2004). The pknE gene lies 301 bp downstream from the protein phosphatase 1/2A/2B homologue encoded by prpA. Both pknE and prpA inactivation mutants produce aberrant heterocysts (Zhang et al., 1998). Overexpression of the putative Ser/Thr kinase encoded by pknE from its native promoter inhibited heterocyst development, possibly through inhibition of HetR (Saha & Golden, 2011). Another protein that appears to affect HetR, PatA, contains a phophoacceptor domain with homology to the response regulator CheY, although a corresponding histidine kinase has not been isolated. It appears to attenuate the negative regulation of heterocyst differentiation by PatS and HetN (Orozco et al., 2006), while promoting the activity, and limiting the accumulation, of HetR (Risser & Callahan, 2008). Protein phosphorylation has also been implicated in the later stages of development, in particular during the process of heterocyst maturation. Formation of the minor heterocyst-specific glycolipid (HGL) involves two protein kinases of the hstK family, Pkn30 and Pkn44, which contain both an N-terminal serine/threonine kinase domain and a C-terminal histidine kinase domain (Shi et al., 2007). PrpJ1 has been shown to regulate the synthesis of the major HGL (Jang et al., 2007). Alr0117 and HepK (All4496) both encode putative two-component system sensory histidine kinases that appear to be involved in the induction of hepA, one of the genes responsible for synthesis of the heterocyst polysaccharide layer (Ning & Xu, 2004; Ramı́rez et al., 2005; Xu et al., 2003). The manganese-dependent serine/threonine protein phosphatase of the PPP family DevT (Alr4674) accumulates in mature heterocysts, is not regulated by NtcA and appears to have a role in the later steps of heterocyst differentiation (Espinosa et al., 2010). In this study, the isolation and characterization of a gene encoding a putative PP2C-type protein phosphatase, all1758, is described. METHODS Bacterial strains and growth conditions. Strains used in this study are detailed in Table 1. Growth of Escherichia coli and Anabaena sp. strain PCC 7120 and its derivatives; concentrations of antibiotics; induction of heterocyst formation in BG-110 medium, which lacks a combined-nitrogen source; regulation of PpetE expression; and conditions for photomicroscopy were as previously described (Risser & Callahan, 2007). Images were processed in Adobe http://mic.sgmjournals.org Photoshop CS2. To avoid complications from the vacuolization phenotype observed with strains UHM183 and UHM184 upon growth in BG-11 liquid medium, which contains a source of combined nitrogen, these strains were transferred from solid BG-11 medium to liquid BG-110 medium for induction of heterocyst differentiation. Plasmids were conjugated from E. coli into Anabaena strains as described by Elhai & Wolk (1988). Construction of plasmids. Plasmids used in this study are detailed in Table 1 and oligonucleotides in Table 2. Constructs derived by PCR were sequenced to verify the integrity of the sequence. Plasmid pST112 was used to delete most of the coding region of all1758. An 852 bp region containing the first 30 bp of the all1758 coding region and upstream DNA was amplified from the chromosome (with primers 1758 up F and 1758 up R) and fused to an 848 bp region containing the last 30 bp of the all1758 coding region and downstream DNA (using primers 1758 down F and 1758 down R) via overlap extension PCR. The 1700 bp fragment was cloned into pRL277 as a BglII–SacI fragment using restriction sites introduced on the primers to generate pST112. Plasmid pST114 was used to replace most of the coding region of all1758 with an V interposon. The 1700 bp fragment used for construction of pST112 was moved into the EcoRV site of pBluescript SK+ (Stratagene) to generate pST110. The 1700 bp fragment was moved from pST110 into pRL278 as a BglII–SacI fragment to create pST113. To generate pST114, the 2082 bp V interposon, which confers resistance to spectinomycin and streptomycin (Fellay et al., 1987), was introduced into pST113 at a SmaI site generated during overlap extension PCR. pST141 is a mobilizable shuttle vector containing the putative promoter and coding regions of all1758. An 1866 bp fragment was amplified from the chromosome (using primers Pall1758 BamHI F and all1758 SacI R) and cloned into pAM504 as a BamHI–SacI fragment using restriction sites engineered on the primers to generate pST141. The translational all1758–gfp reporter fusion under the control of the all1758 promoter was created using primers Pall1758 BamHI F and all1758 SmaI R to amplify the 1866 bp fragment from the chromosome. The fragment was moved as a BamHI–SmaI fragment into pSMC232, a replicating plasmid used to generate translational fusions to gfp (Risser & Callahan, 2008), to give pST150. To create the gfp transcriptional reporter for all1758, a 474 bp region upstream of all1758 was amplified from the chromosome with primers Pall1758 SacI F and all1758 15 bp up SmaI R and cloned into pAM1956 as a SacI–SmaI fragment to create pST151. To create the gfp transcriptional reporter for all1759, a 304 bp region upstream of all1759 was amplified from the chromosome using primers Pall1759SacI-F and Pall1759-SmaI-R and moved into the EcoRV site of pBluescript SK+ to generate pST252. The fragment was moved from pST252 as a SacI–SmaI fragment into pAM1956 to create pST249. Two plasmids carrying a PpetE–all1758 transcriptional fusion were constructed. Plasmid pST156 is a mobilizable shuttle vector carrying a transcriptional fusion between the petE promoter and all1758. The 1392 bp coding region of all1758 was amplified from the chromosome using primers all1758 OE EcoRI F and all1758 OE BamHI R and ligated into the EcoRV site of pBluescript SK+ to create pST184. The fragment was moved from pST184 as an EcoRI–BamHI fragment into pKH256 (Higa & Callahan, 2010), which is designed to create transcriptional fusions to the petE promoter, to generate pST156. Plasmid pST367 differs from pST156 in the engineered ribosome-binding site that was introduced by using primer all1758 EcoRI rbs F in place of all1758 OE EcoRI F to amplify all1758 from the chromosome. The PCR fragment was moved into the EcoRV site of pBluescript SK+ to generate pST365, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 381 S. K. Tom and S. M. Callahan Table 1. Strains and plasmids used in this study Strain or plasmid Anabaena sp. PCC 7120 UHM103 UHM128 UHM183 UHM184 Plasmids pAM504 pAM505 pAM1951 pAM1956 pDR138 pDR211 pDR320 pPROEX-1 pRL277 pRL278 pSMC127 pSMC148 pSMC188 pSMC232 pST112 pST114 pST141 pST150 pST151 pST156 pST188 pST249 pST367 pST368 Relevant characteristic(s) Wild-type DhetR Dpbp6 Dall1758 Dall1758 with V cassette Pasteur Culture Collection Buikema & Haselkorn (2001) Nayar et al. (2007) This study This study Shuttle vector for replication in E. coli and Anabaena; Kmr Neor Shuttle vector pAM504 with inverted multiple cloning site pAM505 with PpatS–gfp pAM505 bearing promoterless gfp for transcriptional fusions pAM504 carrying PhetR–hetR pAM504 carrying PpetE–patS pAM504 carrying PpetE–hetN Expression vector for generating polyhistidine epitope-tagged proteins; Apr Suicide vector; Smr Spr Suicide vector; Neor pAM504 carrying PhetR–gfp pPROEX-1 carrying hetR pAM504 bearing Pnir for transcriptional fusions pAM504 bearing promoterless gfp for translational fusions Suicide plasmid used to delete all1758 Suicide plasmid used to replace all1758 with an V interposon pAM504 carrying Pall1758–all1758 pSMC232 with Pall1758–all1758 pAM1956 with Pall1758 fused to gfp pAM505 bearing PpetE–all1758 pPROEX-1 carrying all1758 pAM1956 with Pall1759 fused to gfp pAM505 bearing PpetE–all1758 pAM504 bearing Pnir–all1758 Wei et al. (1994) Wei et al. (1994) Yoon & Golden (2001) Yoon & Golden (2001) Nayar et al. (2007) Risser & Callahan (2009) Risser & Callahan (2009) Life Technologies Black et al. (1993) Black et al. (1993) Callahan & Buikema (2001a) Risser & Callahan (2008) Risser & Callahan (2007) Risser & Callahan (2008) This study This study This study This study This study This study This study This study This study This study and subsequently moved from pST365 as an EcoRI–BamHI fragment into pKH256 to generate pST367. The pPROEX-1 (Life Technologies) derivative, pST188, was used to overexpress all1758 in E. coli and facilitate purification. A 1392 bp region containing the coding region for all1758 was amplified from the chromosome using primers all1758-NdeI-F and all1758-NH6NotIR, and moved into the EcoRV site of pBluescript SK+ to create pST192. To generate pST188, the fragment was moved as a NdeI–NotI fragment into the pPROEX-1 backbone derived from pSMC148 (Risser & Callahan, 2008) digested with NdeI–NotI. The resulting open reading frame (ORF), designated all1758(H6), encodes a glycine, then six histidine codons preceding a 16-residue spacer (DYDIPTTENLYFQGAH, which contains a sequence recognized by the Tobacco Etch Virus protease) fused to all1758 followed by a stop codon after the last codon of all1758. The transcriptional fusion Pnir–all1758 present on plasmid pST368 was