Yuji Kobayashi

Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.

Structure and Binding Mode of a Ribosome Recycling Factor (RRF) from Mesophilic Bacterium

X-ray and NMR analyses on ribosome recycling factors (RRFs) from thermophilic bacteria showed that they display a tRNA-like L-shaped conformation consisting of two domains. Since then, it has been accepted that domain I, consisting of a three-helix bundle, corresponds to the anticodon arm of tRNA and domain II and a β/α/β sandwich structure, corresponds to the acceptor arm. In this study, we obtained a RRF from a mesophilic bacterium,Vibrio parahaemolyticus, by gene cloning and carried out an x-ray analysis on it at 2.2 Å resolution. This RRF was shown to be active in an in vitro assay system usingEscherichia coli polysomes and elongation factor G (EF-G). In contrast, the above-mentioned RRFs from thermophilic bacteria were inactive in such a system. Analysis of the relative orientations between the two domains in the structures of various RRFs, including this RRF from mesophilic bacterium, revealed that domain II rotates about the long axis of the helix bundle of domain I. To elucidate the ribosome binding site of RRF, the peptide fragment (RRF-DI) corresponding to domain I of RRF was expressed and characterized. RRF-DI is bound to 70 S ribosome and the 50 S subunit with an affinity similar to that of wild-type RRF. But it does not bind to the 30 S subunit. These findings caused us to reinvestigate the concept of the mimicry of RRF to tRNA and to propose a new model where domain I corresponds to the acceptor arm of tRNA and domain II corresponds to the anticodon arm. This is just the reverse of a model that is now widely accepted. However, the new model is in better agreement with published biological findings.

Simple and efficient syntheses of Boc- and Fmoc-protected 4(R)- and 4(S)-fluoroproline solely from 4(R)-hydroxyproline

Abstract As building blocks of collagen model peptides, Boc- and Fmoc-protected 4(R)- and 4(S)-fluoroproline, which will be widely used in peptide synthesis including solid-phase strategy, were synthesized from the readily available 4(R)-hydroxyproline in higher yield than with conventional methods. To establish the stereospecificity of the Mitsunobu reaction and the subsequent fluorination that were presumed to cause the inversion of configuration at the C-4 position of a proline derivative, the absolute configuration of one of the key products, Boc-4(S)-fluoroproline, was determined by X-ray crystallography.

Crystallization and preliminary X-ray crystallographic studies of a mutant of ribosome recycling factor from Escherichia coli, Arg132Gly

Ribosome recycling factor (RRF) plays a central role during the recycling of ribosomes in the final step of protein biosynthesis in prokaryotes and is therefore a favourable target for the development of new antibiotics. The crystal structure of Escherichia coli RRF has been reported to have an open L-shaped conformation, while other RRFs from thermophilic bacteria have a strict L-shaped conformation [Yun et al. (2000), Acta Cryst. D56, 84-85]. Wild-type E. coli RRF has so far not been crystallized free from bound detergent. Here, a mutant of RRF, Arg132Gly, has been crystallized without any detergent. A complete data set from a crystal of this mutant obtained by the hanging-drop vapour-diffusion method has been collected at 2.2 A resolution using synchrotron radiation at 100 K. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 46.02, b = 49.27, c = 49.37 A, beta = 110.1 degrees. The currently refined structure indicates that RRF has a tRNA-like L-shaped conformation.

Novel Trimeric Adenylate Kinase from an Extremely Thermoacidophilic Archaeon, Sulfolobus solfataricus: Molecular Cloning, Nucleotide Sequencing, Expression in Escherichia coli,…

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.