Roles of the N-terminal motif in improving the activity and soluble expression of phenylalanine ammonia lyases in Escherichia coli.

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.

Cross-Activation of Two Nitrogenase Gene Clusters by CnfR1 or CnfR2 in the Cyanobacterium Anabaena variabilis.

In Anabaena variabilis, the nif1 genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the nif2 genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the cnfR2 promoter or from the Co2+-inducible coaT promoter, activated the expression of both nifB1 and nifB2. Activation of nifB2, but not nifB1, by CnfR1 required NtcA. Thus, expression of the nif1 system requires no heterocyst-specific factor other than CnfR1. In contrast, CnfR2, when it was expressed in heterocysts under the control of the cnfR1 promoter or from the coaT promoter, did not activate the expression of nifB1 or nifB2. Thus, activation of the nif2 system in anaerobic vegetative cells by CnfR2 requires additional factors absent in heterocysts. CnfR2 made from the coaT promoter activated nifB2 expression in anaerobic vegetative cells grown with fixed nitrogen; however, oxygen inhibited CnfR2 activation of nifB2 expression. In contrast, activation of nifB1 and nifB2 by CnfR1 was unaffected by oxygen. CnfR1, which does not activate the nifB2 promoter in heterocysts, activated the expression of the entire nif2 gene cluster from a nifB2::nifB1::nifB2 hybrid promoter in heterocysts, producing functional Nif2 nitrogenase in heterocysts. However, activity was poor compared to the normal Nif1 nitrogenase. Expression of the nif2 cluster in anaerobic vegetative cells of Nostoc sp. PCC 7120, a strain lacking the nif2 nitrogenase, resulted in expression of the nif2 genes but weak nitrogenase activity. IMPORTANCE Cyanobacterial nitrogen fixation is important in the global nitrogen cycle, in oceanic productivity, and in many plant and fungal symbioses. While the proteins that mediate nitrogen fixation have been well characterized, the regulation of this complex and expensive process is poorly understood in cyanobacteria. Using a genetic approach, we have characterized unique and overlapping functions for two homologous transcriptional activators CnfR1 and CnfR2 that activate two distinct nitrogenases in a single organism. We found that CnfR1 is promiscuous in its ability to activate both nitrogenase systems, whereas CnfR2 depends on additional cellular factors; thus, it activates only one nitrogenase system.

Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation.

Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2'-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK restriction endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity.

Reconstruction of a regulated two-cell metabolic model to study biohydrogen production in a diazotrophic cyanobacterium Anabaena variabilis ATCC 29413.

Anabaena variabilis is a diazotrophic filamentous cyanobacterium that differentiates to heterocysts and produces hydrogen as a byproduct. Study on metabolic interactions of the two differentiated cells provides a better understanding of its metabolism especially for improving hydrogen production. To this end, a genome-scale metabolic model for Anabaena variabilis ATCC 29413, iAM957, was reconstructed and evaluated in this research. Then, the model and transcriptomic data of the vegetative and heterocyst cells were applied to construct a regulated two-cell metabolic model. The regulated model improved prediction for biomass in high radiation levels. The regulated model predicts that heterocysts provide an oxygen-free environment and then, this model was used to find strategies for improving hydrogen production in heterocysts. The predictions indicate that the removal of uptake hydrogenase improves hydrogen production which is consistent with previous empirical research. Furthermore, the regulated model proposed activation of some reactions to provide redox cofactors which are required for improving hydrogen production up to 60% by bidirectional hydrogenase.

One-Pot Enzymatic Synthesis of D-Arylalanines Using Phenylalanine Ammonia Lyase and L-Amino Acid Deaminase.

The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic D,L-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure D-arylalanine, a modified AvPAL with high D-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for D-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual L-enantiomer product in reaction solution could be converted into the D-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH3BH3. At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (eeD)) of D-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize D-arylalanine with different groups on the phenyl ring. Among these D-arylalanines, the yield of m-nitro-D-phenylalanine was highest and reached 96%, and the eeD exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.

Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU.

Phenylketonuria (PKU) is a genetic disease characterized by the inability to convert dietary phenylalanine to tyrosine by phenylalanine hydroxylase. Given the importance of gut microbes in digestion, a genetically engineered microbe could potentially degrade some ingested phenylalanine from the diet prior to absorption. To test this, a phenylalanine lyase gene from Anabaena variabilis (AvPAL) was codon-optimized and cloned into a shuttle vector for expression in Lactobacillus reuteri 100-23C (pHENOMMenal). Functional expression of AvPAL was determined in vitro, and subsequently tested in vivo in homozygous PAHenu2 (PKU model) mice. Initial trials of two PAHenu2 homozygous (PKU) mice defined conditions for freeze-drying and delivery of bacteria. Animals showed reduced blood phe within three to four days of treatment with pHENOMMenal probiotic, and blood phe concentrations remained significantly reduced (P < 0.0005) compared to untreated controls during the course of experiments. Although pHENOMMenal probiotic could be cultured from fecal samples at four months post treatment, it could no longer be cultivated from feces at eight months post treatment, indicating eventual loss of the microbe from the gut. Preliminary screens during experimentation found no immune response to AvPAL. Collectively these studies provide data for the use of a genetically engineered probiotic as a potential treatment for PKU.

Role of the nifB1 and nifB2 Promoters in Cell-Type-Specific Expression of Two Mo Nitrogenases in the Cyanobacterium Anabaena variabilis ATCC 29413.

Anabaena variabilis ATCC 29413 has one Mo nitrogenase that is made under oxic growth conditions in specialized cells called heterocysts and a second Mo nitrogenase that is made only under anoxic conditions in vegetative cells. The two large nif gene clusters responsible for these two nitrogenases are under the control of the promoter of the first gene in the operon, nifB1 or nifB2 Despite differences in the expression patterns of nifB1 and nifB2, related to oxygen and cell type, the regions upstream of their transcription start sites (tss) show striking homology, including three highly conserved sequences (CS). CS1, CS2, and the region just upstream from the tss were required for optimal expression from the nifB1 promoter, but CS3 and the 5' untranslated region (UTR) were not. Hybrid fusions of the nifB1 and nifB2 upstream regions revealed that the region including CS1, CS2, and CS3 of nifB2 could substitute for the similar region of nifB1; however, the converse was not true. Expression from the nifB2 promoter region required the CS1, CS2, and CS3 regions of nifB2 and also required the nifB2 5' UTR. A hybrid promoter that was mostly nifB2 but that had the region from about position -40 to the tss of nifB1 was expressed in heterocysts and in anoxic vegetative cells. Thus, addition of the nifB1 promoter region (from about position -40 to the tss of nifB1) in the nifB hybrid promoter supported expression in heterocysts but did not prevent the mostly nifB2 promoter from also functioning in anoxic vegetative cells. IMPORTANCE: In the filamentous cyanobacterium Anabaena variabilis, two Mo nitrogenase gene clusters, nif1 and nif2, function under different environmental conditions in different cell types. Little is known about the regulation of transcription from the promoter upstream of the first gene of the cluster, which drives transcription of each of these two large operons. The similarity in the sequences upstream of the primary promoters for the two nif gene clusters belies the differences in their expression patterns. Analysis of these nif promoters in strains with mutations in the conserved sequences and in strains with hybrid promoters, comprising parts from nif1 and nif2, provides strong evidence that each promoter has key elements required for cell-type-specific expression of the nif1 and nif2 gene clusters.

Structure and Function of Cyanobacterial DHDPS and DHDPR.

Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the divergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis. The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR diverged after cyanobacteria evolved.

Protein engineering of a bacterial N-acyl-d-glucosamine 2-epimerase for improved stability under process conditions.

