Susumu Masuda

Specificity crosstalk among group 1 and group 2 sigma factors in the cyanobacterium Synechococcus sp. PCC7942: In vitro specificity and a phylogenetic analysis.

The chromosome of the cyanobacterium Synechococcus sp. PCC7942 contains at least one group 1 (rpoD1) and three group 2 (rpoD2, rpoD3 and rpoD4) sigma factor genes. In this study, we have analysed the structure of rpoD3 and rpoD4 and have shown that these genes are dispensable for growth at normal physiological conditions. An RNA polymerase core enzyme of the cyanobacterial strain was purified, reconstituted with the recombinant sigma factors (the rpoD1, rpoD3 and rpoD4 gene products), and the resultant holoenzymes were examined in vitro for transcription specificity. All of the holoenzymes recognized canonical promoters of Escherichia coli as well as cyanobacterial rrnA, cpcB1A1 P1a and rpoD1 promoters, although the three holoenzymes had some preference for specific promoters. These results suggest that group 1 as well as group 2 sigma factors of cyanobacteria may direct transcription initiation from the eubacterial consensus‐type promoters containing the Pribnow −10 element, and we postulate that specificity crosstalk is a common characteristic among eubacterial group 1 and group 2 sigma factors. Phylogenetic analyses revealed that most group 2 sigma factors were positioned in one of four distinct clusters. The implication of the phylogenetic tree is also discussed in this paper.

Induction of a Group 2 σ-Factor, RPOD3, by High Light and the Underlying Mechanism in Synechococcus elongatus PCC 7942

Among the σ70 family bacterial σ factors, group 2 σ factors have similar promoter recognition specificity to group 1 (principal) σ factors and express and function under specific environmental and physiological conditions. In general, the cyanobacterial genome encodes more than four group 2 σ factors, and the unicellular Synechococcus elongatus PCC 7942 (Synechococcus) has five group 2 σ factors (RpoD2–6). In this study, we analyzed expression of group 2 σ factors of Synechococcus at both mRNA and protein levels, and we showed that the rpoD3 expression was activated only by high light (1,500 μmol photons m–2 s–1) among the various stress conditions examined. After high light shift, rpoD3 mRNA accumulated transiently within the first 5 min and diminished subsequently, whereas RpoD3 protein increased gradually during the first several hours. We also found that the rpoD3 deletion mutant rapidly lost viability under the same conditions. Analysis of the rpoD3 promoter structure revealed the presence of an HLR1 (high light-responsive element 1) sequence, which was suggested to be responsible for the high light-induced transcription under the control of the NblS (histidine kinase)-RpaB (response regulator) two-component system (Kappell, A. D., and van Waasbergen, L. G. (2007) Arch. Microbiol. 187, 337–342), at +6 to +23 with respect to the transcriptional start site. Here we demonstrated that recombinant RpaB protein specifically bound to HLR1 of the rpoD3 and hliA genes in vitro, and overexpression of a truncated RpaB variant harboring only the phosphoreceiver domain derepressed the transcription in vivo. Thus, we have concluded that phosphorylated RpaB are repressing the rpoD3 and hliA transcription under normal growth conditions, and the RpaB dephosphorylation induced by high light stress results in transcriptional derepression.

Possible Involvement of Leaf Gibberellins in the Clock-Controlled Expression of XSP30, a Gene Encoding a Xylem Sap Lectin, in Cucumber Roots

Root-produced organic compounds in xylem sap, such as hormones and amino acids, are known to be important in plant development. Recently, biochemical approaches have revealed the identities of several xylem sap proteins, but the biological functions and the regulation of the production of these proteins are not fully understood. XYLEM SAP PROTEIN 30 kD (XSP30), which is specifically expressed in the roots of cucumber (Cucumis sativus), encodes a lectin and is hypothesized as affecting the development of above-ground organs. In this report, we demonstrate that XSP30 gene expression and the level of XSP30 protein fluctuate in a diurnal rhythm in cucumber roots. The rhythmic gene expression continues for at least two or three cycles, even under continuous light or dark conditions, demonstrating that the expression of this gene is controlled by a circadian clock. Removal of mature leaves or treatment of shoots with uniconazole-P, an inhibitor of gibberellic acid (GA) biosynthesis, dampens the amplitude of the rhythmic expression; the application of GA negates these effects. These results suggest that light signals perceived by above-ground organs, as well as GA that is produced, possibly, in mature leaves, are important for the rhythmic expression of XSP30 in roots. This is the first demonstration of the regulation of the expression of a clock-controlled gene by GA.

The complete nucleotide sequence of the gene (rpoD1) encoding the principal σ factor of the RNA polymerase from the cyanobacterium Synechococcus sp. strain PCC7942

The complete nucleotide sequence of rpoD1 gene from Synechococcus PCC7942 has been determined. The nucleotide data have indicated the presence of an open reading frame of 1155 base pairs encoding a polypeptide which shares the framework structure for principal sigma factors of eubacterial strains.

A receptor-binding peptide from human interleukin-6: Isolation and a proton nuclear magnetic resonance study

In order to locate the receptor-binding region of human interleukin-6 (IL-6), twelve peptide fragments were prepared by digestion of IL-6 with lysylendopeptidase. A significant activity of the receptor-binding was observed only for a peptide Ile88-Lys121, although the activity was estimated at 10(4)-fold less than that of intact IL-6. Solution structure of the peptide Ile88-Lys121 was analyzed by using two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The results indicate the presence of alpha-helices in the regions Leu93-Phe106 and Glu110-Ser119. On the basis of the NMR data, we also prepared two peptides. Four-fold less binding activity than that of the peptide Ile88-Lys121 was observed for the peptide Ile88-Arg105, but no activity for the peptide Glu110-Lys121. These results suggest that the helical peptide Ile88-Arg105 composes a part of the receptor-binding region.

Analysis of Enzyme Production by Submerged Culture of Aspergillus oryzae Using Whole Barley

We have reported on high enzyme production by submerged culture of Aspergillus kawachii using barley with the husk (whole barley). To elucidate the mechanism underlying this high enzyme production, we performed a detailed analysis. Aspergillus oryzae RIB40 was submerged-cultured using whole barley and milled whole barley. Enzyme production was analyzed in terms of changes in medium components and gene expression levels. When whole barley was used, high production of glucoamylase and α-amylase and high gene expression levels of these enzymes were observed. Low ammonium concentrations were maintained with nitrate ion uptake continuing into the late stage using whole barley. These findings suggest that the sustainability of nitrogen metabolism is related to high enzyme production, and that a mechanism other than that associated with the conventional amylase expression system is involved in this relationship.

A Z-DNA binding protein isolated from D. Radiodurans

A DNA binding protein isolated from D. radiodurans changes CD-spectrum of Z-form poly(dG-dC) X poly(dG-dC). We have found that a positive band at 268 nm is converted close to that of B-form in the presence of the protein. Concomitantly, a negative band at 295 nm shown by Z-form poly(dG-dC) X poly (dG-dC) was weakened by the protein but not by albumin. Such changes in the CD-spectra were not induced by the protein and by albumin when they were mixed with Z- or B-form poly(dG-me5dC) X poly(dG-me5dC) or with B-form poly(dG-dC) X poly(dG-dC). The protein formed a complex preferentially with Z-form poly(dG-dC) X poly(dG-dC).

Production of 4,5‐Dihydro‐4,5‐dihydroxyphthalate from Phthalate by Mutant Strain of Pseudomonas testosteroni M4‐1

Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate.