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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.

Intraperitoneal administration of lipopolysaccharide to mice induced a marked reduction of CD5+ B cells in the peritoneal cavity. The reduction was not induced by intravenous, subcutaneous, or oral administration of lipopolysaccharide. The reduction continued for about 10 days after the injection, and the CD5+ B-cell count recovered to the normal state about 14 days after the injection. The reduction of peritoneal CD5+ B cells might be caused by apoptotic cell death. Injection of lipopolysaccharide did not result in production of antibody to lipopolysaccharide. On the other hand, intraperitoneal injection of heat-killed bacteria did not induce a reduction of peritoneal CD5+ B cells and elicited the definite production of antibody to lipopolysaccharide.

Introduction of the romA gene cloned from Enterobacter cloacae into Escherichia coli K-12 resulted in almost complete inhibition of OmpF expression and a concomitant increase in resistance to quinolones, beta-lactams, chloramphenicol, and tetracyclines. In addition, the romA gene reduced the susceptibility to these multiple drugs even in the OmpF porin-deficient mutants of E. coli K-12. Results indicate the presence of romA-sensitive penetration pathway(s) for these multiple drugs other than the OmpF porin in E. coli.

The genes determining the biosynthesis of type 2 (K2) capsular polysaccharide [3----beta Glc1,4----beta Man(1,3----beta GlcUA) 1,4----alpha Glc1----] of Klebsiella pneumoniae Chedid (O1:K2), which is highly virulent for mice, were cloned and introduced into Escherichia coli HB101 and into four noncapsulated mutants derived from K. pneumoniae reference strains of K1, K7, K9, and K28. The recombinant plasmid pCPS7B06 carried 23 kb of a chromosomal DNA fragment of strain Chedid and encoded a part of the Klebsiella cps gene cluster. However, pCPS7B06 encoded enough genetic information for the production of Klebsiella K2 capsular polysaccharide on the cell surfaces of four noncapsulated mutants of K. pneumoniae. On the other hand, both pCPS7B06 and pROJ3 carrying the rmpA gene locus derived from a resident large plasmid of Chedid were required for the biosynthesis of Klebsiella K2 capsular polysaccharide on the cell surface of E. coli HB101. The insertion inactivation analysis using Tn5 revealed that the cps gene cluster occupied more than 15 kb of the chromosome of Chedid. We conclude that rmpA, which has been known to enhance the biosynthesis of colanic acid in E. coli, is also involved in the biosynthesis of Klebsiella capsular polysaccharide in E. coli HB101.

The antibody response to P-fimbriae ofEscherichia coli in patients with upper urinary tract infections was investigated. In the sera of patients with pyelonephritis obtained at the initial visit to hospital (3 to 7 days after the onset of symptoms), a high incidence of antibodies to P-fimbriae was detected (12 out of 14 patients). P-fimbriatedEscherichia coli strains were isolated from urine samples in all of these antibody-positive patients. Antibodies detected by ELISA using purified antigen were essentially IgG and specifically recognized P-fimbriae. These antibodies inhibited completely, or in some cases partially, mannose-resistant hemagglutination with P-fimbriatedEscherichia coli.

Recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide was manufactured genetically by transforming Escherichia coli K-12 with various rfb genes capable of synthesizing the mannose homopolymer. Recombinant lipopolysaccharide exhibited levels of anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity much higher than those exhibited by the original rough-type lipopolysaccharide from E. coli K-12 or lipopolysaccharide possessing the heteropolysaccharide from E. coli O111. Immunological activities of recombinant lipopolysaccharide were as strong as those of wild-type lipopolysaccharide possessing the mannose homopolymer. Characteristic activities of wild-type lipolysaccharide possessing the mannose homopolymer were exhibited by recombinant lipopolysaccharide. The abilities of lipopolysaccharide to activate B cells polyclonally and to produce cytokines did not seem to be related to the presence of the mannose homopolymer. Therefore, it was suggested that the mannose homopolymer in the O-specific polysaccharide might exclusively enhance anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity among various lipid A activities.

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.

The introduction of a newly cloned Enterobacter cloacae chromosomal gene romA, into Escherichia coli and E. cloacae resulted in enhancement of resistance to quinolones, beta-lactams, chloramphenicol, and tetracycline. The primary effect of romA on a multicopy vector in E. coli was almost complete inhibition of OmpF expression in the outer membrane. From the experiments with ompR and envZ mutants or with ompF-lacZ and ompC-lacZ fusion plasmids, it was concluded that this inhibition is posttranscriptional. The introduction of romA on a multicopy vector into strains with micF deletion elicited only a moderate decrease in OmpF protein expression. This indicates that reduction of OmpF expression by romA is partly mediated posttranscriptionally by the activation of micF. Moreover, the overexpression of RomA protein from an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter resulted in nearly complete inhibition of expression of OmpC and OmpA, as well as OmpF. Taken together with an observation in a recent study that overexpressed OmpC inhibited the synthesis of OmpA and LamB, a possible inhibitory mechanism at the translational stage of the synthesis of outer membrane proteins should also be considered. By Southern hybridization, romA was generally detected in the chromosomes of all E. cloacae strains tested but not in the E. coli K-12 chromosome. Sequence data show that there is an open reading frame specifying 368 amino acids residues including a putative signal peptide. RomA appears to belong to the outer membrane protein family since it was extractable from an outer membrane preparation, but no sequence homology to other outer membrane proteins was detected.

