Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer.

Many transmembrane receptors are oligomeric proteins. Binding of a ligand may alter the oligomeric state of the receptor, induce structural changes within the oligomer, or both. The bacterial aspartate chemoreceptor Tar forms a homodimer in the presence or absence of ligands. Tar mediates attractant and repellent responses by modulating the activity of the cytoplasmic kinase CheA. In vivo intersubunit suppression was used to show that certain combinations of full-length and truncated mutant Tar proteins complemented each other to restore attractant responses to aspartate. These results suggest that heterodimers with only one intact cytoplasmic domain are functional. The signaling mechanism may require interactions between dimers or conformational changes within a single cytoplasmic domain.

Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate.

The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.

[Primary arterial switch operation for complete transposition of the great arteries (type I) of a neonate weighing 1,378 g].

A 2-day-old female baby, delivered by emergent cesarean section at 35 weeks of gestational age with a birth weight of 1,378 g, was referred to our institute for intensive care of heart failure. By echocardiography and cardiac catheterization, the patient was diagnosed with isolated complete transposition of the great arteries. Primary arterial switch operation was performed at 13 days of age. No technical difficulty arose, imposed by the small size of cardiovascular structure. On the 5th postoperative day, surgical repair of intestinal perforation was performed. Convalescence thereafter was uneventful. She returned home on the 64th postoperative day with the body weight of 2,310 g. We conclude that primary arterial switch operation can be a feasible surgical option even in a neonate with very low birth weight.

Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium.

The proximal end of the flagellar basal body consists of a structure called the MS ring complex: two (M and S) rings with different thicknesses closely apposed and a rod extending from the center of the S ring. It has been shown that the MS ring complex consists of multiple copies of single protein FliF (molecular mass 61 kDa). We analyzed the domains of FliF to elucidate how a single protein can be used to construct a complicated particle with several distinct sub-structures. Tryptic digestion of the MS ring complex gave rise to a structure which lacked most of the M ring portion by electron microscopy and showed a major band at 25 kDa by SDS/gel electrophoresis. Amino acid sequence analysis of this band showed that both terminal regions of FliF have been digested, leaving a semi-stable peptide starting from Phe120 and ending at around 400. In addition, we constructed a truncated fliF gene which encodes a FliF lacking 103 amino acid residues from the C terminus. Amplification of the truncated FliF gave rise to a ring complex lacking the rim of the M ring. From these results we assign both terminal regions of FliF to the M ring. Possible domain structures of FliF corresponding to the S ring and the rod are also discussed.

M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF.

The flagellar basal body, a major part of the flagellar motor, consists of a rod and four rings. When the fliF gene of Salmonella typhimurium, which was previously shown to code for the component protein of the M ring, was cloned and overexpressed in Escherichia coli, the FliF subunits formed ring structures in the cytoplasmic membrane. Electron microscopic observation of the purified ring structures revealed that each was composed of two adjacent rings and a short appendage extending from the center of the rings. Antibodies raised against the purified FliF protein decorated both the M and S rings of the intact basal body. We conclude that the FliF protein is the subunit protein of the M ring, and of the S ring and of part of the proximal rod of the flagellar basal body.

Color-coding reveals tandem repeats in the Escherichia coli genome.

Genomic DNA contains a wide variety of repetitive sequences. In Escherichia coli, there have been several classes of repetitive sequences reported, some of which cluster as tandem repeats. We propose a novel method for analyzing symbolic sequences by two-dimensional pattern formation with color-coding. We applied this method for searching tandem repeats in the E. coli genome and found approximately 50 repeats with periods longer than 30 bases. The longest repeat has a period of 1267 bases.

Self-assembly of the filament capping protein, FliD, of bacterial flagella into an annular structure.

A bacterial flagellum has a cap structure at the tip of the external filament. The cap is composed of the FliD protein (Mr, 49 x 10(3)), and plays an essential role in the polymerization of the filament protein, flagellin, which is believed to be transported through a central channel in the flagellum. A fliD-deficient mutant becomes non-motile because it lacks flagellar filaments and leaks flagellin monomer out into the medium. We have constructed a FliD-overproducing plasmid and purified the protein. The purified FliD at high concentration formed a large complex (Mr, ca. 600 x 10(3)) under physiological conditions. The complex was found by electron microscopy to be ring shaped. Image analysis revealed that the complex consisted of five substructures arranged in a pentagonal shape. Its outer diameter, approximately 10 nm, was about the same as that of the cap at the tip of the wild-type flagella. When the annular structure was added to the culture medium of a Salmonella fliD mutant, almost all of the cells became able to swim. Overall, about ten molecules of FliD self-assemble into an annular structure in vitro, forming the functional capping structure by incorporating flagellin at the tip of the flagellar filament in vivo.