Enzymatic cascade reactions, i.e. the combination of several enzyme reactions in one pot without isolation of intermediates, have great potential for the establishment of sustainable chemical processes. However, many cascade reactions suffer from cross-inhibitions and enzyme inactivation by components of the reaction system. This study focuses on the two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-d-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE) in combination with an N-acetylneuraminate lyase (NAL) from Escherichia coli. AvaAGE epimerizes N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), which then reacts with pyruvate in a NAL-catalyzed aldol condensation to form Neu5Ac. However, AvaAGE is inactivated by high pyruvate concentrations, which are used to push the NAL reaction toward the product side. A biphasic inactivation was observed in the presence of 50-800mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis revealed that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE. Because ATP is an allosteric activator of the epimerase and the binding of the nucleotide is crucial for its catalytic properties, saturation mutagenesis at position K160 was performed to identify the most compatible amino acid exchanges. The best variants, K160I, K160N and K160L, showed no inactivation by pyruvate, but significantly impaired kinetic parameters. For example, depending on the mutant, the turnover number kcat was reduced by 51-68% compared with the wild-type enzyme. A mechanistic model of the Neu5Ac synthesis was established, which can be used to select the AvaAGE variant that is most favorable for a given process condition. The results show that mechanistic models can greatly facilitate the choice of the right enzyme for an enzymatic cascade reaction with multiple cross-inhibitions and inactivation phenomena.

Homologous regulators, CnfR1 and CnfR2, activate expression of two distinct nitrogenase gene clusters in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

The cyanobacterium Anabaena variabilis has two Mo-nitrogenases that function under different environmental conditions in different cell types. The heterocyst-specific nitrogenase encoded by the large nif1 gene cluster and the similar nif2 gene cluster that functions under anaerobic conditions in vegetative cells are under the control of the promoter for the first gene of each cluster, nifB1 or nifB2 respectively. Associated with each of these clusters is a putative regulatory gene called cnfR (patB) whose product has a C-terminal HTH domain and an N-terminal ferredoxin-like domain. CnfR1 activates nifB1 expression in heterocysts, while CnfR2 activates nifB2 expression. A cnfR1 mutant was unable to make nitrogenase under aerobic conditions in heterocysts while the cnfR2 mutant was unable to make nitrogenase under anaerobic conditions. Mutations in cnfR1 and cnfR2 reduced transcripts for the nif1 and nif2 genes respectively. The closely related cyanobacterium, Anabaena sp. PCC 7120 has the nif1 system but lacks nif2. Expression of nifB2:lacZ from A. variabilis in anaerobic vegetative cells of Anabaena sp. PCC 7120 depended on the presence of cnfR2. This suggests that CnfR2 is necessary and sufficient for activation of the nifB2 promoter and that the CnfR1/CnfR2 family of proteins are the primary activators of nitrogenase gene expression in cyanobacteria.

CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria.

Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide ß-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.

Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats.

Akinetes are resting spore-like cells formed by some heterocyst-forming filamentous cyanobacteria for surviving long periods of unfavourable conditions. We studied the development of akinetes in two model strains of cyanobacterial cell differentiation, the planktonic freshwater Anabaena variabilis ATCC 29413 and the terrestrial or symbiotic Nostoc punctiforme ATCC 29133, in response to low light and phosphate starvation. The best trigger of akinete differentiation of Anabaena variabilis was low light; that of N. punctiforme was phosphate starvation. Light and electron microscopy revealed that akinetes of both species differed from vegetative cells by their larger size, different cell morphology and large number of intracellular granules. Anabaena variabilis akinetes had a multilayer envelope; those of N. punctiforme had a simpler envelope. During akinete development of Anabaena variabilis, the amount of the storage compounds cyanophycin and glycogen increased transiently, whereas in N. punctiforme, cyanophycin and lipid droplets increased transiently. Photosynthesis and respiration decreased during akinete differentiation in both species, and remained at a low level in mature akinetes. The clear differences in the metabolic and morphological adaptations of akinetes of the two species could be related to their different lifestyles. The results pave the way for genetic and functional studies of akinete differentiation in these species.