created using the primers all1758 rbs EcoRI F and all1758 OE BamHI R to amplify all1758 from the chromosome. The fragment was ligated into the EcoRV site of pBluescript SK+ to generate pST366. To create pST368, the fragment was moved as an EcoRI–BamHI fragment into pSMC188, a replicating plasmid used to generate transcriptional fusions to the nirA promoter (Risser & Callahan, 2007) Strain construction. Deletion of the all1758 coding region was performed as previously described (Borthakur et al., 2005), using 382 Source or reference plasmid pST112 or pST114 and Anabaena sp. strain PCC 7120 to yield strains UHM183 and UHM184, respectively. The resultant strains were screened via colony PCR with primers all1758 flank up F and all1758 flank down R, which anneal outside the chromosomal region introduced onto pST112 and pST114 for deletion of all1758, and tested for sensitivity and resistance to the appropriate antibiotics. Primers all1758 39 F and all1757 59 R were used in a PCR to test if recombination between plasmid pST141, which harbours Pall1758– all1758, and the chromosomal locus of all1758 of strain UHM183 had occurred. Overexpression and purification of recombinant All1758 and phosphatase assays. Recombinant All1758 was produced from E. coli BL21(DE3) transformed with pST188 using Ni-NTA affinity chromatography (Qiagen) as described previously (Risser & Callahan, 2007) with the following exception: 4 ml wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 20 %, v/v, glycerol) was added to the column twice, followed by four 500 ml volumes of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 20 % glycerol), each diluted fourfold directly into a total volume of 1.5 ml capture buffer (50 mM NaH2PO4, 300 mM NaCl) upon elution from the column. Assays for phosphatase activity were conducted as described by Ruppert et al. (2002). The substrate, 4-nitrophenol phosphate (pNPP), was dissolved in pNPP buffer containing 10 mM Tris/HCl, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 Microbiology 158 all1758 and heterocyst development Table 2. Oligonucleotides used in this study Primer no. Primer name Sequence (5§ to 3§) 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 1758 up F 1758 up R 1758 down F 1758 down R all1758 flank up F all1758 flank down R all1758 39 F all1757 59 R Pall 1758 BamHI F all1758 SacI R all1758 SmaI R Pall1758 SacI F all1758 15 bp up SmaI R Pall1759-SacI-F Pall1759-SmaI-R all1758 OE EcoRI F all1758 OE BamHI R all1758-NdeI-F all1758-NH6-NotIR all1758 EcoRI rbs F all1758 rbs EcoRI F asr5349 RT F asr5349 RT R asr5350 RT F asr5350 RT R all1757 RT F all1757 RT R all1759 RT F all1759 RT R rnpB RT F rnpB RT R AGATCTGGTGGTGCGATCGCTTGGAG GCCACGACTGTCCCCGGGTGCCTGTTGTTATTATCACG ACGGACAACAATAATCCCGGGCGGTGCTGACAG GAGGCTCGCAGCCGCCCAGCTGTATCTAC GCCCAAGCACGAAATACAG CTTCCCGGTAAACTGTTGG GCTAGAACCTGGTGATACAG ACAGACTGAGAGTTACGTGC TATATGGATCCTAGAAGCTCTCTTGAGTGGC ATATAGAGCTCCCCAGTCCCTTTTATACTCG ATATACCCGGGATTCGATCTGTAAGACCAC TATATGAGCTCTAGAAGCTCTCTTGAGTGGC ATATACCCGGGAAAGGGGTTAATTTTAGATTGAC TATATGAGCTCCCAAAAAAAGGCGCTGAGTG ATATACCCGGGTAGTTGTTACGAGTTATGAG TATATGAATCCATGTGCCTGTGC CTCCATTTTC TATATGGATCCCCCAGTCCCTTTTATACTCG ATATACATATGCCTGTGCCTCCATTTTCCTCTCAAC TTACTGCGGCCGCTTTTCGATCTGTAAGACCACTAAAGTC TATATGAATTCAGGAGGTGATTGTGCCTGTGCCTCCATTTTC TATATAAGGAGGAATTCTGTGCCTGTGCCTCCATTTTC AACTAAATCTAGTGAGCATGG AAGCATATAAAAGGTGATGGT ATGGCATTCATCAAGATACAG AACGCTTGACTCTTGATATTG CTGCCGTCAGTACTGTTAC AGTCCAGGCTTGCTCTTTAG GGTTCCGTCGTCAAACTAACG CACGCCAGTTGTTTGTACTG ATAGTGCCACAGAAAAATACCG AAGCCGGGTTCTGTTCTCTG pH 8.5, 1 mM DTT and 50 mM NaCl with addition of either 2 mM MnCl2 or 5 mM MgCl2 and supplemented with either 10 or 100 mM cAMP or cGMP. As a test for hydrolysis of pNPP by the putative phosphatase activity of All1758, 2 mg of the recombinant protein was added to the cocktail at a final reaction volume of 1 ml. Absorbance at 400 nm was monitored as a measure of hydrolysis of pNPP at 25 uC every 30 s for the first 30 min after addition of protein to the cocktail, then at 45 min, and at 1, 2, 5 and 24 h. Acetylene reduction, glycolipid and exopolysaccharide assays. Acetylene reduction assays on three independent cultures were performed as previously described (Borthakur et al., 2005). Glycolipid extraction for TLC and staining of exopolysaccharides were also done as previously described (Callahan & Buikema, 2001b). RESULTS Measurement of cell size. Reported values for cell sizes were obtained by measuring the ‘height’ of 100 randomly distributed cells of Anabaena sp. strains PCC 7120 and UHM184. Height