ABSTRACT
The cyanobacterium Anabaena sp. strain PCC 7120 exhibits dehydration tolerance. The regulation of gene expression in response to dehydration is crucial for the acquisition of dehydration tolerance, but the molecular mechanisms underlying dehydration responses remain unknown. In this study, the functions of the response regulator OrrA in the regulation of salt and dehydration responses were investigated. Disruption of orrA abolished or diminished the induction of hundreds of genes in response to salt stress and dehydration. Thus, OrrA is a principal regulator of both stress responses. In particular, OrrA plays a crucial role in dehydration tolerance because an orrA disruptant completely lost the ability to regrow after dehydration. Moreover, in the OrrA regulon, avaKa encoding a protein of unknown function was revealed to be indispensable for dehydration tolerance. OrrA and AvaK are conserved among the terrestrial cyanobacteria, suggesting their conserved functions in dehydration tolerance in cyanobacteria.
Subject(s)
Anabaena , Cyanobacteria , Humans , Gene Expression Regulation, Bacterial , Dehydration , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Anabaena/genetics , Anabaena/metabolism , Cyanobacteria/geneticsABSTRACT
Cyanobacteria acclimatize to nitrogen deprivation by changing cellular metabolism. The nitrogen-regulated response regulator A (NrrA) is involved in regulation of carbon metabolism in response to nitrogen deprivation. However, it has not been elucidated whether these regulatory functions of NrrA are particular to a few model strains or are general among diverse cyanobacteria. In this study, we showed that regulation and functions of NrrA were highly conserved among ß-cyanobacteria, which included physiologically and ecologically diverse strains. All ß-cyanobacteria had the nrrA gene, while it was absent in α-cyanobacteria. The canonical NtcA-dependent promoter sequence was found upstream of the nrrA genes in most ß-cyanobacteria, and its expression was indeed induced by nitrogen deprivation. Biochemical and physiological analyses of NrrA from phylogenetically distinct cyanobacteria indicated that regulation of NrrA activity and NrrA functions, namely activation of glycogen catabolism, were also common to ß-cyanobacteria. These results support the conclusion that NrrA plays an important role in acclimatization to nitrogen deprivation, and that activation of glycogen catabolism is a primitive response to nitrogen deprivation in ß-cyanobacteria.
Subject(s)
Bacterial Proteins , Cyanobacteria/genetics , Cyanobacteria/metabolism , Gene Expression Regulation, Bacterial , Glycogen/metabolism , Nitrogen/metabolism , PII Nitrogen Regulatory Proteins/metabolism , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Regulatory Networks , Inverted Repeat Sequences , PII Nitrogen Regulatory Proteins/genetics , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Two-Hybrid System TechniquesABSTRACT
Oxygenic photosynthesis is driven by photosystems I and II (PSI and PSII, respectively). Both have specific antenna complexes and the phycobilisome (PBS) is the major antenna protein complex in cyanobacteria, typically consisting of a core from which several rod-like subcomplexes protrude. PBS preferentially transfers light energy to PSII, whereas a PSI-specific antenna has not been identified. The cyanobacterium Anabaena sp. PCC 7120 has rod-core linker genes (cpcG1-cpcG2-cpcG3-cpcG4). Their products, except CpcG3, have been detected in the conventional PBS. Here we report the isolation of a supercomplex that comprises a PSI tetramer and a second, unique type of a PBS, specific to PSI. This rod-shaped PBS includes phycocyanin (PC) and CpcG3 (hereafter renamed "CpcL"), but no allophycocyanin or CpcGs. Fluorescence excitation showed efficient energy transfer from PBS to PSI. The supercomplex was analyzed by electron microscopy and single-particle averaging. In the supercomplex, one to three rod-shaped CpcL-PBSs associate to a tetrameric PSI complex. They are mostly composed of two hexameric PC units and bind at the periphery of PSI, at the interfaces of two monomers. Structural modeling indicates, based on 2D projection maps, how the PsaI, PsaL, and PsaM subunits link PSI monomers into dimers and into a rhombically shaped tetramer or "pseudotetramer." The 3D model further shows where PBSs associate with the large subunits PsaA and PsaB of PSI. It is proposed that the alternative form of CpcL-PBS is functional in harvesting energy in a wide number of cyanobacteria, partially to facilitate the involvement of PSI in nitrogen fixation.