High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli.

N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained.

Role of RNA secondary structure and processing in stability of the nifH1 transcript in the cyanobacterium Anabaena variabilis.

UNLABELLED: In the cyanobacterium Anabaena variabilis ATCC 29413, aerobic nitrogen fixation occurs in micro-oxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of the large nif1 gene cluster, which is primarily under the control of the promoter for the first gene, nifB1. Strong expression of nifH1 requires the nifB1 promoter but is also controlled by RNA processing, which leads to increased nifH1 transcript stability. The processing of the primary nifH1 transcript occurs at the base of a predicted stem-loop structure that is conserved in many heterocystous cyanobacteria. Mutations that changed the predicted secondary structure or changed the sequence of the stem-loop had detrimental effects on the amount of nifH1 transcript, with mutations that altered or destabilized the structure having the strongest effect. Just upstream from the transcriptional processing site for nifH1 was the promoter for a small antisense RNA, sava4870.1. This RNA was more strongly expressed in cells grown in the presence of fixed nitrogen and was downregulated in cells 24 h after nitrogen step down. A mutant strain lacking the promoter for sava4870.1 showed delayed nitrogen fixation; however, that phenotype might have resulted from an effect of the mutation on the processing of the nifH1 transcript. The nifH1 transcript was the most abundant and most stable nif1 transcript, while nifD1 and nifK1, just downstream of nifH1, were present in much smaller amounts and were less stable. The nifD1 and nifK1 transcripts were also processed at sites just upstream of nifD1 and nifK1. IMPORTANCE: In the filamentous cyanobacterium Anabaena variabilis, the nif1 cluster, encoding the primary Mo nitrogenase, functions under aerobic growth conditions in specialized cells called heterocysts that develop in response to starvation for fixed nitrogen. The large cluster comprising more than a dozen nif1 genes is transcribed primarily from the promoter for the first gene, nifB1; however, this does not explain the large amount of transcript for the structural genes nifH1, nifD1, and nifK1, which are also under the control of the distant nifB1 promoter. Here, we demonstrate the importance of a predicted stem-loop structure upstream of nifH1 that controls the abundance of nifH1 transcript through transcript processing and stabilization and show that nifD1 and nifK1 transcripts are also controlled by transcript processing.

Analysis and elucidation of phosphoenolpyruvate carboxylase in cyanobacteria.

Phosphoenolpyruvate carboxylase (PEPC) a cytosolic enzyme of higher plants is also found in bacteria and cyanobacteria. Genetic and biochemical investigations have indicated that there are several isoforms of PEPC belonging to C3; C3/C4 and C4 groups but, the evolution of PEPC in cyanobacteria is not yet understood. The present study opens up an opportunity to understand the isoforms and functions of PEPC in cyanobacteria. The variations observed in PEPC among lower and higher orders of cyanobacteria, suggests convergent evolution of PEPC. There is a specific PEPC phosphorylation residue 'serine' at the N-terminus and PEPC determinant residue 'serine' at the C-terminal that facilitates high affinity for substrate binding. These residues were unique to higher orders of cyanobacteria, but, not in lower orders and other prokaryotes. The different PEPC forms of cyanobacteria were investigated for their kinetic properties with phosphoenolpyruvate as the substrate and the findings corroborated well with the in silico findings. In vitro enzymatic study of cyanobacteria belonging to three different orders demonstrated the role of aspartate as an allosteric effector, which inhibited PEPC by interacting with the highly conserved residues in the active site. The differences in mode of inhibition among the different order, thus, give a fair picture about the cyanobacterial PEPCs. The higher orders appear to possess the sequence coordinates and functionally conserved residues similar to isoforms of C4 type higher plants, whereas isoforms of PEPC of the lower orders did not resemble either that of C3 or C4 plants.

Simultaneous gene inactivation and promoter reporting in cyanobacteria.

Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.

