PubMed Health⌕ Search

Biomedical subjects

C Ueguchi

Publications and source records attributed to C Ueguchi.

At least 37 records · Page 2Linked to original sources
PubMed1998

The yhhP gene encoding a small ubiquitous protein is fundamental for normal cell growth of Escherichia coli.

H-NS is a major constituent of the Escherichia coli nucleoid, whereas sigmaS is a stress-induced sigma factor. An hns null mutation affects the cellular content of sigmaS in such a way that a remarkable accumulation of sigmaS is observed in the logarithmic growth phase, which results in enhanced expression of a number of sigmaS-dependent genes, including the katE gene. We isolated an extragenic mutation that affects the expression of the katE-lacZ fusion gene in the deltahns background. The relevant gene was identified as yhhP, which encodes a small polypeptide of 81 amino acids. Lesion of this gene seemed to affect the stability of sigmaS. A deletion analysis of yhhP revealed that this small protein plays a fundamental role in the general physiology of E. coli. The yhhP-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells. Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.

Amino Acid Sequence↗
PubMed1997

Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS.

The Escherichia coli nucleoid protein, H-NS, functions as a global regulator for expression of a wide variety of genes. We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-binding, respectively. However, identification of the presumed dimerization domain of H-NS and its functional significance was elusive. To address this particular issue, we first examined a set of N-terminally or C-terminally truncated forms of H-NS, in terms of their so-called dominant-negative effect on the in vivo function of the wild-type H-NS. The results showed that certain truncated forms exhibit such a dominant-negative effect, but others did not. As judged by the results of the dominant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization. This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS. Furthermore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, which was expected to be specifically defective in dimerization. This mutant had an amino acid substitution at position 30 (Leu30 to Pro) in N91 consisting of the N-terminal 91 amino acids of H-NS. This mutant was indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS. When this particular single amino acid substitution was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a transcriptional repressor. These findings collectively provided us with evidence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor.

Amino Acid Sequence↗
PubMed1997

Cloning of a cDNA encoding a putative metal-transporting P-type ATPase from Arabidopsis thaliana.

Metal-transporting P-type ATPases were recently proposed to constitute a newly emerged sub-family of cation-transporting P-type ATPases, and are known to occur widely in prokaryotes and eukaryotes. However, no instance has been reported for higher plants. A cDNA clone encoding a metal-transporting P-type ATPase was thus searched for, if present, and was identified in Arabidopsis thaliana. The amino acid sequence, predicted from the determined nucleotide sequence for the cloned cDNA, shows all the critical features common to known metal-transporting P-type ATPases. This plant P-type ATPase has a typical metal-binding motif at its N-terminal portion. The newly isolated Arabidopsis gene, named PAA1, provides us with the first instance of putative metal-transporting P-type ATPases in higher plants. Some results of genomic analyses for this gene are also presented.

Adenosine Triphosphatases↗
PubMed1996

Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS.

The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hns mutations which resulted in derepression of the proVWX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.

Amino Acid Sequence↗
PubMed1996

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli.

The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. The dnaJ- cbpA- double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ- cbpA- mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD, the gene that encodes the major sigma 70. The biological implications of this finding are examined and discussed.

Bacterial Proteins↗
PubMed1996

H-NS regulates OmpF expression through micF antisense RNA in Escherichia coli.

H-NS is a major constituent of the Escherichia coli nucleoid. Expression of the major outer membrane proteins, OmpC and OmpF, is influenced by hns mutations such that OmpC expression increases whereas OmpF expression decreases irrespective of the osmolarity of the medium (K. A. Graeme-Cook, G. May, E. Bremer, and C. F. Higgins, Mol. Microbiol. 3:1287-1294, 1989). In this study we show that the effect of an hns::neo mutation on OmpF expression is largely diminished in a deletion mutant carrying the micF gene that encodes the ompF mRNA-specific antisense RNA. In addition, the micF transcript levels in the hns::neo mutation are high compared with transcript levels in wild-type cells. On the basis of these results, we provide evidence for a link between OmpC/OmpF expression and the regulatory function of H-NS. We suggest that H-NS most likely affects OmpC expression directly at the level of transcription, but OmpF expression is indirectly regulated by micF antisense RNA.

Bacterial Outer Membrane Proteins↗
PubMed1995

An Escherichia coli curved DNA-binding protein whose expression is affected by the stationary phase-specific sigma factor sigma S.

