Role of the RuvAB protein in avoiding spontaneous formation of deletion mutations in the Escherichia coli K-12 endogenous tonB gene

The endogenous tonB gene of Escherichia coli was used as a target for spontaneous deletion mutations which were isolated from ruvAB-, recG-, and ruvC- cells. The rates of tonB mutation were essentially the same in ruv+, ruvAB-, recG-, and ruvC- cells. We analyzed tonB mutants by sequencing. In the ruv+, recG-, and ruvC- strains, the spectra were different from those obtained from the ruvAB- cells, where deletions dominated followed by IS insertions, base substitutions, and frameshifts, in that order. We then analyzed the tonB-trp large deletion, due to simultaneous mutations of the trp operon, and found that the frequency in ruvAB- was higher than those in ruv+, recG-, and ruvC- cells. To characterize deletion formation further, we analyzed all the tonB mutants from one colicin plate. Seven deletions were identified at five sites from the 45 tonB mutants of ruv+ cells and 24 deletions at 11 sites from the 43 tonB mutants of ruvAB- cells. Thus, the ruvAB- strain is a deletion mutator. We discuss the role of RuvAB in avoiding deletions.     

Role of charged residues of pharaonis phoborhodopsin (sensory rhodopsin II) in its interaction with the transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppR(O)(-)(like)) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppR(O)(-)(like) decay rate and K(D) value for binding, implying that the carboxyl group in question is from Asp-193. The pK(a) of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193(p)()(pR) is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pK(a). In addition, we discuss the importance of Arg-162(p)()(pR) with respect to the binding activity.     

Activity regulation of tyrosinase by using photoisomerizable inhibitors

Enzymatic activity of tyrosinase was controlled on the basis of cis-trans photoisomerization of inhibitors, 4-azobenzene carboxylic acid (ACA) and 4,4'-azobenzene dicarboxylic acid (ADCA). In the case of ACA, the cis form inhibited tyrosinase-catalyzed oxidation of L-tyrosine more strongly than the trans form. On the contrary, in the case of ADCA, the cis form was less inhibitory. The oxidation rate was controlled reversibly by light irradiation in the course of the reaction. In the presence of ACA, UV light irradiation, which isomerized trans to cis form, decelerated the tyrosine oxidation, while visible light irradiation, which isomerized backward, accelerated the reaction. In contrast, in the presence of ADCA, UV light accelerated and visible light decelerated the reaction.     

Diet mixing and its effect on polyphagous grasshopper nymphs

We examined the effects of diet mixture on the nymphal performance of a polyphagous grasshopper Parapodisma subastris Huang by developing the first-stadium nymphs with either single or various combinations of plants occurring in their natural habitat. Intrinsic quality in terms of nymphal survival differed largely across the 16 plant species. However, even combinations of the six worst quality plants (survival 10% or less in each) greatly improved nymphal survival when compared to that of superior quality plants (more than 70% survival). In contrast, the addition of either of two inferior plants (more than 10% and less than 40% survival) to the superior plant affected neither the survival nor the adult mass. Thus, diet mixture can be particularly important when only low quality plants are available. The adaptive significance of diet mixture was discussed in relation to the habitat flora and foraging habits of the grasshopper in the present study.     

Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.     

YM-254890 analogues, novel cyclic depsipeptides with Galpha(q/11) inhibitory activity from Chromobacterium sp. QS3666

The structure elucidation and biological activity of novel YM-254890 (1) analogues and semi-synthetic derivatives are described. Three natural analogues, YM-254891 (2), YM-254892 (3), and YM-280193 (4), were isolated from the fermentation broth of Chromobacterium sp. QS3666, and two hydrogenated derivatives, YM-385780 (5) and YM-385781 (6), were synthesized from YM-254890. Their structures were determined by one- and two-dimensional NMR studies and mass spectrometry. Among these compounds, two natural analogues 2-3 which possessed acyl groups at beta-HyLeu-1 and one derivative 6 whose conformation was similar to that of 1 showed comparable Galpha(q/11) inhibitory activity to that of 1. This indicates that the acyl beta-HyLeu residue plays an important role in activity and also that the alpha,beta-unsaturated carbonyl group of the N-MeDha residue is not critical to activity. The other hydrogenated derivative 5 had significantly less activity, which could be attributed to conformational differences.     