Subject(s)
Anabaena/metabolism , Models, Molecular , Photosystem I Protein Complex/metabolism , Phycobilisomes/metabolism , Protein Conformation , Cell Fractionation , Cluster Analysis , Immunoblotting , Microscopy, Electron , Spectrometry, FluorescenceABSTRACT
The heterocystous cyanobacterium Anabaena sp. strain PCC 7120 grows as linear multicellular filaments that can contain hundreds of cells. Heterocysts, which are specialized cells for nitrogen fixation, are regularly intercalated among photosynthetic vegetative cells, and these cells are metabolically dependent on each other. Thus, multicellularity is essential for diazotrophic growth of heterocystous cyanobacteria. In Anabaena sp. strain PCC 7120, the fraF gene, which is required to limit filament length, is induced by nitrogen deprivation. The fraF transcripts extend to the fraE gene, which lies on the opposite DNA strand and could possess dual functionality, mRNAs for fraF and antisense RNAs for fraE. In the present study, we found that NrrA, a nitrogen-regulated response regulator, directly regulated expression of fraF. Induction of fraF by nitrogen deprivation was abolished by the nrrA disruption. NrrA specifically bound to the promoter region of fraF, and recognized an inverted repeat sequence. Thus, it is concluded that NrrA controls expression of mRNAs for fraF and antisense RNAs for fraE in response to nitrogen deprivation.
Subject(s)
Anabaena/genetics , Anabaena/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Nitrogen/metabolism , RNA, Antisense/biosynthesis , Transcription Factors/metabolism , Anabaena/growth & development , Bacterial Proteins/genetics , Gene Knockout Techniques , Nitrogen Fixation , RNA, Antisense/genetics , Transcription Factors/geneticsABSTRACT
The filamentous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 accumulates sucrose as a compatible solute against salt stress. Sucrose-phosphate synthase activity, which is responsible for the sucrose synthesis, is increased by salt stress, but the mechanism underlying the regulation of sucrose synthesis remains unknown. In the present study, a response regulator, OrrA, was shown to control sucrose synthesis. Expression of spsA, which encodes a sucrose-phosphate synthase, and susA and susB, which encode sucrose synthases, was induced by salt stress. In the orrA disruptant, salt induction of these genes was completely abolished. The cellular sucrose level of the orrA disruptant was reduced to 40% of that in the wild type under salt stress conditions. Moreover, overexpression of orrA resulted in enhanced expression of spsA, susA, and susB, followed by accumulation of sucrose, without the addition of NaCl. We also found that SigB2, a group 2 sigma factor of RNA polymerase, regulated the early response to salt stress under the control of OrrA. It is concluded that OrrA controls sucrose synthesis in collaboration with SigB2.
Subject(s)
Anabaena/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Sucrose/metabolism , Anabaena/drug effects , Anabaena/genetics , Bacterial Proteins/genetics , Gene Deletion , Gene Expression Profiling , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Osmotic Pressure , Sigma Factor/metabolism , Sodium Chloride/metabolismABSTRACT
The filamentous, heterocystous cyanobacterium Anabaena sp. strain PCC 7120 is one of the simplest multicellular organisms that show both morphological pattern formation with cell differentiation (heterocyst formation) and circadian rhythms. Therefore, it potentially provides an excellent model in which to analyze the relationship between circadian functions and multicellularity. However, detailed cyanobacterial circadian regulation has been intensively analyzed only in the unicellular species Synechococcus elongatus. In contrast to the highest-amplitude cycle in Synechococcus, we found that none of the kai genes in Anabaena showed high-amplitude expression rhythms. Nevertheless, ~80 clock-controlled genes were identified. We constructed luciferase reporter strains to monitor the expression of some high-amplitude genes. The bioluminescence rhythms satisfied the three criteria for circadian oscillations and were nullified by genetic disruption of the kai gene cluster. In heterocysts, in which photosystem II is turned off, the metabolic and redox states are different from those in vegetative cells, although these conditions are thought to be important for circadian entrainment and timekeeping processes. Here, we demonstrate that circadian regulation is active in heterocysts, as shown by the finding that heterocyst-specific genes, such as all1427 and hesAB, are expressed in a robust circadian fashion exclusively without combined nitrogen.
