Biomedical subjects

Shigeki Ehira

Publications and source records attributed to Shigeki Ehira.

12 recordsLinked to original sources

PubMed2006

AnCrpA, a cAMP receptor protein, regulates nif-related gene expression in the cyanobacterium Anabaena sp. strain PCC 7120 grown with nitrate.

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.

Anabaena↗

PubMed2006

NrrA directly regulates expression of hetR during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 requires NtcA, the global nitrogen regulator in cyanobacteria, and HetR, the master regulator of heterocyst differentiation. Expression of hetR is upregulated by nitrogen deprivation, and its upregulation depends on NtcA. However, it has not yet been revealed how NtcA regulates the expression of hetR. In the experiments presented here, it was confirmed that NrrA (All4312), a nitrogen-responsive response regulator, was required for the upregulation of hetR. The use of the nitrogen-responsive transcription initiation sites (TISs) for the hetR gene depended upon NrrA. NrrA bound specifically to the region upstream of TISs located at positions -728 and -696 in vitro. Overexpression of nrrA resulted in enhanced hetR expression and heterocyst formation. A molecular regulatory cascade is proposed whereby NtcA upregulates the expression of nrrA upon limitation of combined nitrogen in the medium and then NrrA upregulates the expression of hetR, leading to heterocyst differentiation.

Anabaena↗

PubMed2006

Mutations in four regulatory genes have interrelated effects on heterocyst maturation in Anabaena sp. strain PCC 7120.

Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.

Anabaena↗

PubMed2006

NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120.

The heterocyst is a specialized cell for nitrogen fixation in the filamentous cyanobacteria, and its development is triggered by limitation of combined nitrogen in the medium. During heterocyst development, patterns of gene expression change dramatically. We identified seven genes encoding transcriptional regulators that were upregulated by nitrogen deprivation in Anabaena PCC 7120, using an Anabaena oligonucleotide microarray. Among them, the nrrA gene, which encodes a response regulator of the OmpR family with a DNA-binding domain, has shown the most prominent induction after nitrogen deprivation. Expression of nrrA increased all through the filaments within 3 h of nitrogen deprivation and became higher in proheterocysts than in vegetative cells after 12 h. Sequence analysis of the promoter region of nrrA indicated that the induction of nrrA depended on NtcA, which is the global nitrogen regulator in cyanobacteria. In the nrrA deletion mutant, heterocyst development was delayed and the induction of hetR, which is the master gene in regulation of heterocyst development, was diminished up to 24 h nitrogen deprivation. It is concluded that nrrA facilitates heterocyst development.

Anabaena↗

PubMed2006

Signal transduction genes required for heterocyst maturation in Anabaena sp. strain PCC 7120.

How heterocyst differentiation is regulated, once particular cells start to differentiate, remains largely unknown. Using near-saturation transposon mutagenesis and testing of transposon-tagged loci, we identified three presumptive regulatory genes not previously recognized as being required specifically for normal heterocyst maturation. One of these genes has a hitherto unreported mutant phenotype. Two previously identified regulatory genes were further characterized.

Anabaena↗

PubMed2005

Role of the 5'-UTR in accumulation of the rbpA1 transcript at low temperature in the cyanobacterium Anabaena variabilis M3.

The expression of the rbp genes, which encode RNA-binding proteins with a single RNA-recognition motif and a glycine-rich sequence, is known to increase at low temperature in cyanobacteria. We previously showed that their regulation involved both transcription and mRNA stability. In the present study, various reporter constructs with deletions and mutations were used to analyze this regulation, revealing that at least the following three elements are involved. First, a putative enhancer element is located within the upstream gene. Second, the rbpA1 transcript is dramatically stabilized by a large stem-loop structure located at the 5' terminus. Third, the transcript is also destabilized by a downstream box located within the coding region.

5' Untranslated Regions↗

PubMed2005

NaCl enhances cellular cAMP and upregulates genes related to heterocyst development in the cyanobacterium, Anabaena sp. strain PCC 7120.

Cellular cAMP was rapidly increased in the nitrogen-fixing cyanobacterium, Anabaena sp. PCC 7120, by the addition of 200 mM NaCl to the culture medium. Other alkaline-metal chlorides such as KCl or LiCl caused a lesser increase. The increase in cellular cAMP was transient and diminished when an adenylate cyclase, CyaC, which contains the conserved domains of the bacterial two-component regulatory system, was disrupted. DNA microarray analysis showed that expression of a gene cluster containing all5347 and alr5351 (hglE) was upregulated by NaCl in the wild-type strain but not in the cyaC mutant. Primer extension analysis indicated that transcription levels of all5347 and hglE were rapidly increased in response to the NaCl addition, and that these genes have NaCl-dependent transcription start sites. It was concluded that NaCl induced expression of genes related to heterocyst envelope formation in this cyanobacterium, possibly via a CyaC-cAMP signal transduction system.

