Sodium‐powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan‐binding region

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error‐prone PCR to introduce single point mutations into the sodium‐powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium‐channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C‐terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion‐channel blockers.     

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche

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HIV/AIDS in Asia: The shape of epidemics and their molecular epidemiology

The Asia-Pacific region is a home to 60% of the population in the world and to approximately one quarter of people with HIV/AIDS. Close to a million of people has been infected and a half million people died of AIDS annually in Asia, becoming the second largest epicenter of global AIDS epidemic. Molecular epidemiology has been useful tool to track a course of HIV spread. In-depth knowledge from the studies on molecular epidemiology elucidates the dynamics of HIV spread and the interrelationship of epidemics in the different regions in Asia. Foundation items: Grant support from Ministry of Health, Labour and Welfare and Ministry of Education, Science and Technology in Japan; Japanese Foundation for AIDS Prevention.     

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway

The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model.     

Comparative study of sequential expression of the organizer-related genes in normal Cynops pyrrhogaster embryos and mesodermalized ectoderm

An artificially mesodermalized ectoderm (mE) of early Cynops pyrrhogaster gastrula acquires the organizer property; the mE is able to induce the secondary axis. The expression of organizer-related genes was investigated during the mesodermalizing process of the mE. The expression of C. pyrrhogaster organizer-related genes, such as bra, gsc, lim-1, chd and noggin, were analyzed. Cynops pyrrhogaster shh expression was also investigated. The organizer-related genes were activated by 12 h after the mesoderm-inducing stimulus. It was noted that there was a temporal gap in the expression of each gene. The expression of bra and gsc seemed to be more quickly activated during the mesodermalizing process. While expression of lim-1 and noggin was activated later than that of bra and gsc, lim-1 expression was earlier than chd and noggin expression. Shh expression was activated later than lim-1/noggin. The present study suggests the possibility that the bra/gsc, lim-1, chd, noggin and shh genes are expressed one by one in that order during the mesodermalizing of the presumptive ectoderm. It also indicates that the sequence is not always consistent with that of the whole embryo during normal embryogenesis. The meaning of the discrepancy will be discussed in connection with the cascade of certain genes expressed during the mesodermalizing process.     

Role of the putative membrane-bound endo-1,4-beta-glucanase KORRIGAN in cell elongation and cellulose synthesis in Arabidopsis thaliana

A temperature-sensitive, elongation-deficient mutant of Arabidopsis thaliana was isolated. At the non-permissive temperature of 31 degrees C, the mutation impaired tissue elongation; otherwise, tissue development was normal. Hypocotyl cells that had established cell walls at 21 degrees C under light-dark cycles ceased elongation and swelled when the mutant was shifted to 31 degrees C and darkness, indicating that the affected gene is essential for cell elongation. Analysis of the cell walls of mutant plants grown at 31 degrees C revealed that the cellulose content was reduced to 40% and the pectin content was increased to 162% of the corresponding values for the wild type grown at the same temperature. The increased amounts of pectin in the mutant were bound tightly to cellulose microfibrils. No change in the content of hemicellulose was apparent in the 31 degrees C-adapted mutant. Field emission-scanning electron microscopy suggested that the structure of cellulose bundles was affected by the mutation; X-ray diffraction, however, revealed no change in the crystallite size of cellulose microfibrils. The regeneration of cellulose microfibrils from naked mutant protoplasts was substantially delayed at 31 degrees C. The recessive mutation was mapped to chromosome V, and map-based cloning identified it as a single G-->A transition (resulting in a Gly(429)-->Arg substitution) in KORRIGAN, which encodes a putative membrane-bound endo-1,4-beta-glucanase. These results demonstrate that the product of this gene is required for cellulose synthesis.     

Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M(r) of 47? omitted?457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.