Subject(s)
Anabaena/genetics , Anabaena/metabolism , Circadian Clocks , Circadian Rhythm , Gene Expression Regulation, Bacterial , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Circadian Rhythm Signaling Peptides and Proteins/biosynthesis , Circadian Rhythm Signaling Peptides and Proteins/genetics , Gene Expression , Nitrogen Fixation/geneticsABSTRACT
BACKGROUND: Thioredoxins (Trxs) play a crucial role in the oxidative stress response. RESULTS: A redox-sensing transcriptional regulator, RexT, controls expression of TrxA2, and TrxA2 regulates the DNA binding activity of RexT. CONCLUSION: The RexT-TrxA2 regulatory system regulates gene expression in response to redox state. SIGNIFICANCE: This is the first report on a transcriptional regulator of the trx gene in cyanobacteria. Thioredoxins are ubiquitous proteins that catalyze thiol-disulfide redox reactions. They have a crucial role in the oxidative stress response as well as the redox regulation of metabolic enzymes. In cyanobacteria, little is known about the regulation of trx gene expression despite the importance of thioredoxins in cellular functions. In the present study, transcriptional regulation of the trx genes under oxidative stress conditions was investigated in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120. When cells were exposed to H(2)O(2), only the trxA2 gene (all1866) of seven trx genes was induced. Disruption of the rexT gene (alr1867), encoding a transcriptional regulator of the ArsR family, resulted in increased expression of trxA2. RexT bound to the region downstream of the transcription initiation site of trxA2. The DNA binding activity of RexT was impaired by H(2)O(2) through the formation of an intramolecular disulfide bond, which induced expression of the trxA2 gene. The inactivated DNA binding activity of RexT was restored by reduced TrxA2. Hence, RexT is considered as a redox-sensing transcriptional repressor of trxA2. These results support the idea that the RexT-TrxA2 regulatory system is important for the oxidative stress response in this cyanobacterium.
Subject(s)
Anabaena/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Thioredoxins/genetics , Transcription Factors/metabolism , Anabaena/chemistry , Anabaena/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , Hydrogen Peroxide/metabolism , Molecular Sequence Data , Oxidation-Reduction , Oxidative Stress , Protein Binding , Thioredoxins/chemistry , Thioredoxins/metabolism , Transcription Factors/geneticsABSTRACT
Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium in which certain vegetative cells differentiate into heterocysts that are specialized cells for nitrogen fixation. Heterocysts are unable to carry out photosynthesis and depend on vegetative cells for carbohydrate to generate ATP and reductants required for nitrogen fixation. Thus, carbohydrate metabolism is very important for nitrogen fixation in the filamentous cyanobacteria; however, its regulatory mechanism remains unknown. In the present study, a nitrogen-regulated response regulator NrrA, which is a transcriptional regulator involved in heterocyst differentiation, was shown to control glycogen catabolism. The transcript levels of genes involved in glycogen catabolism, such as glgP1 and xfp-gap1-pyk1-talB operon, were decreased by the nrrA disruption. Moreover, glycogen accumulation and depression of nitrogenase activities were observed in this disruptant. NrrA bound specifically to the promoter region of glgP1, encoding a glycogen phosphorylase, and to the promoter region of sigE, encoding a group 2 σ factor of RNA polymerase. SigE activated expression of the xfp operon, encoding enzymes of glycolysis and the pentose phosphate pathway. It is concluded that NrrA controls not only heterocyst differentiation but also glycogen catabolism in Anabaena sp. strain PCC 7120.