Assembly characteristics of flagellar cap protein HAP2 of Salmonella: decamer and pentamer in the pH-sensitive equilibrium.

The cap of the bacterial flagellum is an oligomeric assembly of HAP2 protein (also called FliD), tightly attached to the tip of the flagellar filament. Flagellar growth does not occur in fliD-deficient mutants because flagellin monomers transported through the central channel of the flagellum leak out without polymerizing at the distal end. The structure of the cap complex is not known yet. An in vitro assembly of HAP2 proteins was found to have a pentagonal shape, while its molecular mass corresponded roughly to that of a dodecamer. To characterize the structure and assembly behavior of the complex formed in vitro in more detail, the stoichiometry of the complex and the association equilibrium have been studied. Crosslinking experiments now clearly show that the HAP2 complex is decameric. The assembly equilibrium is mainly between the monomer and decamer with a minor population of intermediate oligomers involved, and is highly dependent on the solution pH as well as the salt concentration: the fraction of the decamer sharply rises as the pH decreases from 8.5 to 8.0; the physiological concentration of salt partially suppresses the decamer formation. A preferential crosslinking within a pentameric unit together with a bipolar feature of the complex particle observed by electron microscopy suggests that the decamer is a bipolar pair of pentamers. Because of the polar nature of the filament cap structure, the pentamer is suggested to be the cap complex with its decamer forming surface involved in interactions with the filament.

Geometry of the flagellar motor in the cytoplasmic membrane of Salmonella typhimurium as determined by stereo-photogrammetry of quick-freeze deep-etch replica images.

The precise geometry of the flagellar basal structure anchored in the cytoplasmic membrane was determined by digital stereo-photogrammetry of the images captured by quick-freeze deep-etch replica electron microscopy. In order to examine the structure on the periplasmic side of the membrane, we analyzed the MS ring complexes of Salmonella typhimurium overproduced in the cytoplasmic membrane of Escherichia coli. The rod, the S ring, and the shoulder of the M ring were exposed to the periplasm. On the cytoplasmic side of the membrane, small bumps corresponding to the cytoplasmic rod were discernible. We also examined the intact inner surface of the cells of polyhook mutant which was prepared by a new protocol and found the bell-shaped structure extending from the membrane towards the cytoplasm. It was identified as the C ring, since it was located at the base of the polyhook. Various dimensions of the MS ring complex and the C ring projecting from the membrane were determined by digital stereo-photogrammetry, and a three-dimensional model of the total basal structure is presented.

Radial mass analysis of the flagellar filament of Salmonella: implications for the subunit folding.

X-ray fiber diffraction patterns of the R-type straight flagellar filament of Salmonella typhimurium SJW1655 strain showed layer-lines with an axial spacing of 1/437 A-1, which could be resolved only due to very small disorientation angles (< 2 degrees) of the filaments in oriented sol specimens. Although the equatorial layer-line was situated between the relatively strong first layer-lines right above and below it, these small disorientation angles and a new method of two-dimensional angular deconvolution allowed us to determine the equatorial layer-line intensities quite accurately. The equatorial data were phased by using the amplitude difference between the native flagellar filament and its heavy atom derivatives. One of the heavy-atom derivatives was prepared by introducing a cysteine residue by site-directed mutagenesis and applying a mercury compound. From the equatorial structure factors, the radial density distribution of the filament was calculated at 11 A resolution. A prominent feature was two pairs of high density peaks at radii of around 25 and 45 A and a deep density trough between them, which corresponds to the concentric double tubular structure in the core region that has been found in the density map recently deduced by helical image reconstruction from electron micrographs of frozen hydrated filaments. The molecular masses were estimated for four radial segments that correspond to the morphological domains identified in the map of helical image reconstruction. Then the domains were assigned to sequence positions by correlating the estimated masses with those of proteolytic fragments of flagellin. The assignment is consistent with the distributions of secondary structures and in particular alpha-helical coiled-coils that were predicted from the sequence. It also helps to understand how the polymerization behaviour is affected by truncation of the disordered terminal regions of flagellin and why mutations in a specific region are responsible for changes in the polymorphic shape of the filament.

Activin A: serum levels and immunohistochemical brain localization in rats given diets deficient in L-lysine or protein.

