Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from Anabaena sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant. The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR. The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.     

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.     

The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.     

Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential

Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields.     

鱼腥藻PCC 7120乙酰辅酶A合成酶All0769的缺失降低异形胞频率

研究鉴定了All0769为鱼腥藻PCC 7120中乙酰辅酶A合成酶,通过CRISPR/Cpf1系统敲除鱼腥藻PCC7120中的乙酰辅酶A合成酶(由all0769编码),探究了乙酰辅酶A合成酶在异形胞分化中的调控机制。结果所示:All0769能在体外反应中催化乙酰辅酶A的生成。在供氮环境下,敲除all0769会影响藻细胞生长速率。而无论环境中是否存在化合氮,Δall0769突变株的乙酰辅酶A和α-酮戊二酸含量均显著减少。在供氮环境下,Δall0769突变株中检测到(26.17±1.55) nmol/mg protein的乙酰辅酶A,而在野生型中检测出(43.04±1.09) nmol/mg的乙酰辅酶A。Δall0769突变株的α-酮戊二酸[(1.41±0.24) nmol/mg protein]低于野生型的α-酮戊二酸[(2.13±0.05) nmol/mg protein]。在缺氮环境下,Δall0769突变株中检测到(10.00±2.81) nmol/mg protein的乙酰辅酶A,而在野生型中检测出(29.82±4.04) nmol/mg protein的乙酰辅酶A。Δall07...     

The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.     

hetC, a gene coding for a protein similar to bacterial ABC protein exporters, is involved in early regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120.

Transposon-generated mutant C3 of Anabaena sp. strain PCC 7120 is unable to form heterocysts upon deprivation of combined nitrogen but forms a pattern of spaced, weakly fluorescent cells after 2 days of deprivation. Sequence analysis of chromosomal DNA adjacent to the ends of transposon Tn5-1058 in mutant C3 showed a 1,044-amino-acid open reading frame, designated hetC, whose predicted protein product throughout its C-terminal two-thirds has extensive similarity to the HlyB family of bacterial protein exporters. Its N-terminal third is unique and does not resemble any known protein. hetC lies 1,165 bp 5' from the previously described gene hetP. Reconstruction of the C3 mutation and its complementation in trans with a wild-type copy of hetC confirmed that hetC has an essential regulatory role early in heterocyst development. hetC is induced ca. 4 h after nitrogen stepdown, hours after induction of hetR. Expression of hetC depends on HetR and may depend on HetC. Highly similar sequences are present 5' from the initiation codons and in the 3' untranslated regions of hetC and of two heterocyst-specific genes, devA and hetP.     

patS minigenes inhibit heterocyst development of Anabaena sp. strain PCC 7120

The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition. patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling. patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P(petE), P(patS), or P(rbcL) promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not. In contrast to the full-length patS gene, P(hepA)-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif. Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.     

Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120

Heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 occurs at regular intervals under nitrogen starvation and is regulated by a host of signaling molecules responsive to availability of fixed nitrogen. The heterocyst differentiation inhibitor PatS contains the active pentapeptide RGSGR (PatS-5) at its C-terminus considered the minimum PatS fragment required for normal heterocyst pattern formation. PatS-5 is known to bind HetR, the master regulator of heterocyst differentiation, with a moderate affinity and a submicromolar dissociation constant. Here we characterized the affinity of HetR for several PatS C-terminal fragments by measuring the relative ability of each fragment to knockdown HetR binding to DNA in electrophoretic mobility shift assays and using isothermal titration calorimetry (ITC). HetR bound to PatS-6 (ERGSGR) >30 times tighter (K(d) = 7 nM) than to PatS-5 (K(d) = 227 nM) and >1200 times tighter than to PatS-7 (DERGSGR) (K(d) = 9280 nM). No binding was detected between HetR and PatS-8 (CDERGSGR). Quantitative binding constants obtained from ITC measurements were consistent with qualitative results from the gel shift knockdown assays. CW EPR spectroscopy confirmed that PatS-6 bound to a MTSL spin-labeled HetR L252C mutant at a 10-fold lower concentration compared to PatS-5. Substituting the PatS-6 N-terminal glutamate to aspartate, lysine, or glycine did not alter binding affinity, indicating that neither the charge nor size of the N-terminal residue's side chain played a role in enhanced HetR binding to PatS-6, but rather increased binding affinity resulted from new interactions with the PatS-6 N-terminal residue peptide backbone.