Subject(s)
Anabaena/metabolism , Bacterial Proteins/metabolism , Cyanobacteria/metabolism , Gene Expression Regulation, Bacterial , Glycogen/metabolism , Sigma Factor/metabolism , Trans-Activators/metabolism , Anabaena/chemistry , Bacteria/metabolism , Base Sequence , Carbohydrates/chemistry , Glycogen/chemistry , Glycogenolysis , Models, Biological , Molecular Sequence Data , Mutation , Nitrogen/chemistry , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium in which certain vegetative cells differentiate into heterocysts, which are specialized cells for nitrogen fixation. Heterocysts are unable to carry out photosynthesis and are supplied with carbohydrate required for nitrogen fixation from neighbouring vegetative cells. Thus, filament integrity is very important for diazotrophic growth of the heterocystous cyanobacteria. The pknH gene (alr1336), encoding a putative Ser/Thr protein kinase, was upregulated in heterocysts after nitrogen deprivation. Its expression was developmentally regulated by the hetR gene. Expression levels of genes involved in heterocyst maturation, such as hepA, hglE and nifH, in the pknH disruptant were similar to those of the wild-type strain. The disruptant was able to form heterocysts with nitrogenase activity, but most heterocysts were detached from filaments. Hence, the pknH disruptant showed a growth defect in the medium without combined nitrogen. It is concluded that the pknH gene is not involved in the development of heterocyst function but is involved in maintaining connections between heterocysts and vegetative cells.
Subject(s)
Anabaena/enzymology , Anabaena/growth & development , Bacterial Proteins/genetics , Gene Expression Regulation, Developmental , Protein Serine-Threonine Kinases/genetics , Anabaena/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Protein Serine-Threonine Kinases/metabolismABSTRACT
C-Aryl 5a-carba-ß-d-glucopyranose derivatives were synthesized and evaluated for inhibition activity against hSGLT1 and hSGLT2. Modifications to the substituents on the two benzene rings resulted in enhanced hSGLT2 inhibition activity and extremely high hSGLT2 selectivity versus SGLT1. Using the created superimposed model, the reason for the high hSGLT2 selectivity was speculated to be that additional substituents occupied a new space, in a different way than known inhibitors. Among the tested compounds, the ethoxy compound 5h with high hSGLT2 selectivity exhibited more potent and longer hypoglycemic action in db/db mice than our O-carbasugar compound (1) and sergliflozin (2), which could be explained by its improved PK profiles relative to those of the two compounds. These results indicated that 5h might be a promising drug candidate for the treatment of type 2 diabetes.