Salmonella 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 4 degrees C 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 O-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.

The chromosomally encoded beta-lactamase gene of Klebsiella oxytoca E23004, a strain resistant to cefoperazone and aztreonam, was cloned and expressed in Escherichia coli HB101. The molecular mass and pI of this enzyme were 28 kilodaltons and 7.4, respectively. Although the beta-lactamase of K. oxytoca hydrolyzed many cephalosporins, including broad-spectrum drugs, the nucleotide sequence and deduced amino acid sequence lacked homology with chromosomal class C beta-lactamase genes (ampC) of E. coli or Citrobacter freundii. Rather, about 45% nucleotide sequence homology and 40% deduced amino acid sequence homology were observed between the K. oxytoca beta-lactamase and TEM-1, a class A beta-lactamase which does not efficiently hydrolyze cephalosporins. Values of Km, relative Vmax, and relative Vmax/Km for the K. oxytoca beta-lactamase indicated that the enzyme is a penicillinase but that it can hydrolyze cefoperazone effectively and other broad-spectrum cephems weakly. Hence, the chromosomal beta-lactamase of K. oxytoca E23004 belongs to class A but differences in its amino acid sequence provide a broader spectrum of activity.

We extracted an R-form lipopolysaccharide (LPS) by the phenol-water method from Klebsiella sp. strain LEN-111 (O3-:KI-) and followed the changes in ultrastructure of the LPS during the extraction procedure. When the LPS was obtained from the water phase of an extract by addition of 2 volumes of 10 mM MgCI2-ethanol, it consisted of membrane pieces with a hexagonal lattice structure with a lattice constant of 14 to 15 nm. The lattice structure of the LPS was disrupted into short rods with sodium dodecyl sulfate, but the same hexagonal lattice structure was again formed by precipitation with 2 volumes of 10 mM MgCI2-ethanol. The LPS preparation after two cycles of treatment by the phenol-water method, which contained no detectable amounts of proteins, kept an unaltered ability to form the hexagonal lattice structure. Extensive treatment with pronase and extraction with chloroform did not impair the ability of the LPS preparation to form the lattice structure. When the other salts, NaCI, CaCI2 or Zn(CH3COO)2, were used for precipitation of the LPS with ethanol in place of MgCI2, the LPS did not form the hexagonal lattice structure. However, if the LPS precipitated with NaCI-ethanol was converted to the magnesium salt form after it was electrodialyzed, it formed the same hexagonal lattice structure as the LPS precipitated with MgCI2-ethanol. From these results, it was concluded that the R-form LPS has the ability of in vitro self-assembly into a hexagonal lattice structure in the presence of Mg2+ without the help of other components such as proteins and free lipids from outer membrane.

Intraperitoneal administration of lipopolysaccharide to mice induced a marked reduction of CD5+ B cells in the peritoneal cavity. The reduction was not induced by intravenous, subcutaneous, or oral administration of lipopolysaccharide. The reduction continued for about 10 days after the injection, and the CD5+ B-cell count recovered to the normal state about 14 days after the injection. The reduction of peritoneal CD5+ B cells might be caused by apoptotic cell death. Injection of lipopolysaccharide did not result in production of antibody to lipopolysaccharide. On the other hand, intraperitoneal injection of heat-killed bacteria did not induce a reduction of peritoneal CD5+ B cells and elicited the definite production of antibody to lipopolysaccharide.

Recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide was manufactured genetically by transforming Escherichia coli K-12 with various rfb genes capable of synthesizing the mannose homopolymer. Recombinant lipopolysaccharide exhibited levels of anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity much higher than those exhibited by the original rough-type lipopolysaccharide from E. coli K-12 or lipopolysaccharide possessing the heteropolysaccharide from E. coli O111. Immunological activities of recombinant lipopolysaccharide were as strong as those of wild-type lipopolysaccharide possessing the mannose homopolymer. Characteristic activities of wild-type lipolysaccharide possessing the mannose homopolymer were exhibited by recombinant lipopolysaccharide. The abilities of lipopolysaccharide to activate B cells polyclonally and to produce cytokines did not seem to be related to the presence of the mannose homopolymer. Therefore, it was suggested that the mannose homopolymer in the O-specific polysaccharide might exclusively enhance anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity among various lipid A activities.

A rapid and easy method for staining lipopolysaccharides with ethidium bromide is described. Lipopolysaccharides could be visualized by ethidium bromide with almost the same sensitivity as found with the silver-staining method in less than 30 min. The ethidium bromide-staining method was particularly suitable for staining lipopolysaccharides possessing acidic O-specific polysaccharides, which were poorly visualized by silver staining.

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.