AcrAB Efflux System: Expression and Contribution to Fluoroquinolone Resistance in Klebsiella spp. (1/92)

Seven Klebsiella pneumoniae and four Klebsiella oxytoca clinical isolates with different levels of resistance to ciprofloxacin were studied. Mutations in the topoisomerase genes were found in almost all strains, but the contribution of a multidrug efflux system homologous to AcrAB in Escherichia coli was also observed. Overexpression of this efflux system was demonstrated by immunoblotting with antibodies against E. coli AcrA.  (+info)

Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution. (2/92)

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS. The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.  (+info)

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics. (3/92)

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), His(125), His(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His(125) might be the residue required for albicidin binding. Mutation of His(125) to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His(125) totally abolished binding activity. Mutation of His(125) to arginine and tyrosine had no effect. These results indicate that His(125) plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.  (+info)

Enterobacterial repetitive intergenic consensus 1R PCR assay for detection of Raoultella sp. isolates among strains identified as Klebsiella oxytoca in the clinical laboratory. (4/92)

The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of Raoultella and the two genetic groups of Klebsiella oxytoca, was applied to 19 clinical isolates identified as K. oxytoca. By this method, as confirmed by species-specific gene sequencing, two Raoultella ornithinolytica and two unclassifiable K. oxytoca isolates were identified.  (+info)

Structural rationalization for the lack of stereospecificity in coenzyme B12-dependent diol dehydratase. (5/92)

Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca is apparently not stereospecific and catalyzes the conversion of both (R)- and (S)-1,2-propanediol to propionaldehyde. To explain this unusual property of the enzyme, we analyzed the crystal structures of diol dehydratase in complexes with cyanocobalamin and (R)- or (S)-1,2-propanediol. (R)- and (S)-isomers are bound in a symmetrical manner, although the hydrogen-bonding interactions between the substrate and the active-site residues are the same. From the position of the adenosyl radical in the modeled "distal" conformation, it is reasonable for the radical to abstract the pro-R and pro-S hydrogens from (R)- and (S)-isomers, respectively. The hydroxyl groups in the substrate radicals would migrates from C(2) to C(1) by a suprafacial shift, resulting in the stereochemical inversion at C(1). This causes 60 degrees clockwise and 70 degrees counterclockwise rotations of the C(1)-C(2) bond of the (R)- and (S)-isomers, respectively, if viewed from K+. A modeling study of 1,1-gem-diol intermediates indicated that new radical center C(2) becomes close to the methyl group of 5'-deoxyadenosine. Thus, the hydrogen back-abstraction (recombination) from 5'-deoxyadenosine by the product radical is structurally feasible. It was also predictable that the substitution of the migrating hydroxyl group by a hydrogen atom from 5'-deoxyadenosine takes place with the inversion of the configuration at C(2) of the substrate. Stereospecific dehydration of the 1,1-gem-diol intermediates can also be rationalized by assuming that Asp-alpha335 and Glu-alpha170 function as base catalysts in the dehydration of the (R)- and (S)-isomers, respectively. The structure-based mechanism and stereochemical courses of the reaction are proposed.  (+info)

Recognition of two genetic groups in the Klebsiella oxytoca taxon on the basis of chromosomal beta-lactamase and housekeeping gene sequences as well as ERIC-1 R PCR typing. (6/92)