Subject(s)
Cyclohexanols/chemistry , Diabetes Mellitus, Type 2/drug therapy , Glucose/analogs & derivatives , Hypoglycemic Agents/chemistry , Sodium-Glucose Transporter 2 Inhibitors , Administration, Oral , Animals , Area Under Curve , Blood Glucose/analysis , Cyclohexanols/pharmacokinetics , Cyclohexanols/therapeutic use , Glucose/pharmacokinetics , Glucose/therapeutic use , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/therapeutic use , Mice , Mice, Obese , Sodium-Glucose Transporter 2/metabolism , Structure-Activity RelationshipABSTRACT
5a-Carba-ß-D-glucopyranose derivatives were synthesized and identified as novel SGLT2-selective inhibitors. These inhibitors exhibited potent SGLT2 inhibition with high selectivity over SGLT1. Among the tested compounds, 6f indicated the most potent hSGLT2 inhibition and the highest selectivity over hSGLT1. Moreover, the pharmacokinetics data also showed that 6h, which had the same aglycon structure as sergliflozin-active (3-active), had a threefold longer half-life time (T(1/2)) than sergliflozin (3) with a high distribution volume in db/db mice. Subsequently, 6h lowered blood glucose levels as much as 3 and showed longer hypoglycemic action than 3 in db/db mice.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Glucose/analogs & derivatives , Sodium-Glucose Transporter 2 Inhibitors , Animals , Glucose/chemical synthesis , Glucose/chemistry , Glucose/pharmacology , Male , Mice , Mice, Obese , Molecular Conformation , Molecular Sequence Data , Sodium-Glucose Transporter 2/metabolism , Stereoisomerism , Structure-Activity Relationship , Tissue DistributionABSTRACT
The survival of the terrestrial cyanobacterium Nostoc sp. HK-01 was tested as part of the Tanpopo mission experiment, which was conducted both outside and inside the International Space Station (ISS). The selection of Nostoc sp. HK-01 was based on the results of on-ground experiments that demonstrated that the cyanobacterium can survive simulated space environments. This study verified cell survival after exposure to the outside environment in low Earth orbit (LEO). We examined the cellular tolerance of Nostoc sp. HK-01 simultaneously outside and inside of the ISS over a 3-year period. After the experiments were conducted, we confirmed cell viability by fluorescein diacetate (FDA). Cell growth abilities for 3 years without sunlight in space-vacuum-exposed cells were not significantly different from those of cells kept in the dark of control cells in the ISS and on the ground. Though a few light-exposed cells in space vacuum survived outside the ISS after 3 years as judged by FDA staining assay, the survival could not be verified by testing the growth ability due to an insufficient number of cells. To the best of our knowledge, this is the first pure strain of Nostoc sp. HK-01 that survived in a space environment on the inside and outside of the ISS with and without sunlight for more than 3 years (1126 days).
Subject(s)
Nostoc , Cell Survival , Earth, Planet , VacuumABSTRACT
We have previously identified two target genes (slr1667 and slr1668) for transcriptional regulation by a cAMP receptor protein, SYCRP1, in a cAMP-dependent manner. For this study we investigated the localizations of products of slr1667 and slr1668 (designated cccS and cccP, respectively) biochemically and immunocytochemically, and examined the phenotypes of their disruptants. CccS protein was detected in the culture medium and the acid-soluble fraction containing proteins derived from outside the outer membrane. Disruptants of cccS and cccP showed a more or less similar pleiotropic phenotype. Several proteins secreted into the culture medium or retained on the outside of the outer membrane were greatly reduced in both disruptants compared with the wild type. Electron microscopy revealed that the cccS disruptant lacked the thick pili responsible for motility and that the cccP disruptant had almost no discernible thick pili on its cell surface. Both disruptants largely secreted far greater amounts of yellow pigments into the culture medium than did the wild type. Furthermore, the disruptions reduced the amount of UV-absorbing compound(s) extractable from the exopolysaccharide layer. These results suggest that the cccS and cccP genes are involved in the construction of cell surface components in Synechocystis sp. strain PCC 6803.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Synechocystis/genetics , Bacterial Outer Membrane Proteins/genetics , Fimbriae, Bacterial/ultrastructure , Gene Expression Regulation, Bacterial , Genes, Bacterial , Mutation , Open Reading Frames , Phenotype , Pigments, Biological/metabolism , Synechocystis/metabolism , Synechocystis/ultrastructureABSTRACT
The filamentous cyanobacterium Anabaena sp. PCC 7120 fixes dinitrogen facultatively. Upon depletion of combined nitrogen, about 10% of vegetative cells within the filaments differentiate terminally into nitrogen-fixing cells. The heterocyst has been studied as a model system of prokaryotic cell differentiation, with major focus on signal transduction and pattern formation. The fate of heterocyst differentiation is determined at about the eighth hour of induction (point of no return), well before conspicuous morphological or metabolic changes occur. However, little is known about how the initial heterocysts are selected after the induction by nitrogen deprivation. To address this question, we followed the fate of every cells on agar plates after nitrogen deprivation with an interval of 4 h. About 10% of heterocysts were formed without prior division after the start of nitrogen deprivation. The intensity of fluorescence of GFP in the transformants of hetR-gfp increased markedly in the future heterocysts at the fourth hour with respect to other cells. We also noted that the growing filaments consisted of clusters of four consecutive cells that we call quartets. About 75% of initial heterocysts originated from either of the two outer cells of quartets at the start of nitrogen deprivation. These results suggest that the future heterocysts are loosely selected at early times after the start of nitrogen deprivation, before the commitment. Such early candidacy could be explained by different properties of the outer and inner cells of a quartet, but the molecular nature of candidacy remains to be uncovered.