Direct shoot regeneration from cotyledonary nodes as a marker for genomic groupings within the Asiatic Vigna (subgenus Ceratotropis {Piper} Verdc.) species

In vitro culture of cotyledonary node (cn)explants, decapitated and intact seedlings of four different AsiaticVigna species (germinated and subcultured in MS-B5 mediumwith 1.0 mgl^–1 BA) resulted in axillaryshoot formation only from the epigeal V. radiata and thehypogeal but allotetraploid V. glabrescens. The hypogealspecies V. angularis and V. umbellatafailed to exhibit this response. Shoot decapitation invivopromoted similar response only in V. radiata, indicatingthat in vitro culture in BA-supplemented medium isrequiredby V. glabrescens to achieve the same response.Histological observations of the cn explants from 4-d-oldin-vitro-germinated seedlings at d 0(inoculation day), d 4 and d 8 (after inoculation) revealed the formation ofprimary axillary shoots (pas) in both V. radiata andV. glabrescens. Secondary axillary branching at the baseofthe pas was observed at 8 d. Further examination by scanningelectron microscope (SEM) confirmed the presence of shoot buds enveloped by atleast two leaf primordia in the explants at d 0 in all epigeal species namelyV. radiata, V. mungo and V.aconitifolia together with the hypogeal but allotetraploidV. glabrescens. Consistently, these structures were absentin V. angularis and V. umbellata.Present histological and SEM observations support the previous findings ofAvenido and Hattori (Plant Cell Tissue Organ Cult, 1999) that the induction ornon-induction of shoots directly from the nodes of invitro-cultured cn explants is a new marker corresponding to thegenomic grouping within the Asiatic Vigna species. Basedonhybridization studies, Dana (Breeding Methods for Improvement of Crops, 1980)designates AA, A₁A₁ and A₁A₁/- toall epigeal, hypogeal and the hypogeal but allotetraploid AsiaticVigna species, respectively. Moreover, these findingsproved that the differential in vitro regenerationresponse(i.e., response to BA) arises from inherent anatomical and developmentaldifferences, and is supportive of the genomic grouping within subgenusCeratotropis of the genus vigna.     

Cochlodinium fulvescens sp. nov. (Gymnodiniales, Dinophyceae), a new chain-forming unarmored dinoflagellate from Asian coasts

Cellular morphology and the phylogenetic position of a new unarmored photosynthetic dinoflagellate Cochlodinium fulvescens Iwataki, Kawami et Matsuoka sp. nov. were examined by light microscopy and molecular phylogenetic analyses based on partial large subunit ribosomal DNA (LSU rDNA) and small subunit ribosomal DNA (SSU rDNA) sequences. The cells of C. fulvescens closely resemble C. polykrikoides, one of the most harmful red tide forming dinoflagellates, due to it possessing a cingulum encircling the cell approximately twice, a spherical nucleus positioned in the anterior part of the cell and an eyespot‐like orange pigmented body located in the dorsal side of the epicone, as well as formation of cell‐chains. However, this species is clearly distinguished from C. polykrikoides based on several morphological characteristics, namely, cell size, shape of chloroplasts and the position of narrow sulcus situated in the cell surface. The sulcus of C. fulvescens is located at the intermediate position of the cingulum in the dorsal side, whereas that of C. polykrikoides is situated immediately beneath the cingulum. LSU rDNA phylogenies indicated that C. fulvescens is clearly distinct from, but closely related to C. polykrikoides among dinoflagellates.