Yoshichika Arakawa

Salmonella typhimurium SR-form lipopolysaccharide (LPS), consisting of a single repeating unit of the O-antigenic polysaccharide, linked to the R-core consisting of oligosaccharide that is, in turn, linked to lipid A, formed crystals whose shapes were hexagonal plates, discoids, and solid columns when precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl(2) and kept in 70% ethanol containing 250 mM MgCl(2) at 4 C for 10 days, Among these crystals, the basic form is considered to be the hexagonal plates. Analyses of hexagonal plate crystals showed that they consist of hexagonal lattices with a lattice constant (a axis) of 4.62 Angstrom and longitudinal axis (c axis) of approximately 100 Angstrom. In X-ray diffraction patterns in the low-angle region, crystals of S, typhimurium SR-form LPS exhibited much less distinct reflections when compared with crystals of synthetic Escherichia coli-type lipid A. In contrast to the previous finding that S, minnesota S-form LPS possessing the O-antigenic polysaccharide does not crystallize under the same experimental conditions as used in the present study, the presence of a single repeating unit of the O-antigenic polysaccharide does not inhibit crystallization.

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive alpha-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous alpha-glycosyltransferases, four strictly conserved regions, I, II, III, and IV,were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of alpha-glycosidic linkage From alpha-linked nucleotide diphospho-sugar donor, Hydrophobic cluster analysis revealed a conserved domain at the N termini of these alpha-glycosyltransferases. This domain was similar to that previously reported for beta-glycosyltransferases. Thus, this domain is likely to be involved in the formation of beta-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction, Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.

We have previously reported that purified Shiga-like toxins (SLT), SLT-I and SLT-II coupled with liposomes induced a substantial amount of anti-SLT-I and anti-SLT-II IgG antibody production, respectively, in mice. The levels of anti-SLT antibody in the sera of SLT-liposome-immune mice correlated well with the protection against subsequent challenge with SLT, In this study, mice were immunized intraperitoneally with the mixture of SLT-I-liposome and SLT-II-liposome and protection against, oral infection with cytotoxin-producing Escherichia coli O157:H7 was evaluated. All of the mice that received immunization with the mixture of SLT-I-liposome and SLT-II-liposome were protected against subsequent intravenous challenge with 10 LD50 Of either SLT-I or SLT-II, Eight weeks after primary immunization, mice were inoculated intragastrically with 10(9) CFU of E, coli O157:H7 strain 96-60. All SLT-liposome-immune mice tested survived without any apparent symptom while control mice died within 5 days. In addition, as shown by other antigen-liposome conjugates, SLT-liposome induced undetectable anti-SLT IgE antibody production while they induced substantial amounts of anti-SLT IgG antibodies. These results suggest that SLT-liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin-producing E, coli infection.

Low molecular weight thiol compounds have been found to be strong inhibitors of metallo-β-lactamase (IMP-1) produced by Serratia marcescens TN9106, which was expressed by Echerichia coli JM109 cells. Mercaptoacetic acid and 2-mercaptopropionic acid strongly and competitively inhibited IMP-1 with K_i of 0.23 and 0.19 μM, respectively. 2-Mercaptoethanol reversibly inhibited IMP-1 but did not show simple competitive inhibition.

RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli. In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a ΔompF mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1^- mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.

We applied PCR to the rapid detection of the metallo-beta-lactamase gene, bla(IMP), in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 mu g/ml) were subjected to PCR analyses with the bla(IMP)-specific primers, since the bla(IMP)-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two bla(IMP)-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among bla(IMP)-positive strains. These results imply that the bla(IMP) gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging bla(IMP)-positive clinical isolates which demonstrate consistent resistance to beta-lactams.

Nine Klebsiella oxytoca strains which demonstrated resistance to the combination of sulbactam and cefoperazone were isolated from geographically separate hospitals in Japan in 1995, Among them, K, oxytoca SB23 showed high level resistance to sulbactam-cefoperazone (MIC, >128 mu g/ml) and aztreonam (MIG, 128 mu g/ml). The sulbactam-cefoperazone resistance was not transferred from strain SB23 to Escherichia coli CSH2 by conjugation. beta-Lactamase RbiA, produced by strain SB23, was purified, and the molecular mass was estimated to be 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic parameters for RbiA revealed that cefoperazone and aztreonam were hydrolyzed efficiently by this enzyme, Moreover, ceftazidime and imipenem were also hydrolyzed weakly by RbiA, although strain SB23 did not show any resistance to these agents, Clavulanate, sulbactam, and tazobactam failed to block the hydrolysis of cefoperazone by RbiA, The structural gene of RbiA (bla(RBI)) was cloned and sequenced, and the deduced amino acid sequence of RbiA demonstrated high-level similarities to those of the beta-lactamases found in K, oxytoca D488, E23004, and plasmid-mediated MEN-I, which have been classified into Bush functional group 2be, Although RbiA demonstrates high-level molecular similarity to the enzymes in group 2be, from an enzymological point of view, this enzyme might be differentiated from the enzymes in that group, Hybridization analysis revealed that beta-lactamase genes highly similar to bla(RBI) were generally encoded on the chromosome of the sulbactam-cefoperazone-resistant clinical isolates of K. oxytoca tested in the study, despite their different derivations, This observation suggests that sulbactam-cefoperazone-resistant K, oxytoca strains which produce RbiA-type beta-lactamases have been proliferating in many hospitals in Japan.