Subject(s)
Anabaena/cytology , Amino Acid Sequence , Anabaena/genetics , Anabaena/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Culture Media/metabolism , Fluorescence , Gene Deletion , Gene Dosage , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Developmental , Gene Silencing , Genes, Bacterial , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Multigene Family , Mutation , Nitrogen/metabolism , Nitrogen Fixation/genetics , Phencyclidine/analogs & derivatives , Phencyclidine/metabolism , Promoter Regions, Genetic , Sequence AlignmentABSTRACT
Cellular cAMP level increased dramatically upon rehydration following dehydration for 24h in Anabaena sp. PCC 7120, but not in disruptant of an adenylate cyclase gene, cyaC. Oxygen consumption in the cyaC disruptant upon rehydration was higher than that in wild-type strain. Determination of lipid peroxidation and protein carbonylation of the cells revealed greater oxidative stress in the cyaC disruptant than in the wild-type strain during rehydration. Addition of cAMP or KCN to the cyaC disruptant decreased cellular oxygen consumption upon rehydration and oxidative damage. These results suggest that respiration upon rehydration is regulated by cAMP and that the higher respiration activity results in more oxidative damage in cyaC disruptant.
Subject(s)
Anabaena/metabolism , Cyclic AMP/metabolism , Oxidative Stress , Oxygen/metabolism , Water/metabolism , Adenylyl Cyclases/genetics , Anabaena/drug effects , Anabaena/genetics , Cyclic AMP/genetics , Lipid Peroxidation , Potassium Cyanide/pharmacologyABSTRACT
DNA-binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystissp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy () for SYCRP1 for those sites. The values were calculated by summing DeltaDeltaG values derived from systematic single base-pair substitution experiments (symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a value <3.9kcal.mol(-1) located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a DeltaDeltaG(total) value <3.9kcal.mol(-1). The best correlation coefficient between and the observed DeltaDeltaG(total) for binding of SYCRP1 to those sites was 0.78. These results suggest that the DeltaDeltaG values derived from systematic single base-pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.
Subject(s)
Bacterial Proteins/chemistry , Receptors, Cyclic AMP/chemistry , Synechocystis/genetics , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Genome, Bacterial , Molecular Sequence Data , Point Mutation , Receptors, Cyclic AMP/genetics , ThermodynamicsABSTRACT
The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.
Subject(s)
Anabaena/genetics , Anabaena/metabolism , Bacterial Proteins/genetics , Genes, Bacterial , Polysaccharides, Bacterial/biosynthesis , Sigma Factor/genetics , Acclimatization/genetics , Base Sequence , DNA Primers/genetics , DNA, Bacterial/genetics , Disasters , Ecosystem , Nostoc/genetics , Nostoc/metabolism , Phylogeny , Water/metabolismABSTRACT
Target genes for a cAMP receptor protein, AnCrpA, were screened using an Anabaena oligonucleotide microarray and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Several gene expressions, including some involved in nitrogen fixation, were downregulated in the ancrpA disruptant when cells were grown with nitrate. Electrophoretic mobility shift assays (EMSAs) revealed that AnCrpA bound to the 5' upstream region of nifB, all1439, hesA, all5347, hglE and coxBII in the presence of cAMP, and all of them are related with nitrogen fixation. A possible AnCrpA-binding site in the 5' upstream region of nifB was predicted using hidden Markov model (HMM) software based on the result of in vitro selection of AnCrpA-binding sequences, and the binding was confirmed by EMSA. Thus, AnCrpA regulates the expressions of gene clusters related to nitrogen fixation in the presence of nitrate.