refers to the dimension perpendicular to the longitudinal axis of the filament, halfway between cell poles. Significance of differences in height distributions was established using an unpaired t-test with a twotailed P-value of less than 0.0001. Approximate cell volumes were calculated by treating cells as spheres, using the mean cell height as the radius in the formula volumesphere54/3pr3. RNA isolation and RT-PCR. Total RNA was extracted as previously described (Risser & Callahan, 2008). For reversetranscription PCR (RT-PCR), 0.5 mg total RNA was used for the synthesis of cDNA with reverse transcriptase and the corresponding reverse primers for subsequent use in PCR, which was carried out for 27 cycles using primers 23–32 as previously described (Risser & Callahan, 2008). http://mic.sgmjournals.org Mutation of all1758 prevents diazotrophic growth In an effort to identify genes necessary for diazotrophic growth of Anabaena sp. strain PCC 7120, a genetic screen using transposon mutagenesis both to increase and to mark the sites of mutations was conducted as previously described (Nayar et al., 2007). In one mutant the transposon had disrupted ORF all1758 in the annotated genome sequence, suggesting that all1758 was necessary for diazotrophic growth. To verify a cause-and-effect relationship between inactivation of all1758 and the phenotype of the mutant, an V interposon conferring spectinomycin and streptomycin resistance was used to replace nucleotides +31 to +1358 relative to the GTG translational initiation Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 383 S. K. Tom and S. M. Callahan codon of all1758 in the wild-type strain. The resulting mutant, UHM184, contained the first and last 30 nucleotides of all1758 flanking the V interposon and exhibited impaired diazotrophic growth similar to that of the isolated transposon mutant. Complementation of UHM184 with a plasmid bearing all1758 preceded by 497 bp upstream of all1758, including the 474 bp intergenic region between the end of upstream gene all1759 and the start of all1758, restored diazotrophic growth to the mutant and suggested that inactivation of all1758 and not polar effects of the transposon insertion was the cause of the mutant phenotype. The all1758 gene is located between two genes transcribed in the same orientation and predicted to have a role in cell division, ftsX (all1757) and ftsY (all1759). To verify that inactivation of all1758 was solely responsible for the phenotypes of the mutants, nucleotides +31 to +1358 were cleanly deleted from the chromosome of the wild-type. The resulting strain, UHM183, had an in-frame deletion of all1758 and differed from UHM184 only by the absence of the V interposon. The phenotype of this strain was similar to that of UHM184. Strain UHM183 was complemented in the same manner as for UHM184, and there was no evidence for recombination between the plasmid and the chromosomal locus of all1758, as indicated by the lack of a PCR product using primers corresponding to the region of all1758 that is deleted from the mutant chromosome and the coding region of all1757. Lastly, transcripts of all1757 and all1759 were detected by RT-PCR in RNA isolated from strains UHM184 and the wild-type (data not shown). Taken together, these results indicated that a functional copy of all1758 was required for diazotrophic growth of Anabaena sp. strain PCC 7120. The predicted 463 amino acid sequence encoded by all1758 was compared to sequences in the NCBI Protein Database. Residues 45–200 appeared to comprise a GAF domain, which is named after the types of proteins containing the domain (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA). Many of these proteins bind cyclic nucleotides. Residues 270–463 were similar in sequence to serine/threonine protein phosphatases of the PP2C superfamily, which comprises metallo-phosphatases that employ two metal ions in the catalytic site. PP2C proteins generally have a catalytic domain of approximately 290 amino acids that includes eight absolutely conserved residues within 11 conserved motifs (Kennelly, 2001; Shi et al., 2007). However, the predicted protein sequence of All1758 lacked the invariant glycine present in motif 5 and, instead, had an alanine in its place. In an attempt to demonstrate phosphatase activity of All1758, the 52 kDa protein with an N-terminal polyhistidine epitope tag was purified from E. coli strain BL21(DE3) by affinity chromatography. Attempts to characterize phosphatase activity using pNPP as a substrate were unsuccessful even after addition of either 10 or 100 mM cAMP or cGMP separately with the pNPP substrate, in an effort to support the potential role of the GAF domain in phosphatase function. 