Outer membrane of Salmonella typhimurium. Electron spin resonance studies.

The supramolecular structure of the outer membrane of Salmonella typhimurium that produces an Rc-type lipopolysaccharide was studied by adding spin-labeled fatty acid probes to membranes as well as model bilayers. Lipopolysaccharide of this organism apparently formed a bilayer structure in 0.2 M NaCl/0.01 M MgCl2, and the electron spin resonance spectra suggested that the motion of the segments of hydrocarbon chains near the carboxyl end was quite restricted even at high temperature; this is presumably due to the anchoring of more than a dozen fatty acid residues to a single backbone structure. In the presence of Mg2+, we could produce lipoplysaccharide-phospholipid mixed bilayers contining up to 50% (by weight) lipoplysaccharide. Their spectra showed no sign of major heterogeneity, and the maximum hyperfine splitting values were considerably larger than in phospholipid-only liposomes; these results suggest that the two components are finely interspersed and that the mobility of phospholipid hydrocarbons is severely restricted by the hydrocarbon chains of lipopolysaccharide. In spite of the presence of lipoplysaccharide in an amount equal to or exceeding that of phospholipids, the outer membrane produced spectra remarkably similar to those of the inner membrane, which does not contain lipoplysaccharide, and there was little sign of immobilization by lipopolysaccharides. Signals corresponding to the pure lipoplysaccharide phase were not detected, either. These results suggest that the phospholipids and lipopolysaccharides are segregated into separate domains in the outer membrane, and the fatty acid probes enter almost exclusively into the phospholipid domains. This conclusion was fully corroborated by determining, through the exchange broadening of line width, the total area of the domains that accommodated the spin label probes.     

Fine-structure map of the histidine transport genes in Salmonella typhimurium.

Afine-structure genetic map of the histidine transport region of the Salmonella typhimurium chromosome was constructed. Twenty-five deletion mutants were isolated and used for dividing the hisJ and hisP genes into 8 and 13 regions respectively. A total of 308 mutations, spontaneous and mutagen induced, have been placed in these regions by deletion mapping. The histidine transport operon is presumed to be constituted of genes dhuA, hisJ, and hisP, and the regulation of the hosP and hisJ genes by dhuA is discussed. The orientation of this operon relative to purF has been established by three-point crosses as being: purF duhA hisJ hisP.     

Outer membrane of Salmonella XIV. Reduced transmembrane diffusion rates in porin-deficient mutants

Rates of diffusion of a β-lactam antibiotic, cephaloridine, across the outer membrane of $\text{Salmonella typhimurium}$ cells were measured by determining the rates of its hydrolysis by β-lactamases located in the periplasmic space. It was shown that the permeability coefficient of the outer membrane toward cephaloridine decreased to about one-tenth of that in the wild type, in mutant strains deficient in two “porin” proteins, previously shown to produce transmembrane pores in in vitro reconstitution experiments. In contrast, the loss of the 33,000 dalton outer membrane protein did not have any noticeable effect on the permeability coefficient.     

Biosynthesis of uridine diphosphate N-acetylmannosaminuronic acid in rff mutants of Salmonella tryphimurium.

In Salmonella typhimurium, three groups of genes located in rfb, rfe, and rff clusters are known to be involved in the biosynthesis of the enterobacterial common antigen. We found that enzymatic synthesis of uridine diphosphate N-acetylmannosaminouric acid, the activated form of a constituent sugar of the common antigen, followed the pathway previously described in Escherichia coli (N. Ichihara, N. Ishimoto, and E. Ito, FEBS Lett. 39:46--48, 1974). All of the six rff mutants tested, which fail to synthesize the common antigen, were deficient in one or both of the two enzymes needed for the synthesis of this sugar nucleotide from uridine diphosphate N-acetylglucosamine; these results established the physiological role of the pathway studied for the biosynthesis of N-acetylmannosaminuronic acid residues. The levels of these enzymes were not reduced in rfe mutants or rfb deletion mutants, although they produced no or only traces of the common antigen.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Jatropham derivatives and steroidal saponins from the bulbs of Lilium hansonii.

Two new jatropham derivatives and three new steroidal saponins were isolated from the fresh bulbs of Lilium hansonii, along with previously known compounds. The structures of the new compounds were elucidated, on the basis of spectroscopic data and chemical evidence, and by comparing them with those of known compounds, as (-)-5-hydroxy-3-methyl-3-pyrrolin-2-one (jatropham) 5-O-beta-D-glucopyranosyl-(1----3)-beta-D-glucopyranoside, (2S*,4R*)-1-(3-methyl-2-oxo-3-pyrrolinyl)-4-methyl-5-oxo-2-pyrr olidinecarboxyli c acid, 26-O-beta-D-glucopyranosyl-(25R)-5 alpha-furostan-3 beta,22 zeta-diol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside, (25R)-5 alpha-spirostan-3 beta,12 alpha-diol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside and (25R)-spirost-5-en-3 beta,12 alpha-diol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside, respectively. The stereostructure of jatropham dimer, the plain structure of which was presented previously, was confirmed by X-ray crystallographic analysis. The inhibitory activity on cyclic AMP phosphodiesterase of the steroidal saponins was evaluated.     

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis-syn-cyclobutane thymine dimer (T[c,s]T). ³¹P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[c,s]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V_H domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3′-side of the nucleotides onto the H1 and H3 segments, with the 5′-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.     

AcrD of Escherichia coli is an aminoglycoside efflux pump

AcrD, a transporter belonging to the resistance-nodulation-division family, was shown to participate in the efflux of aminoglycosides. Deletion of the acrD gene decreased the MICs of amikacin, gentamicin, neomycin, kanamycin, and tobramycin by a factor of two to eight, and DeltaacrD cells accumulated higher levels of [(3)H]dihydrostreptomycin and [(3)H]gentamicin than did the parent strain.     

Monophyletic Origin of the Order Chiroptera and Its Phylogenetic Position Among Mammalia, as Inferred from the Complete Sequence of the Mitochondrial DNA of a Japanese Megabat, the Ryukyu Flying Fox (Pteropus dasymallus)

Complete sequences of mitochondrial DNA (mtDNA) are useful for the reconstruction of phylogenetic trees of mammals and, in particular, for inferring higher-order relationships in mammals. In this study, we determined the complete sequence (16,705 bp) of the mtDNA of a Japanese megabat, the Ryukyu flying fox (Pteropus dasymallus). We analyzed this sequence phylogenetically by comparing it with the complete sequence of mtDNAs of 35 mammals in an effort to reevaluate the enigmatic relationship between Megachiroptera and Microchiroptera and the relationships between them and other mammals. Maximum-likelihood analysis of 12 concatenated mitochondrial proteins from 36 mammals strongly suggested the monophyly of the order Chiroptera and its close relationship to Fereuungulata (Carnivora + Perissodactyla + Cetartiodactyla). We estimated that megabats and microbats diverged approximately 58 MyrBP and discussed the origin and early evolution of Chiroptera based on our findings. Received: 28 January 2000 / Accepted: 30 June 2000