Subject(s)
Anabaena/metabolism , Bacterial Proteins/biosynthesis , Cyclic AMP Receptor Protein/metabolism , Gene Expression Regulation, Bacterial/physiology , Nitrogen Fixation/physiology , Regulatory Elements, Transcriptional/physiology , Anabaena/genetics , Bacterial Proteins/genetics , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , Cyclic AMP Receptor Protein/genetics , Gene Deletion , Gene Expression Regulation, Bacterial/drug effects , Markov Chains , Models, Genetic , Multigene Family/physiology , Nitrates/pharmacology , Nitrogen Fixation/drug effects , Protein Binding , SoftwareABSTRACT
PURPOSE: To identify nucleotide sequence variations in the rhodopsin (RHO) gene of Japanese patients with retinitis pigmentosa (RP) in order to search for mutations or haplotypes responsible for RP. METHODS: The entire region of RHO locus including a promoter region and introns was sequenced using blood-derived genomic DNA samples donated by 68 patients with RP and 68 control subjects. RESULTS: We found 39 single nucleotide substitutions including 17 rare substitutions of less than 1% in frequency, one insertion/deletion polymorphism, and one CA-repeat polymorphism in a 7.8 kbp region spanning the promoter, five exons, and four introns of the RHO gene locus. There were no affected subjects with amino acid substitutions in RHO, and there was 1 control subject with a novel substitution (Ala42Thr) who had no symptoms of RP. Fine analysis of single nucleotide polymorphism (SNPs) revealed eight haplotype structures of the Japanese RHO locus. There was no significant difference between RP patients and controls in terms of haplotype frequency. CONCLUSIONS: No mutation causing an amino acid substitution of RHO was observed in 68 Japanese patients with RP, but 1 control subject did have a novel amino acid substitution. The Japanese RHO locus is comprised of eight major haplotypes. The RP-associated haplotype was not identified. The haplotype-tagging SNPs identified in this study will be useful as markers for the linkage-based screening of RP patients.
Subject(s)
Asian People/genetics , Chromosome Mapping , DNA Mutational Analysis , Haplotypes , Retinitis Pigmentosa/genetics , Rhodopsin/genetics , DNA Transposable Elements , Exons , Female , Gene Deletion , Gene Frequency , Genetic Variation , Humans , Introns , Linkage Disequilibrium , Male , Phylogeny , Polymorphism, Single Nucleotide , Promoter Regions, Genetic/geneticsABSTRACT
In 1982 we proposed the presence of a subtype of type 1 diabetes [slowly progressive insulin-dependent diabetes mellitus (SPIDDM)], which was characterized by persistently positive islet cell antibody, late age of onset, noninsulin-dependent diabetes, and slowly progressive beta cell failure. Since then many studies demonstrated that this subtype of type 1 diabetes is prevalent in many ethnic groups and was later called the latent autoimmune diabetes in adults (LADA). Recent epidemiological studies reported that about 10% of patients with apparent type 2 diabetes have at least one autoantibodies against islet-specific antigen with high potential to progress to insulin-dependent state. Between SPIDDM and LADA some differences are reported in terms of some genetic predispositions including HLA class II and class I genes, vitamin D receptor gene, and CTLA4 genes. Common features in SPIDDM and LADA including preserved beta cells at the onset of diabetes and weak T cell response to residual beta cells suggest that these subtypes of type 1 diabetes are suitable candidates for prevention treatment for further progression of beta cell failure.