Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid.

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-kb 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) were 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 sequence similar to the sigma 54-dependent promoter, as well as the usual -35 and -10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the sigma 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 polsaccharide 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 of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis

Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens.

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 blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. 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, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/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 blaIMP 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, > or = 4 to < or = 8 micrograms/ml), had no detectable blaIMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, > or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like metallo-beta-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital

Enhancement of extracapsular polysaccharide synthesis in Klebsiella peneumoniae by RmpA2, which shows homology to NtrC

Enhancement of extracapsular polysaccharide synthesis in Klebsiella pneumoniae by RmpA2

Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12.

Escherichia coli K-12 harboring a part of the structural genes for the Klebsiella K2 capsular polysaccharide (cpsK*) 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 cpsK* 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 cpsK* 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 cpsK* 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 cpsK*. 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

Enhancement of extracapsular polysaccharide synthesis in Klebsiella pneumoniae by RmpA2, which shows homology to NtrC and FixJ.

We determined the complete nucleotide sequence of a 2.1-kb HindIII-EcoRI fragment that was cloned from a resident large plasmid of Klebsiella 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

Mechanisms of antibacterial action of tachyplesins and polyphemusins, a group of antimicrobial peptides isolated from horseshoe crab hemocytes.

Tachyplesins I and II and polyphemusins I and II, cationic peptides isolated from the hemocytes of horseshoe crabs, show bactericidal activities with similar efficiencies for both gram-negative and gram-positive bacteria. Tachyplesin I inhibited bacterial growth irreversibly within 40 min. A subinhibitory concentration of tachyplesin I sensitized gram-negative bacteria to the bactericidal actions of novobiocin and nalidixic acid, although polymyxin B-resistant strains which have altered lipopolysaccharides were susceptible to tachyplesin I. This implies that tachyplesin permeabilizes the outer membrane and that the likely target of its action is outer membrane constituents other than lipopolysaccharides. On the other hand, a defensin-susceptible phoP strain of Salmonella typhimurium was also susceptible to tachyplesin I. Tachyplesin I rapidly depolarized the inverted inner-membrane vesicles of Escherichia coli. These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram-negative bacteria, is associated with tachyplesin-mediated bactericidal activity. The similarity between the actions of tachyplesin and those of defensin was discussed

Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance.

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 blaIMP 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 blaIMP, 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 beta-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 blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP 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 blaIMP 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

A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP.

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 blaIMP, and it was located in the space between an integrase-like gene and an aac(6')-Ib-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 blaIMP 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