A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994, Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the metallo-beta-lactamase gene (bla(IMP))-specific probe, Fifteen strains carrying the metallo-beta-lactamase gene were identified in five hospitals in different geographical areas, Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, greater than or equal to 128 mu g/ml), two strains exhibited low-level imipenem resistance (MIC, less than or equal to 4 mu g/ml), and the rest of the strains were in between. These results revealed that the acquisition of a metallo-beta-lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the metallo-beta-lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital, These findings suggest that the metallo-beta-lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the metallo-beta-lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of metallo-beta-lactamase-producing strains with resistance to various beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of metallo-beta-lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum beta-lactams including carbapenems.

Synthetic Escherichia coli-type lipid A formed hexagonal plate crystals when it was 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 C for 10 days, Analyses of crystals by electron diffraction and synchrotron X-ray diffraction showed that crystals consist of hexagonal lattices with the lattice constant (a side of the lozenge as a unit cell on the basal plane) of 4.62 Angstrom and the longitudinal axis (perpendicular to the basal plane) of 49.3+/-1.3 Angstrom. Results suggest that the previous finding that various kinds of R-form lipopolysaccharides crystallized but free lipid A isolated by acid hydrolysis from Re lipopolysaccharide did not crystallize under the same experimental conditions (Kato et al, J. Bacteriol., 172:1516-1528, 1990) is due to structural changes of lipid A occurring during the procedure of isolation of free lipid A.

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (03(-):K1(-)) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1x0.8 mu m, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl2 acid kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Angstrom and c axis of 79.8 +/-2.6 Angstrom. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl2 at 4 C.

The effects of acidic conditions on activities of seven beta-lactamases-TEM-1 (class A), KOXY (class A), LMP-1 (class B), AmpC (class C), MOX-1 (class C), OXA-5 (class D), and PSE-2 (class D)-and their inhibitors were measured. The enzymatic activities of KOXY, IMP-1, and MOX-1 at pH 5.8 were slightly lower than those at pH 7.5. However, the activities of PSE-2 and OXA-5 were greatly reduced at pH 5.8. All of the p-lactamase inhibitors tested had poorer inhibitory activities at pH 5.8 than at pH 7.5 except clavulanic acid for TEM-1.

A plasmid-mediated metallo-beta-lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The metallo-beta-lactamase gene was identical to the bla(IMP), and it was located in the space between an integrase-like gene and an aac(6')-lb-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase dependent recombination, were also conserved in the flanking regions of the bla(IMP) gene. These findings imply that the metallo-beta-lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.

The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kh BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) mere identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequences similar to the o(54)-dependent promoter, as well as the usual -35 and -10 sequences, mere identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the o(54)-dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polysaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB off. coil. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.

The distribution of strains producing metallo-beta-lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a metallo-beta-lactamase genes similar to the bla(IMP) gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, R. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum beta-lactams. In particular, strains AK9373, AK9371, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, greater than or equal to 64 mu g/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 mu g/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the bla(IMP) gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the metallo-beta-lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, greater than or equal to 4 to less than or equal to 8 mu g/ml), had no detectable bla(IMP) gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, greater than or equal to 2 mu g/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated bla(IMP)-like metallo-beta-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.

A toxin gene (bceT) on a 2.9 kb DNA fragment of Bacillus cereus B-4ac was cloned and expressed in Escherichia coli, and its nucleotide sequence determined, The DNA fragment contained an open reading frame capable of encoding a polypeptide of 336 amino acids with a molecular mass of 41039 Da, The translated product in E. coli exhibited Vero cell cytotoxicity, and was positive in a vascular permeability assay, It also caused fluid accumulation in a ligated mouse ileal loop and was lethal to mice upon injection. These biological activities are considered characteristic of diarrhoeal enterotoxins. We therefore conclude that this gene, designated bceT, encodes one of the enterotoxic proteins of B. cereus which cause food-borne diarrhoea.

