Effects of fructans and probiotics on the inhibition of Klebsiella oxytoca and the production of short-chain fatty acids assessed by NMR spectroscopy.

Generally, the selection of fructans prebiotics and probiotics for the formulation of a symbiotic has been based on arbitrary considerations and in vitro tests that fail to take into account competitiveness and other interactions with autochthonous members of the intestinal microbiota. However, such analyzes may be a valuable step in the development of the symbiotic. The present study, therefore, aims to investigate the effect of lactobacilli strains and fructans (prebiotic compounds) on the growth of the intestinal competitor Klebsiella oxytoca, and to assess the correlation with short-chain fatty acids production. The short-chain fatty acids formed in the fermentation of the probiotic/prebiotic combination were investigated using NMR spectroscopy, and the inhibitory activities were assessed by agar diffusion and co-culture methods. The results showed that Lactobacillus strains can inhibit K. oxytoca, and that this antagonism is influenced by the fructans source and probably associated with organic acid production.

Structure of the calcium-dependent type 2 secretion pseudopilus.

Many Gram-negative bacteria use type 2 secretion systems (T2SSs) to secrete proteins involved in virulence and adaptation. Transport of folded proteins via T2SS nanomachines requires the assembly of inner membrane-anchored fibres called pseudopili. Although efficient pseudopilus assembly is essential for protein secretion, structure-based functional analyses are required to unravel the mechanistic link between these processes. Here, we report an atomic model for a T2SS pseudopilus from Klebsiella oxytoca, obtained by fitting the NMR structure of its calcium-bound subunit PulG into the ~5-Å-resolution cryo-electron microscopy reconstruction of assembled fibres. This structure reveals the comprehensive network of inter-subunit contacts and unexpected features, including a disordered central region of the PulG helical stem, and highly flexible C-terminal residues on the fibre surface. NMR, mutagenesis and functional analyses highlight the key role of calcium in PulG folding and stability. Fibre disassembly in the absence of calcium provides a basis for pseudopilus length control, essential for protein secretion, and supports the Archimedes screw model for the type 2 secretion mechanism.

An extracellular polymeric substance quickly chelates mercury(II) with N-heterocyclic groups.

A strain of Klebsiella oxytoca DSM 29614 is grown on sodium citrate in the presence of 50 mg l-1 of Hg as Hg(NO3)2. During growth, the strain produces an extracellular polymeric substance (EPS), constituted by a mixture of proteins and a specific exopolysaccharide. The protein components, derived from the outer membrane of cells, are co-extracted with the extracellular exopolysaccharide using ethanol. The extracted EPS contains 7.5% of Hg (total amount). This indicates that EPS is an excellent material for the biosorption of Hg2+, through chemical complexation with the EPS components. The binding capacity of these species towards Hg2+ is studied by cyclic voltammetry, and Hg L3-edge XANES and EXAFS spectroscopy. The results found indicate that Hg2+ is mainly bound to the nitrogen of the imidazole ring or other N-heterocycle compounds. The hydroxyl moities of sugars and/or the carboxyl groups of two glucuronic acids in the polysaccharide can also play an important role in sequestring Hg2+ ions. However, N-heterocyclic groups of proteins bind Hg2+ faster than hydroxyl and carboxyl groups of the polysaccharide.

Physicochemical and biological characteristics of the nanostructured polysaccharide-iron hydrogel produced by microorganism Klebsiella oxytoca.

There is an increasing interest in the nanostructured polysaccharide-iron hydrogel produced by Klebsiella oxytoca. Critical physicochemical and biological characteristics of these nanostructures should be revealed for biomedical applications. Accordingly, an iron reducing strain K. oxytoca, which synthesizes biogenic polysaccharide-iron hydrogel nanoparticles, known as Fe (III)-exopolysaccharide (Fe-EPS) was isolated from a mineral spring. For microbiological identification purpose 16S rRNA sequence analysis and different morphological, physiological, and biochemical characteristics of the isolate were studied. Critical physicochemical and biological characteristics of the produced Fe-EPS were evaluated using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray crystallography (XRD), vibrating sample magnetometer (VSM). In addition, for the first time, Fe-EPS which synthesized by K. oxytoca was evaluated by dynamic light scattering (DLS), thermo gravimetric analysis (TGA), and cytotoxicity assay. TEM micrographs showed that the biogenic Fe-EPS is composed of ultra-small (about 1.8 nm) iron oxide nanoparticles (IONs) which are trapped in a polysaccharide matrix. The matrix was about 17% (w/w) of Fe-EPS total weight and provided a large negative charge of -71 mV. Interestingly, Fe-EPS showed a growth promotion effect on hepatocarcinoma cell line (Hep-G2) and 36% increase in the percentage of viability was observed by 24 h exposure to 500 µg ml-1 Fe-EPS.