From Escherichia coli, a DNA-binding protein that preferentially recognizes a curved DNA sequence was isolated and shown to correspond to one that has recently been reported as a binding protein for the replication origin of the E. coli chromosome, named Rob. Here, a rob promoter-lacZ transcriptional fusion was constructed on the chromosome, and used to demonstrate that the expression of rob is notably enhanced at the onset of stationary phase in Luria-broth and also under certain growth conditions in a minimal medium, such as glucose- and phosphate-starvation medium. It was further shown that this growth condition-dependent expression of rob is notably reduced in a null mutant for the stationary phase-specific sigma subunit of RNA polymerase, sigma s, although sigma s-independent expression of rob was significant during the logarithmic growth phase. Furthermore the rob null mutant was found to exhibit, as compared with the wild-type, an altered profile of protein synthesis, particularly at the very late stationary phase.

Amino Acid Sequence↗
PubMed1995

Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli.

The three-dimensional structure of the C-terminal domain (47 residues) obtained from the hydrolysis of H-NS protein with bovine trypsin was determined by NMR measurements and distance geometry calculations. It is composed of an antiparallel beta-sheet, an alpha-helix and a 3(10)-helix which form a hydrophobic core, stabilizing the whole structure. This domain has been found to bind to DNA. Possible DNA binding sites are discussed on the basis of the solution structure of the C-terminal domain of H-NS.

Amino Acid Sequence↗
PubMed1995

Quantitative control of the stationary phase-specific sigma factor, sigma S, in Escherichia coli: involvement of the nucleoid protein H-NS.

In Escherichia coli, recent intensive studies revealed that expression of a certain subset of genes is under the control of the stationary phase-specific sigma factor, sigma S, which is encoded by the rpoS gene. Since sigma S functions predominantly under certain growth conditions, its activity and/or cellular content has accordingly to be tightly controlled, however, the underlying molecular mechanism is at present unclear. We previously demonstrated that expression of the cbpA gene, encoding an analogue of the DnaJ molecular chaperone, is largely dependent on sigma S function. Here we have found that a mutational lesion of the hns gene, which encodes one of the well-characterized nucleoid proteins, H-NS, affects the cellular content of sigma S remarkably and consequently affects the expression of cbpA. Enhanced accumulation of sigma S in hns deletion cells was particularly observed in the logarithmic growth phase and was demonstrated to result from an elevated translational efficiency of rpoS mRNA and also from an increased stability of newly synthesized sigma S. Although H-NS is known to influence the transcription of a number of apparently unlinked genes on the chromosome, in this study we provide a novel instance in which H-NS is deeply implicated in post-transcriptional regulation(s) of the expression of rpoS. As to physiological relevance, it was also demonstrated that hns deletion cells exhibit an extreme thermotolerance even in the logarithmic growth phase, presumably because of the enhanced accumulation of sigma S.

Adaptation, Biological↗
PubMed1995

A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli.

The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. To gain insight into the function of CbpA, we examined the nature of a cbpA null mutation with special reference to those of dnaK and dnaJ null mutations. In particular, the cbpA dnaJ double-null mutant was found to exhibit severe defects in cell growth, namely, a very narrow temperature range for growth, a defect in cell division, and susceptibility to killing by carbon starvation. These phenotypes are very similar to those reported for dnaK null mutants but not to those of dnaJ null mutants. Our results are best interpreted by assuming that CbpA is capable of compensating for DnaJ for cell growth and thus that the function(s) of CbpA is closely related to that of DnaJ.

Cell Division↗
PubMed1994

An analogue of the DnaJ molecular chaperone in Escherichia coli.

Escherichia coli DnaJ functions as a typical molecular chaperone in coordination with other heat shock proteins such as DnaK and GrpE in a variety of cellular processes. In this study, it was found that E. coli possesses an analogue of DnaJ, as judged from not only its primary structure but also its possible function. This protein, named CbpA (for curved DNA-binding protein), was first identified as a DNA-binding protein that preferentially recognizes a curved DNA sequence. Cloning and nucleotide sequencing of the gene encoding CbpA revealed that the predicted product is very similar to DnaJ in amino acid sequence: overall identity is 39%. The cbpA gene functions as a multicopy suppressor for dnaJ mutations. The mutational lesions characteristic of a dnaJ null mutant--namely, temperature sensitivity for growth and defects in lambda phage and mini-F DNA replication--were all restored upon introduction of the cbpA gene on a multicopy plasmid. An insertional mutant of cbpA was also isolated, which showed no noticeable phenotype, particularly with regard to temperature sensitivity for growth. However, when this cbpA::kan allele was combined with the dnaJ null allele, the resultant strain was unable to grow at 37 degrees C, at which strains carrying each mutation alone could grow normally. These genetic results are interpreted as meaning that the function(s) of CbpA in E. coli is closely related to that of DnaJ.

Amino Acid Sequence↗
PubMed1994

A fission yeast gene encoding a protein that preferentially associates with curved DNA.

