No abstract

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure 32-␣-Man- (132) (18,55), and the O3-and O5-specific polysaccharides of Klebsiella strains are identical to the O8 and O9 polysaccharides of E. coli (8,18,29). Mannans of algal origin were found to exert antitumor activity (37). Such an activity could later be attributed also to the mannan-containing LPS of E. coli and Klebsiella strains (13, 37).The genetics of LPS biosynthesis in enteric bacteria is well documented in recent reviews (33,43,48,55). Two mechanisms, block and monomeric, have been described for O-polysaccharide synthesis (49). In the block mechanism, observed for Salmonella typhimurium and related Salmonella serotypes, the oligosaccharide repeating units are assembled on undecaprenol phosphate (antigen carrier lipid [ACL]) under the direction of rfb genes. The first sugar transferred was found to be galactose-1-phosphate, and the corresponding transferase gene was termed rfbP (48). The repeating units are polymerized under the direction of the rfc gene, which may be located outside of or within the rfb gene cluster (41). The chain length is controlled by the rol gene, located between gnd and his (3, 4). The monomeric mechanism, experimentally proven only for E. coli O8 and O9 (18, 55), consists of the direct and sequential transfer of the monosaccharide residues from their nucleotideactivated precursors to the nonreducing end of the growing polysaccharide chain.The synthesis of some O polysaccharides requires the rfe gene. According to this requirement, LPS biosynthesis can also be divided into rfe-dependent and rfe-independent pathways. The rfe gene, first described by Mäkelä et al. (31), was found to be essential for the synthesis of the O polysaccharide in Salmonella strains of O groups C1 and L, and E. coli O8 and O9 (18, 33) and more recently in E. coli O4, O7, O18, O75, and O111 (1, 23a). It was reported to determine the tunicamycinsensitive transfer of N-acetylglucosamine (GlcNAc)-1-phosphate from UDP-GlcNAc to undecaprenol monophosphate

We have studied the fluorescence dynamics of “nonfluorescent” flavoproteins including flavodoxin (FD), its mutants W60F, Y98F, and W60F/Y98F, and riboflavin binding protein (RBP) with the femtosecond fluorescence up-conversion method and have observed the fluorescence quenching dynamics of FD and its mutants for the first time. The strong fluorescence quenching in these flavoproteins seems to be caused by ultrafast electron transfer (ET) from aromatic amino acid residues to the excited flavin chromophore in stacked configuration according to previous transient absorption studies. In the present work, we have made comparative studies on the dynamics of fluorescence quenching due to ET to the excited chromophore in RBP and FD. We have observed also fluorescence dynamics of FD mutants where active electron donors Trp·NH and Tyr·OH are partially (either of them) or completely replaced by inactive phenylalanine and directly demonstrated the ET mechanism of the ultrafast fluorescence quenching in PNS of FD.

The plasma of several different gases has shown a sporicidal activity. From these gases, nitrogen gas was most difficult to produce atomic nitrogen radicals. However, these radicals have a high energy, indicating that nitrogen gas plasma could be used to sterilize microorganisms and inactivate endotoxins. The sterilization mechanism of nitrogen gas plasma is the synergistic effect of a high rising-up voltage pulse, UV irradiation and atomic nitrogen radicals. Thus, the target cells were damaged by degradation, which resulted in death. The biological indicator (BI) used in this study was Geobacillus stearothermophilus ATCC 7953 at a population of 1 x 106 CFU/sheet. Sterility assurance was confirmed by using the Bl. Moreover, endotoxins were successfully inactivated. More than 5 log reduction of endotoxins could be attained with 30 minutes of nitrogen gas plasma exposure. Material functionality influenced by nitrogen gas plasma presented a satisfactory result. No deterioration of polymers could be observed by nitrogen gas plasma exposure.

Volume 33, no. 1, p. 63, column 2: The last line should read "buffer. Fractions with activity were pooled and loaded on a DEAE-Sephacel (Pharmacia) column preequilibrated with 50 mM phosphate buffer (pH 7.0). ,B-Lactamase activity passed through the column without absorption. Fractions with activity were pooled and concentrated

SalmoneUa minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4TC for 10 days.Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5. 85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S.minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the 0-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions. Bacterial lipopolysaccharide (LPS) is the constituent of the outer membrane of gram-negative bacteria and consists of the polysaccharide (O antigen) which is linked to the R core consisting of oligosaccharide, which in turn is linked to the lipid portion termed lipid A (49). LPS strongly elicits a variety of host reactivities through interactions with humoral and cellular factors of the host. It has been widely accepted that the lipid A portion is mostly responsible for the biological activities of LPS (14,17,47), although the polysaccharide or the R core can modify the strength of action in some biological activities. Recently, studies on the relationship between chemical structure and biological activity of lipid A components have progressed greatly, since synthetic preparations of lipid A components and related compounds have been available (16,18,20,22,23,26,32,33,46). Although several studies have been done on the three-dimensional structure of LPS (4,10,11,34,35,39,40,56) and a schematic model of LPS has been proposed (34), the conformation of LPS has not been determined conclusively. We have found (29-31) that certain of the R-form LPSs form ordered two-dimensional hexagonal structures in the presence of MgCl2. During the experiments, we noticed that R-form LPSs from Salmonella minnesota and Escherichia coli K-12 form three-dimensional crystals when they are precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and suspended in 70% ethanol containing 250 mM MgCl2 and incubated at 4°C. We present here analyses of crystals of these R-form LPSs. This report is the first one that has de...

Sixty-three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded p-lactamase gene of Klebsiella pneumoniae and the TEM p-lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds of /?-lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe. P-Lactamase Nucleotide sequence Amino acid sequence(Klebsiella pneumoniae)

The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of m-oxo haem dimer on the cell surface. Gingipain-adhesin complexes are responsible for production of m-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124,[43][44][45][46][47][48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain-adhesin complexes.