384 Pleiotropic phenotype of all1758 null mutants Although all1758 was discovered in a genetic screen designed to isolate mutants incapable of growth on nitrogen-deficient media, disruption of all1758 also affected viability on nitrogen-replete media. Colonies of the mutant were pale green rather than the vibrant green of the wild-type, and after about 3 weeks of growth on BG-11 solid media with either 17 mM nitrate or 2 mM ammonia as a source of fixed nitrogen, growth appeared to cease, and the strain could not be revived by subculturing onto fresh medium. In comparison, the wild-type strain remained viable under the same conditions for more than 3 months. However, the strain could apparently be maintained indefinitely if repeatedly subcultured onto BG-11 solid medium every 2 weeks. To test if loss of viability was the result of a change in the medium over time, non-viable filaments of UHM184 were scraped from a plate culture 3 weeks after its inoculation, and the resulting ‘conditioned’ solid medium was streaked with PCC 7120. Growth of the wild-type strain was indistinguishable from that on unconditioned medium. Cells of UHM184 filaments cultured on BG-11 solid medium were markedly smaller than those of the wild-type grown under the same conditions. The mean (±SD) vegetative cell height (measured perpendicular to the longitudinal axis of the filament) of mutant cells was 2.88±0.25 mm, compared with 4.34±0.23 mm for cells of the wild-type, which corresponded to a more than three-fold reduction in cell volume for the mutant (Fig. 1a, b). This diminutive phenotype persisted until loss of viability and when cells were transferred to liquid medium lacking fixed nitrogen. Growth of the mutant in liquid BG-11 containing nitrate or ammonium as a source of fixed nitrogen was different from that on solid medium. Within 24 h of inoculation into liquid medium, the cytoplasm was found on one side of the cell, and the majority of the cell volume was occupied by what appeared to be a single, large vacuole (Fig. 1c). Alternatively, the plasma membrane may have pulled away from the cell wall in a manner resembling plasmolysis in plant cells. The process was unlikely to be attributable to the formation of a large gas vesicle because mutant cells settled to the bottom of growth vessels more readily than those of the wild-type. Vacuole formation was accompanied by enlargement of cells and resulted in cell lysis within 48 h. To test if cell lysis and/or vacuolization of cells was in response to a substance excreted into the medium by the mutant, conditioned medium separated from cellular debris by centrifugation was inoculated with PCC 7120. Growth of PCC 7120 in medium conditioned by UHM184 was similar to that in unconditioned medium, indicating that lysis of UHM184 was not caused by alteration of the medium by the mutant so as to make it unfit for growth of PCC 7120. The pH of medium conditioned by growth of UHM184 was similar to that of medium conditioned by the wild-type and to unconditioned medium. In addition, the inverse experiment, growing UHM184 in spent medium of strain PCC 7120, did not rescue any of the mutant phenotypes. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 Microbiology 158 all1758 and heterocyst development of degradation of phycobiliproteins, and polar cyanophycin granules associated with mature heterocysts. Note that all of the mutant phenotypes were complemented by introduction of all1758 on a plasmid to strain UHM184 as described above. Strain UHM184 is Fix– and lacks the minor heterocyst-specific glycolipid During the late stages of heterocyst development, deposition of heterocyst-specific polysaccharides contributes to the creation of the micro-oxic environment necessary for the function of nitrogenase, an oxygen-labile enzyme. Heterocysts of strain UHM184 were readily stained by Alcian Blue, indicative of the presence of heterocyst envelope exopolysaccharides (Fig. 2). Thus, polysaccharide synthesis and deposition appears unimpaired in the mutant. Just interior to the layer of exopolysaccharide, a layer of heterocyst-specific glycolipid (HGL) also contributes to the micro-oxic interior of a heterocyst. To determine if HGLs were present in mutant strain UHM184, lipids from strains PCC 7120, UHM184 and UHM103, the last being a hetR mutant that served as a negative control, were extracted after 72 h of combined-nitrogen