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity. As its putative coding sequence, a 1.9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41,597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed1994

An analogue of the DnaJ molecular chaperone whose expression is controlled by sigma s during the stationary phase and phosphate starvation in Escherichia coli.

The Escherichia coli CbpA protein appears to be an analogue of the molecular chaperone, DnaJ, as judged from not only its structure but also its possible function. The expression of cbpA, however, was not significantly affected by up-shift of the growth temperature. Remarkably, it was found that the expression of cbpA was induced under certain growth conditions, such as the entry of cells into stationary phase, or growth in a phosphate-limited medium. Such conditional expression of cbpA was regulated at the transcriptional level in a sigma s-dependent manner. The structure of this sigma s-dependent cbpA promoter was clarified by determining its transcription start site. The cbpA promoter region was found to contain an unusual DNA structure (i.e. DNA curvature). From these results, it was suggested that, in contrast to DnaJ, CbpA may function as a molecular chaperone in an adaptive response to environmental stresses other than heat shock.

Amino Acid Sequence↗
PubMed1994

Importance of stereospecific positioning of the upstream cis-acting DNA element containing a curved DNA structure for the functioning of the Escherichia coli proV promoter.

The mechanism by which the Escherichia coli proV promoter is activated more than 100-fold in response to the medium osmolarity, without the help of any known trans-acting activators, is not yet fully understood. In this context, it has recently begun to be realized that structural features, not the primary sequences, of cis-acting DNA elements may be important for transcriptional regulation in prokaryotes. From this point of view, in this study the proV promoter was characterized by constructing a series of spacer-insertion mutants in a proV-lacZ fusion on the chromosome. Here it was found that the upstream cis-acting sequence must be positioned stereospecifically with respect to the principal -35 and -10 regions for the proV promoter to be fully activated. In this regard, it was suggested that an overall DNA structure, particularly DNA curvature, is an important cis-acting parameter for activation of the proV promoter.

Bacterial Proteins↗
PubMed1993

Sequence of a fission yeast gene encoding a protein with extensive homology to eukaryotic elongation factor-1 gamma.

A polypeptide with an apparent molecular mass of 23 kDa was identified, that exhibited an affinity to a 491-bp DNA derived from one of the Schizosaccharomyces pombe centromeric DNAs (cen1). After determining its N-terminal amino acid (aa) sequence, a Sz. pombe genomic DNA encompassing the coding sequence of the isolated protein was cloned, and a 2.3-kb genomic DNA region sequenced. Further sequence analysis of cDNA clones, originating from this particular genomic region, confirmed the existence of an open reading frame with a short intron, which encodes a 409-aa protein with striking homology to eukaryotic elongation factor-1 gamma.

Amino Acid Sequence↗
PubMed1993

The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor.

The H-NS protein is a major constituent of the Escherichia coli nucleoid structure and is implicated in the compact organization of the chromosome. Based on recent genetic evidence, this protein appears to influence the transcription of a variety of apparently unlinked genes on the chromosome, although the underlying molecular mechanism is not fully understood. In this study, we carried out a series of in vitro transcription assays including purified H-NS with special reference to the osmotically inducible proV promoter of the proVWX operon (or proU), whose expression is known to be derepressed by lesions of the hns (osmZ) gene. Here, H-NS was revealed to selectively inhibit an early step(s) of proV transcription initiation through its direct binding to the promoter region. It was thus demonstrated that H-NS functions directly as a transcriptional repressor. Under the in vitro conditions used, this in vitro inhibitory effect of H-NS was affected by changes in the superhelical density of template DNAs and more significantly by the concentration of potassium (K+) ions. These results are also discussed with regard to the mechanism underlying regulation of the proV promoter in response to the medium osmolarity.

Bacterial Outer Membrane Proteins↗
PubMed1993

Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription.

The Escherichia coli nucleoid protein, H-NS (or H1a), appears to influence the regulation of a variety of unrelated E. coli genes and operons. To gain an insight into the regulation of the hns gene itself, we constructed in this study a hns-lacZ transcriptional fusion gene and inserted a single copy at the att lambda locus on the E. coli chromosome. Expression of hns transcription appeared to be moderately regulated in a growth phase-dependent manner. It also emerged that hns transcription is under negative autoregulation, at least in the logarithmic growth phase. The results of in vitro transcription experiments confirmed that H-NS functions as a repressor for its own transcription. Thus, H-NS was shown to exhibit relatively high affinity for the DNA sequence encompassing the hns promoter region, as compared with a non-specific sequence. These results support the view that the nucleoid protein, H-NS, can function as a transcriptional regulator.

Bacterial Outer Membrane Proteins↗