O-Methyltransferase is shared between the pentose phosphate and shikimate pathways and is essential for mycosporine-like amino acid biosynthesis in Anabaena variabilis ATCC 29413.

The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7-phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in-frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV-induced MAA biosynthesis.

Regulation of nitrogenase gene expression by transcript stability in the cyanobacterium Anabaena variabilis.

The nitrogenase gene cluster in cyanobacteria has been thought to comprise multiple operons; however, in Anabaena variabilis, the promoter for the first gene in the cluster, nifB1, appeared to be the primary promoter for the entire nif cluster. The structural genes nifHDK1 were the most abundant transcripts; however, their abundance was not controlled by an independent nifH1 promoter, but rather, by RNA processing, which produced a very stable nifH1 transcript and a moderately stable nifD1 transcript. There was also no separate promoter for nifEN1. In addition to the nifB1 promoter, there were weak promoters inside the nifU1 gene and inside the nifE1 gene, and both promoters were heterocyst specific. In an xisA mutant, which effectively separated promoters upstream of an 11-kb excision element in nifD1 from the downstream genes, the internal nifE1 promoter was functional. Transcription of the nif1 genes downstream of the 11-kb element, including the most distant genes, hesAB1 and fdxH1, was reduced in the xisA mutant, indicating that the nifB1 promoter contributed to their expression. However, with the exception of nifK1 and nifE1, which had no expression, the downstream genes showed low to moderate levels of transcription in the xisA mutant. The hesA1 gene also had a promoter, but the fdxH gene had a processing site just upstream of the gene. The processing of transcripts at sites upstream of nifH1 and fdxH1 correlated with increased stability of these transcripts, resulting in greater amounts than transcripts that were not close to processing sites.

2-epi-5-epi-Valiolone synthase activity is essential for maintaining phycobilisome composition in the cyanobacterium Anabaena variabilis ATCC 29413 when grown in the presence of a carbon source.

The cyclase 2-epi-5-epi-valiolone synthase (EVS) is reported to be a key enzyme for biosynthesis of the mycosporine-like amino acid shinorine in the cyanobacterium Anabaena variabilis ATCC 29413. Subsequently, we demonstrated that an in-frame complete deletion of the EVS gene had little effect on in vivo production of shinorine. Complete segregation of the EVS gene deletion mutant proved difficult and was achieved only when the mutant was grown in the dark and in a medium supplemented with fructose. The segregated mutant showed a striking colour change from native blue-green to pale yellow-green, corresponding to substantial loss of the photosynthetic pigment phycocyanin, as evinced by combinations of absorbance and emission spectra. Transcriptional analysis of the mutant grown in the presence of fructose under dark or light conditions revealed downregulation of the cpcA gene that encodes the alpha subunit of phycocyanin, whereas the gene encoding nblA, a protease chaperone essential for phycobilisome degradation, was not expressed. We propose that the substrate of EVS (sedoheptulose 7-phosphate) or possibly lack of its EVS-downstream products, represses transcription of cpcA to exert a hitherto unknown control over photosynthesis in this cyanobacterium. The significance of this finding is enhanced by phylogenetic analyses revealing horizontal gene transfer of the EVS gene of cyanobacteria to fungi and dinoflagellates. It is also conceivable that the EVS gene has been transferred from dinoflagellates, as evident in the host genome of symbiotic corals. A role of EVS in regulating sedoheptulose 7-phosphate concentrations in the photophysiology of coral symbiosis is yet to be determined.

[Identification of cyanobacteria from genus Anabaena isolated from the rhizosphere of cotton-plant].

Identification of nucleotide sequence of the gene 16S rRNA of cyanobacteria Anabaena variabilis str.21 (Uzb1) exhibits that the strain from root sphere of cotton plants is 99% homologous to a known strain of Anabaena variabilis (EF488831.1). This data confirms the phylogenetic relationship of the strain of cyanobacteria A. variabilis Uzb1 to other described representatives of genus Anabaena as addition to morphological characteristics (presence of akinets (spores) and heterocysts).