Whilst searching for a molecular method to identify the different species of Raoultella and Klebsiella oxytoca, it was observed that the OXY-1 and OXY-2 beta-lactamase-producing K. oxytoca isolates displayed two distinguishable enterobacterial repetitive intergenic consensus (ERIC)-1R profiles. It was hypothesized that the two groups of chromosomal beta-lactamases might correspond to two groups of strains in the K. oxytoca taxon. To confirm this hypothesis, clinical isolates and reference strains of K. oxytoca were studied by determination of the sequence of their bla(OXY) genes, and of a partial fragment of their 16S rRNA (387 bp) and rpoB (512 bp) genes. The sequence data were phylogenetically analysed by using the parsimony method. Four clinical isolates possessed a bla(OXY-1) gene and nine possessed a bla(OXY-2) gene. The mean percentage of rpoB and 16S rRNA gene identity was > 99% within each group of strains, whereas it was 96.56 +/- 0.24% for rpoB genes and 97.80 +/- 0.22% for 16S rRNA genes between the group of strains harbouring the bla(OXY-1) gene and the group harbouring the bla(OXY-2) gene. The phylogenetic tree resulting from combined analysis of the 16S rRNA and rpoB datasets showed that the K. oxytoca isolates were monophyletic and separated into two clades; these clades included strains with either the bla(OXY-1) gene or the bla(OXY-2) gene. This result was supported with high bootstrap values of 97 and 99%, respectively. Moreover, the two groups of strains displayed distinct ERIC-1R profiles, with bands characteristic of each profile. Thus, the chromosomal bla(OXY) gene sequence is able to delineate not only two groups of beta-lactamases in K. oxytoca, but also two clades in the K. oxytoca taxon, in a manner similar to the sequence of housekeeping genes. These results suggest that K. oxytoca should be divided into two genetic groups, group OXY-1 represented by K. oxytoca strain SL781 (=CIP 104963) and group OXY-2 by K. oxytoca strain SL91l (= CIP 106098).  (+info)

New Klebsiella oxytoca beta-lactamase genes bla(OXY-3) and bla(OXY-4) and a third genetic group of K oxytoca based on bla(OXY-3). (7/92)

The two genetic groups (oxy-1 and oxy-2) previously identified in the Klebsiella oxytoca taxon are recognizable by four independent molecular markers: (i). ERIC-1R profiles, (ii). 16S ribosomal DNA (rDNA) signature sequences, (iii). singular nucleotides in a defined fragment of the rpoB gene, and (iv) the type of the strain's bla(OXY) gene (i.e., bla(OXY-1) or bla(OXY-2)). K. oxytoca strains SG266 and SG271 could not be classified into these genetic groups based on their ERIC-1R profile and bla(OXY) gene sequence. With regard to the gene identity percentages between the bla(OXY-1) and bla(OXY-2) gene groups (86.8% +/- 0.4%) and within a bla(OXY) gene group (>99%), it was concluded that the bla(OXY) gene of strain SG271 was representative of a new bla(OXY) gene group (bla(OXY-3)), since the mean identity percentages between it and the two bla(OXY) gene groups were 85.5% +/- 0.2% and 84.4% +/- 0.4%, respectively. Since the corresponding percentages were 95.0% +/- 0.4% and 86.2% +/- 0.3% for strain SG266, it was impossible to classify its bla(OXY) gene, which was therefore named bla(OXY-4). The 16S rDNA signature sequences of the two strains could be determined only after cloning experiments. The SG266 clones displayed the same signature sequence as that of the genetic group oxy-1, whereas the SG271 clones displayed three different 16S rDNA signature sequences that also differed from those of the two genetic groups. Singular nucleotides were found within the rpoB sequence of the two strains, allowing for their distinction from the two genetic groups. All of these results, combined with those previously obtained by the ERIC-1R PCR method, indicate that strain SG271 is representative of a new K. oxytoca genetic group (oxy-3), whereas strain SG266 could not be classified.  (+info)

Ligation mediated PCR performed at low denaturation temperatures--PCR melting profiles. (8/92)

We show that using low denaturation temperatures (80-88 degrees C) during ligation mediated PCR (LM PCR) of bacterial DNA leads to the amplification of limited sets of the less stable DNA fragments. A set of electrophoretic patterns of such fragments obtained at different denaturation temperatures forms the PCR melting profile (PCR MP). A single pattern obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very specific for the given bacterial genome and may be used for strain characterisation and differentiation. The method may also be used for amplification and isolation of the less stable DNA fragments in a genome.  (+info)