The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.     

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.     

鱼腥藻PCC7120基因组中一个影响异形胞发育和图式形成的新区域

以Tn5 10 87b诱变鱼腥藻PCC712 0 ,筛选不能利用氮气生长、异形胞图式发生变化的突变株。突变株 # 180 1经缺氮诱导 2 4h后无异形胞形成 ,48h后沿藻丝形成少量成熟异形胞 ,占藻细胞总数比例为 2 .8% ,其异形胞发育速度和形成频率均与野生型有显著区别。以ClaⅠ酶切该突变株总DNA、自环化后以电泳冲法转化大肠杆菌 ,回收带有插入位点两侧DNA片段的转座子Tn5 10 87b。经测序确定转座子位于未知功能基因alr0 0 99第 14 8和 14 9碱基之间。为证明突变性状确由alr0 0 99内的插入突变引起而不是由于其他二次突变 ,通过同源双交换将含新霉素抗性基因的C .K2片段定向插入基因组中alr0 0 99的EcoRV位点 ,获得重构的鱼腥藻突变株DR2 14 ,其表型与原突变株 # 180 1相同。以上结果表明alr0 0 99或其下游基因是鱼腥藻PCC712 0基因组中与异形胞发育和图式形成相关的一个新区域。     

HcwA, an autolysin, is required for heterocyst maturation in Anabaena sp. strain PCC 7120.

In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N(2). Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA in Anabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomal hepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resembles N-acetylmuramoyl-L-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of hepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.     

Heterocyst-specific expression of patB,a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.     

A TolC-like protein is required for heterocyst development in Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).     

Anabaena sp. strain PCC 7120 hetY gene influences heterocyst development

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.     

Septum-localized protein required for filament integrity and diazotrophy in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.     

Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

ppGpp Metabolism Is Involved in Heterocyst Development in the Cyanobacterium Anabaena sp. Strain PCC 7120

When deprived of a combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) can form heterocysts capable of nitrogen fixation. The process of heterocyst differentiation takes about 20 to 24 h, during which extensive metabolic and morphological changes take place. Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation (J. Akinyanju, R. J. Smith, FEBS Lett. 107:173–176, 1979; J Akinyanju, R. J. Smith, New Phytol. 105:117–122, 1987). Here we show that all1549 (here designated rel_ana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of rel_ana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549Ωsp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of rel_ana. Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation.     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.