Anabaena↗

PubMed2005

Identification of Low-temperature-regulated ORFs in the cyanobacterium Anabaena sp. strain PCC 7120: distinguishing the effects of low temperature from the effects of photosystem II excitation pressure.

Most organisms have developed various strategies to react rapidly to temperature down-shift and regulate expression of various genes to acclimate to low temperature. In photosynthetic organisms, temperature down-shift in the light results in not only a decrease in growth temperature but also an increase in PSII excitation pressure. Distinguishing the effects of low temperature from the effects of excitation pressure is necessary to understand the mechanism of low-temperature signal transduction. In this report, we analyzed changes in gene expression after three different environmental changes, i.e. temperature down-shift in the light, temperature down-shift in the dark and transfer to the dark, using DNA microarray in the cyanobacterium Anabaena sp. strain PCC 7120. By comparing the expression patterns under the three experimental conditions, we identified 15 open reading frames (ORFs) that were up-regulated by temperature down-shift both in the light and in the dark. These ORFs are considered to be regulated by low temperature, but not by excitation pressure. Six of them have a consensus sequence within the upstream region of their coding region and were indicated also to be up-regulated by tetracycline. Functional or structural changes in the ribosome could affect transcript levels of the low-temperature-regulated ORFs.

Anabaena↗

PubMed2004

Use of segment-based microarray in the analysis of global gene expression in response to various environmental stresses in the cyanobacterium Anabaena sp. PCC 7120.

We prepared microarrays that contain genomic sequences of a heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120. The complete genome of this cyanobacterium codes for about 5,368 protein-coding genes in the main chromosome of 6.4 Mbp. In total, 2,407 DNA segments were selected from the sequencing clones, and amplified by PCR, then spotted on glass slides in duplicate. These microarrays differ from the widely used commercial or custom-made ones for other microorganisms in that each DNA segment was 3-4 kbp long, and contained about 3-4 predicted genes on average. This feature, however, did not decrease the usefulness of the microarrays, since we were able to detect a number of potentially novel genes that are induced in response to nitrogen deprivation, low temperature and drought. In addition, we found some genomic regions in which dozens of contiguous genes are simultaneously regulated. These results suggest that these segment-based microarrays are useful especially for such large genomes as Anabaena, for which the number of genes exceeds either technical or practical limitations.

Anabaena↗

PubMed2003

GenoMap, a circular genome data viewer.

A Tcl/Tk-based application called GenoMap is described, a viewer for genome-wide map of microarray expression data within a circular bacterial genome. An interactive interface facilitates easy identification of the expressed region. This software is also used for drawing genome-wide quantitative data.

Computer Graphics↗

PubMed2003

Conserved temperature-dependent expression of RNA-binding proteins in cyanobacteria with different temperature optima.

The expression of the rbp genes, which encode small RNA-binding proteins with a single RNA-recognition motif, is known to increase at low temperature in Anabaena variabilis M3. The 5'-untranslated region (UTR) of the rbpA1 gene is involved in the cold-regulation. We compared the regulation of the rbp genes in three strains of cyanobacteria having different temperature optima, namely, a mesophilic strain Anabaena sp. PCC 7120, a thermophilic strain Thermosynechococcus elongatus BP-1, and a psychrophilic Antarctic strain Oscillatoria sp. SU1. In Anabaena 7120 and T. elongatus, all the rbp gene sequences are known, and the 5'-UTR sequences of some rbp genes have a high similarity to the 5'-UTR of rbpA1. We found that transcripts as well as protein products of these rbp genes accumulated at low temperature. In addition, the expression of rbp genes increased at low temperature in the Oscillatoria sp. SU1. This suggests that a mechanism of cold-regulation of rbp genes is common among various species of cyanobacteria that belong to different taxa and have different temperature optima.

5' Untranslated Regions↗

PubMed2003

Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A heterocyst is a terminally differentiated cell of cyanobacteria which is specialized in dinitrogen fixation. Heterocyst differentiation in Anabaena sp. strain PCC 7120 is triggered by deprivation of combined nitrogen in the medium. Although various genes that are upregulated during heterocyst differentiation have been reported, most studies to date were limited to individual or a small number of genes. We prepared microarrays in collaboration with other members of the Anabaena Genome Project. Here we report on the genome-wide expression analysis of the responses to nitrogen deprivation in Anabaena. Many unidentified genes, as well as previously known genes, were found to be upregulated by nitrogen deprivation at various time points. Three main profiles of gene expression were found: genes expressed transiently at an early stage (1-3 hr) of nitrogen deprivation, genes expressed transiently at a later stage (8 hr), and genes expressed when heterocysts are formed (24 hr). We also noted that many of the upregulated genes were physically clustered to form 'expressed islands' on the chromosome. Namely, large, continuous genomic regions containing many genes were upregulated in a coordinated manner. This suggests a mechanism of global regulation of gene expression that involves chromosomal structure, which is reminiscent of eukaryotic chromatin remodelling. The possible implications of this global regulation are discussed.

Anabaena↗