deprivation, and separated by TLC for visualization (Fig. 3). Two HGLs have been characterized in the wild-type (Gambacorta et al., 1996; Lambein & Wolk, 1973; Soriente et al., 1995). Although the more abundant, slower-migrating species corresponding to 1-(O-a-D-glucopyranosyl)-3,25-hexacosanediol (the major HGL) was present in strains UHM184 and PCC 7120, the less abundant, faster migrating glycolipid, 1-(O-a-D-glycopyranosyl)-3-keto-25-hexacosanol (the minor HGL), was absent from UHM184. As expected, both HGLs were absent from strain UHM103, which does not make heterocysts. The absence of the minor HGL, which is necessary for a functional heterocyst, could Fig. 1. Phenotype of the all1758 deletion strain, UHM184, under different conditions. Note the diminutive cell size of UHM184 (b) as compared with the wild-type strain Anabaena sp. PCC 7120 (a) cultured on solid BG-11 medium. At 24 h after culture in liquid BG-11 medium, UHM184 develops intracellular vacuoles among all cells of a filament (c). At 24 h after transfer from solid BG-11 medium to liquid BG-110 medium, which lacks a source of combined nitrogen, heterocysts form in PCC 7120 (d) but not in UHM184 (e). After 48 h of nitrogen starvation, UHM184 exhibits heterocyst patterning among cells of diminutive size (f). Arrowheads indicate heterocysts. Bars, 10 mm. UHM184 was incapable of growth in the absence of fixed nitrogen, and morphologically distinct heterocysts were not observed until 48 h after removal of fixed nitrogen, about twice the time for the wild-type (Fig. 1d, e, f). The periodic pattern of heterocyts along filaments was similar to that of the wild-type. Heterocysts of UHM184 had characteristic reduced levels of autofluorescence, indicative http://mic.sgmjournals.org Fig. 2. Heterocyst-specific exopolysaccharide of UHM184. Bright-field images of heterocyst-specific exopolysaccharides stained by Alcian Blue in strains PCC 7120 (a) and UHM184 (b) at 48 h after removal of combined nitrogen. Arrowheads indicate heterocysts. Bars, 10 mm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 385 S. K. Tom and S. M. Callahan account for the inability of UHM184 to grow under diazotrophic conditions. Typically, strains affected in HGL synthesis lack both the minor and the major HGL, with one exception. A double kinase mutant was shown by RT-PCR to lack expression of two glycolipid synthesis genes, asr5349 and asr5350 (Shi et al., 2007). Conversely, transcripts of asr5349 and asr5350 were detected by RT-PCR in RNA isolated from strain UHM184 (data not shown). Strains that lack one or both of the HGLs cannot fix nitrogen in the presence of molecular oxygen (Fox2 phenotype) but can do so in its absence. Other strains that are deficient in more than just the creation of microoxic heterocysts cannot fix under either condition (Fix2 phenotype). The nitrogenase activity of strain UHM184 at 48 h post-induction was assessed by acetylene reduction assays under both oxic and anoxic conditions to determine if it was a Fox2 or Fix2 strain. Under oxic conditions, rates of acetylene reduction were 0.350±0.064 nmol ethylene h21 ml 21 (OD750 unit)21 for PCC 7120 and undetectable for strains UHM184, UHM103 (a Fix2 strain) and UHM128 (a previously characterized Fox2 strain: Leganés et al., 2005; Nayar et al., 2007). Under anoxic conditions, rates of acetylene reduction were 0.073±0.038 nmol ethylene Fig. 3. HGLs of strain UHM184. Lipid extracts from the strains indicated were separated by TLC 72 h after removal of combined nitrogen. Lane 1, the all1758 mutant, UHM184, which lacks the minor HGL; lane 2, PCC 7120; lane 3, the hetR mutant, UHM103, which lacks both the minor and the major HGLs. The minor [1-(Oa-D-glycopyranosyl)-3-keto-25-hexacosanol] and the major [1-(Oa-D-glucopyranosyl)-3,25-hexacosanediol] HGLs are indicated by the top and bottom arrows, respectively. Lipid extracts were deposited at the origin, denoted by an asterisk. 