The minimum inhibitory concentrations (MIC) of ceftazidime (CAZ), piperacillin (PIPC), sulbenicillin (SBPC), ceftizoxime (CZX), ceftriaxone (CTRX), cefpirome (CPR), latamoxef (LMOX) and cefminox (CMNX) were determined for eight strains of CAZ-resistant Klebsiella pneumoniae isolated from clinical samples collected in Japan between July and September 1994. MICs were measured by the broth microdilution method as described by the Japan Society of Chemotherapy. MICs of over 400 μg/ml were obtained with CAZ in two of the eight strains. The implications of these and other results obtained on the eight CAZ-resistant Klebsiella pneumoniae strains in the present study are discussed. © 1995, Japanese Society of Chemotherapy. All rights reserved.

A 1954-bp DNA fragment containing the bla(MOX-1) gene, identified on a large resident plasmid (pRMOX-1) of Klebsiella pneumoniae NU2936, was sequenced and an open reading frame (ORF) coding for a 390-amino-acid (aa) MOX-1 was found. The total deduced aa sequence of MOX-1 shared considerable homology with that of AmpC-type class C beta-lactamases of Gram(-) bacteria, especially of Pseudomonas aeruginosa PAO1 [51.3%; 63.8% at the nucleotide (nt) level]. However, the regulatory gene ampR and a 38-bp AmpR-binding region were not present upstream from bla(MOX-1), although the expression of P. aeruginosa ampC is directly regulated by AmpR. Possible -35 and -10 regions, a Shine-Dalgarno (SD) sequence and terminators were identified which are peculiar to bla(MOX-1). On the other hand, a sequence highly homologous (91.6%) to the region upstream from dhfrX in the In7 integron carried by plasmid pDG0100 was found upstream from bla(MOX-1) at nt 1 to 488. No significant difference was detected between the promoter activities of bla(MOX-1) in ampD(-) and smpD(+) strains of Enterobacter cloacae, as measured by the chloramphenicol acetyltransferase (CAT) assay. These results clearly show that bla(MOX-1) belongs to the group of ampC-related bla genes and that it is expressed constitutively, independently of transcriptional regulators such as AmpR, AmpG and AmpD. Homology analysis among AmpC enzymes or ampC genes implied that integration of the chromosomal ampC gene into a large resident plasmid, followed by transconjugation, was involved in the evolution of bla(MOX-1).

Lidocaine hydrochloride (LH), a local anesthetic, and acetylsalicylate (AcSAL), show antibacterial activity for both gram-negative and gram-positive bacteria. Kinetic studies indicated that antibacterial activity of LH was different from that of AcSAL. A subinhibitory concentration of LH and AcSAL enhanced the sensitivity of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to novobiocin and nalidixic acid. The synergistic effect of AcSAL with novobiocin and nalidixic acid was higher than that of LH. The effect of both drugs on the membrane potential of inner membrane was also studied using inverted membrane vesicles of bacteria. Both LH and AcSAL depolarized the membrane potential after the vesicles were energized with nicotinamide adenine dinucleotide. However, unlike AcSAL, pre-treatment of vesicles with LH had no effect on the generation of membrane potential. These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram-negative bacteria, is associated with antibacterial activity of LH and AcSAL. The difference in actions of LH and AcSAL was discussed.

A clinical isolate of Serratia marcescens (TN9106) produced a metallo beta-lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum beta-lactams. The bla(IMP) gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying bla(IMP) and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various 13-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that bla(IMP) was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, bla(IMP) was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the metallo beta-lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of bla(IMP) was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial metallo beta-lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum beta-lactams.

Various chemotypes (Re, Rd(2), Rd(1)P(-), Rd(1), RcP(-), Rc, Rb-3, Rb-2, Rb-1, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl2, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Angstrom. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.