When a L-lysine (Lys)-deficient diet is given to rats, Lys in plasma and brain declines and rats will then select a Lys solution from among other L-amino acids (AAs). The recording of single-unit activity in the lateral hypothalamic area of these rats suggested that neural plasticity occurred, specifically responding to the deficient nutrient, Lys, centrally and during ingestion of AA. Possible neurotrophic factors in serum from rats with or without deficiency of either protein or Lys was assayed by Hydra japonica. An increase in serum inhibin and activin A was observed in rats fed a Lys-sufficient and nonprotein diet, respectively. However, serum activin A-like activity was severely suppressed under Lys deficiency. Additionally, the immunohistochemical distribution of activin A in the brain was found in the nucleus tractus solitarius, the area postrema, and the arcuate nucleus. These facts indicate that ingestion of Lys-deficient or nonprotein diet caused a change in serum levels of activin A as a possible neurotrophic factor. This release may elicit plasticity in the sensitivity of neurons to deficient AA in the nuclei that could selectively drive ingestive behavior for its particular AA (e.g., Lys) to maintain AA homeostasis.

Feasibility of 2nd generation STS retinal prosthesis in dogs.

We developed a 2(nd) generation suprachoroidal transretinal stimulation (STS) system with a 49 channel electrode array and implanted in 2 dogs. One month after surgery, all electrodes were functioning and the ocular fundus was normal in both dogs. The results indicate the 2(nd) generation STS retinal prosthesis is feasible and can be considered for clinical use.

Analysis of mutations in the transmembrane region of the aspartate chemoreceptor in Escherichia coli.

Site-specific mutagenesis was used to replace an alanine with a lysine residue and to create a deletion of seven amino acids into the first transmembrane region (TMI region) of the aspartate chemoreceptor in Escherichia coli. The mutations resulted in the loss of aspartate chemotaxis on tryptone motility plates. However, both mutant proteins were able to associate with the membrane and to bind aspartate. They were both refractory to methylation or to modification of the C-terminal region of the protein by the cheB gene product. These results suggested that the integrity of the TMI domain of the protein was required to maintain the function of the cytoplasmic portion of the receptor. The Lys-19 mutant retained the ability to generate a repellent response. Analysis of suppressor mutations of the Lys-19 mutation suggested that formation of an ion pair or specific changes in a 40 amino acid stretch in the cytoplasmic region of the protein (from amino acid 264 to amino acid 303) could suppress the effects of the Lys-19 mutation. The TMI region of the protein may be involved in transmembrane transmission of signals from the periplasmic portion of the cell to the cytoplasmic portion of the Tar protein.

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.).

The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.

Glycerol and ethylene glycol: members of a new class of repellents of Escherichia coli chemotaxis.

By using the chemical-in-plug method, we found that glycerol and ethylene glycol caused negative chemotaxis in wild-type cells of Escherichia coli; the threshold concentration was about 10(-3) M for both chemicals. As with other known repellents, the addition of glycerol or ethylene glycol induced a brief tumble response in wild-type cells but not in generally nonchemotactic mutants. Experiments with mutants defective in various methyl-accepting chemotaxis proteins (MCPs) revealed that the presence of any one of three kinds of MCPs (MCP I, MCP II, or MCP III) was necessary to give a tumble response to these repellents. Consistently, it was found that the methylation-demethylation system of MCPs was involved in the adaptation of the cells to these repellents. The effect of glycerol or ethylene glycol was not enhanced by lowering the pH of the medium, and glycerol did not alter the membrane potential of the cells. All of these results suggest that glycerol and ethylene glycol are members of a new class of repellents which produce a tumble response in the cells by perturbing the MCPs in the membrane.

Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol.

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.

Overproduction of the bacterial flagellar switch proteins and their interactions with the MS ring complex in vitro.

The flagellar switch proteins (FliG, FliM, and FliN) of Salmonella typhimurium were overproduced in Escherichia coli and partially purified in soluble form. They were mixed with purified MS ring complexes (which consist of subunits of FliF protein) to examine their interactions in vitro. The degree of interaction was estimated by ultracentrifugation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the band density on the gel, we estimated that FliG bound to the MS ring complex at an approximately 1:1 molar ratio (FliG:FliF), whereas FliM did so only at a 1:5 molar ratio (FliM:FliF). FliN did not bind to the MS ring complex by itself or in the presence of the other switch proteins. A possible configuration of the switch proteins is discussed.

Mapping of the pin locus coding for a site-specific recombinase that causes flagellar-phase variation in Escherichia coli K-12.

Although the vh2 mutation almost entirely prevents phase variation in Salmonella spp., an Escherichia coli strain that carried the Salmonella H1 and H2 region, including the vh2 mutation, showed phase variation. From this strain, EJ1076, a number of mutants defective in phase variation were isolated, and the symbol pin was assigned to their mutant gene. The pin locus was mapped between purB and trp near purB by interrupted matings using Tn10 sites inserted near pin. The locus was cotransduced with purB by P1 vir at a frequency of around 0.33. All the mutations tested were clustered at this locus. Three E. coli K-12 strains probably derived via different lines from the wild type have been tested for the presence of pin+ by introducing the two Salmonella H regions; two were pin+, and one was a pin mutant.