386 h21 ml 21 (OD750 unit)21 for strain UHM128 and undetectable for strains UHM184 and UHM103. Thus, the phenotype of UHM184 was Fix2, suggesting that lack of a functional copy of all1758 disrupted more than just the creation of micro-oxic conditions in heterocysts. The all1758 ORF is constitutively expressed and autoregulates its own expression Expression of all1758 was assessed both temporally and spatially using the gene for green fluorescent protein (GFP) as a reporter. A uniform level of bright florescence was observed in both vegetative cells and heterocysts when a shuttle vector carrying the putative promoter region of all1758 fused to gfp was introduced into the wild-type strain. Similar levels of expression were seen with and without nitrate in the medium (Fig. 4a). However, no GFPdependent fluorescence was observed when the same plasmid was introduced into strain UHM184, which has all1758 deleted from the chromosome (Fig. 4b). No GFP-dependent fluorescence was observed from the wild-type strain harbouring a plasmid bearing all1758 translationally fused to gfp under the control of its native promoter in the presence or absence of nitrate (data not shown). UHM184 bearing the same plasmid also remained non-fluorescent, similar to the wild-type, although the cell size had returned to the wild-type dimensions, apparently Fig. 4. A functional all1758 gene is required for expression of all1758 but not for patterned expression of patS. (a, b) Expression of all1758 as visualized with a transcriptional Pall1758–gfp fusion on plasmid pST151 in strains PCC 7120 (a) or UHM184 (b) at 24 h after removal of combined nitrogen. (c, d) Expression of patS in PCC 7120 (c) and UHM184 (d) as visualized using the transcriptional fusion patS–gfp on plasmid pAM1951 at 14 h after induction of heterocyst formation. Micrographs show bright-field (left panels) and fluorescence (right panels) images acquired using identical microscope and camera settings. Arrowheads indicate heterocysts. Bars, 10 mm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 Microbiology 158 all1758 and heterocyst development due to complementation by the fusion protein encoded on the plasmid. In addition, overexpression of all1758 from the copper-inducible petE promoter in the wild-type strain had no apparent effect on heterocyst differentiation or cell size, whereas extra copies of patS or hetN expressed from the petE promoter on a plasmid in strain UHM184 prevented heterocyst differentiation, but the diminutive cell phenotype remained (data not shown). Overexpression of all1758 from the nirA promoter, which is induced under both nitrate-replete conditions or conditions of nitrogen starvation (Frı́as et al., 1997), yielded the same results as those observed with use of the petE promoter (data not shown). all1758 is not required for the timing of pattern formation A null mutation in all1758 delays morphological differentiation of heterocysts. Prior to changes in the morphology of differentiating cells, the cells of filaments that will differentiate are specified by the generation of a pattern of gene expression. This pattern can be visualized using fusions of the promoters of hetR or patS to gfp (Callahan & Buikema, 2001a; Yoon & Golden, 2001). Strain UHM184 carrying a plasmid with the promoter of either hetR or patS fused to gfp exhibited a pattern of GFP-dependent fluorescence that was similar to that observed in the wild-type carrying the same plasmid (Fig. 4c, d). Both the number of cells between fluorescing cells and the timing of the appearance of fluorescence were similar. The only noticeable difference was the smaller size of the cells in filaments of UHM184. Thus, all1758 was not required for formation of the pattern that specifies which cells will differentiate into heterocysts. Bypass of mutation of all1758 by extra copies of hetR In an attempt to ascertain where in the regulatory network controlling heterocyst differentiation all1758 may have been acting, plasmid-borne copies of hetR were put into UHM184 and the resulting strain was examined for phenotypic differences. The strain with extra copies of hetR had multiple contiguous heterocysts and reduced spacing between groups of heterocysts in media with or without nitrate as a fixed nitrogen source (data not shown). The same Mch (multiple contiguous heterocysts) phenotype was observed when the plasmid was introduced into the wild-type strain. Extra copies of hetR bypassed the delay in morphological differentiation of heterocysts, but the diminutive cell phenotype and other associated phenotypes remained. DISCUSSION Phosphorylation of proteins is widely used in the regulation of various cellular processes. All1758 is predicted to be a http://mic.sgmjournals.org PP2C phosphatase, which is a member of the PPM family of serine/threonine phosphatases that are dependent on the presence of the divalent cation Mg2+ for catalytic function. However, repeated attempts to demonstrate the role of All1758 as a phosphatase were unsuccessful. The simplest explanation for the lack of discernible phosphatase activity is the absence of one of the eight absolutely conserved residues in the protein sequence