We determined the complete nucleotide sequence of a 2.1-kb HindIII-EcoRI fragment that was cloned from a resident large plasmid of Klabsiella pneumoniae Chedid, a highly virulent and mucoviscous strain of the O1:K2 serotype. This fragment encoded an ability to enhance K2 capsular polysaccharide synthesis in K. pneumoniae, and a 636-bp open reading frame (rmpA2) was found. The 411-bp rmpA reported to be involved in the virulence and mucoid phenotypes of K. pneumoniae by Nassif et al. (Mol. Microbiol. 3:1349-1359, 1989) was a part of rmpA2. Eighty percent homology in nucleotide sequence was found between rmpA2 and rmpA in the corresponding regions. The central domain of the deduced amino acid sequence of RmpA2 showed considerable homology to the central domains of NtrC of K. pneumoniae and Escherichia coli, to which the sigma factor of RNA polymerase binds. The C-terminal domain of RmpA2 also demonstrated considerable homology with the putative helix-turn-helix motifs of LuxR of Vibrio fischeri and FixJ of Rhizobium meliloti. Moreover, RmpA2 also showed some homology in its N- and C-terminal regions to those of RcsA, a transcriptional activator for colanic acid synthesis in E. coli. On the other hand, a sequence upstream of rmpA2 was found to be highly homologous to insertion sequence 3 of members of the family Enterobacteriaceae. Southern hybridization analysis suggested that rmpA2 exists on the large plasmids of all mucoviscous virulent K2 strains but not on those of the slightly mucoviscous avirulent strains. Freeze substitution electron microscopy and fluorescent-antibody staining with anti-K2 serum revealed that K. pneumoniae Chedid has a dense and thick capsule (180 nm) with dense extracapsular substance, whereas K. pneumoniae K2-215, one of the slightly mucoviscous and avirulent strains, has a capsule which is looser and thinner (120 nm) than that of strain Chedid and no extracapsular substance. Introduction of rmpA2 into K2-215 as well as reference strains K. pneumoniae K9 and K72 resulted in a change of the colony phenotype to highly mucoviscous through abundant production of extracapsular substance which reacted with anti-K2, -K9, or -K72, respectively, as did their parental strains. From these results, it is suggested that RmpA2 belongs to the family of transcriptional regulators and confers a highly mucoviscous phenotype on cells of various serotypes of K. pneumoniae by enhancing extracapsular polysaccharide synthesis.

Klebsiella pneumoniae NU2936 was isolated from a patient and was found to produce a plasmid-encoded beta-lactamase (MOX-1) which conferred resistance to broad spectrum beta-lactams, including moxalactam, flomoxef, ceftizoxime, cefotaxime, and ceftazidime. Resistance could be transferred from K. pneumoniae NU2936 to Escherichia coli CSH2 by conjugation with a transfer frequency of 5 x 10(-7). The structural gene of MOX-1 (bla(MOX-1)) was cloned and expressed in E. coli HB101. The MIC of moxalactam for E. coli HB101 producing MOX-1 was >512 mug/ml. The apparent molecular mass and pl of this enzyme were calculated to be 38 kDa and 8.9, respectively. Hg2+ and Cu2+ failed to block enzyme activity, and the presence of EDTA in the reaction buffer did not reduce the enzyme activity. However, clavulanate and cloxacillin, serine beta-lactamase inhibitors, inhibited the enzyme activity competitively (K(i)s = 5.60 and 0.35 muM, respectively). The kinetic study of MOX-1 suggested that it effectively hydrolyzed broad-spectrum beta-lactams. A hybridization study confirmed that bla(MOX-1) is encoded on a large resident plasmid (pRMOX1; 180 kb) of strain NU2936. By deletion analysis, the functional region was localized within a 1.2-kb region of the plasmid. By amino acid sequencing, 18 of 33 amino acid residues at the N terminus of MOX-1 were found to be identical to those of Pseudomonas aeruginosa AmpC. These findings suggest that MOX-1 is a plasmid-mediated AmpC-type beta-lactamase that provides enteric bacteria resistance to broad-spectrum beta-lactams, including moxalactam.

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three-dimensional crystals 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 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516-1528 (1990)), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 angstrom. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the {1011BAR} planes of solid hexagonal column crystals.

Escherichia coli K-12 harboring a part of the structural genes for the Klebsiella K2 capsular polysaccharide (cps(K)*) expresses a large amount of K2 capsular polysaccharide as a thick capsule in the presence of plasmids carrying rmpA and rcsB. We have previously shown that expression of the Klebsiella K2 capsule in E. coli HB101 harboring cps(K)* depends on the presence of rmpA, a regulatory gene from a large plasmid of Klebsiella pneumoniae Chedid (O1:K2). E. coli K-12 JM109, however, produces only a small amount of K2 capsular polysaccharide, even in the presence of plasmids carrying rmpA as well as the cps(K)* structural genes. Introduction of the rcsB gene, a positive regulator of colanic acid capsule synthesis in E. coli K-12 which was cloned from HB101 on a plasmid, into JM109 cells carrying cps(K)* and rmpA, results in the expression of a thick K2 capsule. By Northern (RNA) hybridization analysis, rcsB has been found to enhance transcription of a long strand of mRNA (longer than 14 kb) from cps(K)*. These E. coli transformants which produce a thick K2 capsule also express colanic acid production at high levels. Therefore, rcsB can act as a positive regulator of Klebsiella K2 capsule production and two capsular polysaccharides can be expressed in E. coli simultaneously. With a somewhat different strain background, we have found that both of the colanic acid regulators, rcsA and rcsB, contribute to the basal level of Klebsiella K2 capsule expression but that the presence of multicopy rcsB in either an rcsB or an rcsA mutant of E. coli is sufficient to increase the expression of K2 capsular polysaccharide. These results suggest further parallels between the regulation of colanic acid synthesis in E. coli and the regulation of Klebsiella K2 capsule synthesis.