of all1758. PP2C proteins generally have a catalytic domain of approximately 290 amino acids that includes eight absolutely conserved residues within 11 conserved motifs (Kennelly, 2001; Shi et al., 2007). All1758 has an alanine residue, a conservative substitution, in lieu of the glycine residue reported as invariant in motif 5. Alternatively, phosphatase activity of the protein encoded by all1758 may not be measurable under the conditions of our assay due, for instance, to substrate specificity, lack of a cofactor or improper folding of the recombinant protein. The minor HGL, which is essential for provision of microaerobic conditions necessary for the activity of nitrogenase within the heterocyst, was absent in a strain lacking all1758. However, the absence of nitrogen fixation in the mutant strain even under anoxic conditions suggests that all1758 is required for some cellular process(es) related to fixation of nitrogen not related to diminution of molecular oxygen in heterocysts. Indeed, a diverse and seemingly unrelated range of phenotypes resulted from mutation of all1758. For instance, the delay in heterocyst formation in UHM184 may suggest the involvement of all1758 in the timing of heterocyst morphology development. The diminutive cell size and associated decrease in cell volume apparent upon mutation of all1758 may be indicative of a change in the timing of cell division in the mutant, which would prevent the cell from attaining a normal and/or enlarged cell size. Inhibition of cell division has been shown to prevent differentiation of heterocysts, and heterocysts have twice the DNA content of vegetative cells (Sakr et al., 2006a, b). The increased rate of cell division in strains lacking all1758 may account for the defect in heterocyst differentiation in the mutant. The location of all1758 between the cell division genes ftsX and ftsY may implicate all1758 as a cell division gene as well. In E. coli, for example, the cell division gene ftsE (b3463) lies between ftsX (b3462) and ftsY (b3464). Alternatively, a defect in a process specific to heterocyst differentiation may influence cell division. Also, the decrease in cell volume may have perturbed the concentration of specific intracellular components, resulting in the observed phenotypes. The relationship between cell division and heterocyst differentiation remains enigmatic. Unpatterned expression of all1758 in all cells was evident under both nitrate-replete and nitrate-depleted conditions using a GFP transcriptional reporter fusion, suggesting that the gene is constitutively expressed. In contrast, GFPdependent fluorescence was not observed from an all1758 translational fusion in all background strains tested. However, the fusion protein did appear to complement strain UHM184, suggesting the fusion protein was Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 05:26:46 387 S. K. Tom and S. M. Callahan functional. The lack of detectable fluorescence may indicate that levels of All1758 are very low in cells, so the associated fluorescence from the fusion protein was below the level of detection, or the GFP portion of the protein may have been non-functional. This phenomenon is not new. In our hands, plasmid constructs bearing hetR–gfp and hetF–gfp translational fusions in hetR and hetF backgrounds, respectively, also complement the inactivated gene, yet visible fluorescence is lacking (Risser & Callahan, 2008). kinase domain and a His kinase domain are regulated by NtcA in the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 188, 4822– 4829. Alterations in the levels of expression of other genes involved in heterocyst differentiation have been shown to affect cell size. Inactivation of all2874, encoding a diguanylate cyclase, results in a light intensity-dependent variability in heterocyst frequency and reduction in vegetative cell size (Neunuebel & Golden, 2008). Conversely, a strain lacking hetF has enlarged cells, and extra copies of hetF on a plasmid result in a diminutive cell phenotype (Risser & Callahan, 2008). In contrast to the correlation between smaller cell size and a delay in heterocyst differentiation in the all1758 mutant, a strain with extra copies of hetF forms more heterocysts than the wild-type. Overexpression of patA also results in enlarged cells (Young-Robbins et al., 2010). There is no evidence for the phosphorylation of HetF or PatA, but PatA is similar in sequence to response regulators of twocomponent transduction systems (Liang et al., 1992). 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