Predicting Homogeneous Pilus Structure from Monomeric Data and Sparse Constraints.

Type IV pili (T4P) and T2SS (Type II Secretion System) pseudopili are filaments extending beyond microbial surfaces, comprising homologous subunits called "pilins." In this paper, we presented a new approach to predict pseudo atomic models of pili combining ambiguous symmetric constraints with sparse distance information obtained from experiments and based neither on electronic microscope (EM) maps nor on accurate a priori symmetric details. The approach was validated by the reconstruction of the gonococcal (GC) pilus from Neisseria gonorrhoeae, the type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae, and pseudopilus of the pullulanase T2SS (the PulG pilus) from Klebsiella oxytoca. In addition, analyses of computational errors showed that subunits should be treated cautiously, as they are slightly flexible and not strictly rigid bodies. A global sampling in a wider range was also implemented and implied that a pilus might have more than one but fewer than many possible intact conformations.

XAS analysis of a nanostructured iron polysaccharide produced anaerobically by a strain of Klebsiella oxytoca.

A strain of Klebsiella oxytoca, isolated from acid pyrite-mine drainage, characteristically produces a ferric hydrogel, consisting of branched heptasaccharide repeating units exopolysaccharide (EPS), with metal content of 36 wt%. The high content of iron in the EPS matrix cannot be explained by a simple ferric ion bond to the sugar skeleton. The bio-generated Fe-EPS is investigated by X-ray absorption spectroscopy. Fe K-edge XANES analysis shows that iron is mostly in trivalent form, with a non-negligible amount of Fe(2+) in the structure. The Fe EXAFS results indicate that iron in the sample is in a mineralized form, prevalently in the form of nano-sized particles of iron oxides/hydroxides, most probably a mixture of different nano-crystalline forms. TEM shows that these nanoparticles are located in the interior of the EPS matrix, as in ferritin. The strain produces Fe-EPS to modulate Fe-ions uptake from the cytoplasm to avoid iron toxicity under anaerobic conditions. This microbial material is potentially applicable as iron regulator.

Biofilm growth of individual and dual strains of Klebsiella oxytoca from the dairy industry on ultrafiltration membranes.

Formation of biofilms in dairy membrane plants causes membrane pore blocking, product contamination and subsequent economic loss. To investigate the biofilm growth, two Klebsiella oxytoca strains, K. B006 and TR002, previously isolated from New Zealand dairy membrane plants, were grown both individually and combined on three types of ultrafiltration (UF) membranes in different concentrations of whey medium in biofilm reactors (CBR 90, BioSurface Technologies, Bozeman, USA). Biofilms of both the individual and combined strains grew on the membrane surfaces to levels of 4.9-7.99 log colony-forming units (CFU) cm(-2) measured by standard plate counting after removing the cells by sonication. More biofilm grew on used polyethersulfone (PES) membranes than on new PES and polyvinylidene fluoride (PVDF) membranes. Both strains formed good biofilms, although K. B006 formed a denser biofilm than TR002. This corresponded to our previous study on the attachment of these organisms, where K. B006 attached in greater numbers than K. TR002. The dual strains produced a higher biofilm density than single strains on the new membranes. Biofilm density tended to increase with increased whey concentration. The saturated biofilm was approximately 10(8) CFU cm(-2). PES membranes appeared to support biofilm growth less readily than did PVDF membranes and therefore may be the preferred material for UF membranes to reduce problems with microbial colonisation. Used membranes were more readily colonised with biofilm than were new membranes. Therefore, selecting a membrane type and monitoring membrane age will help manage biofilm development during UF.

In vitro multimerization and membrane insertion of bacterial outer membrane secretin PulD.

Synthesis of the Klebsiella oxytoca outer membrane secretin PulD, or its membrane-associated core domain, in a liposome-supplemented Escherichia coli in vitro transcription-translation system resulted in the formation of multimers that appeared as typical dodecameric secretin rings when examined by negative-stain electron microscopy. Cryo-electron microscopy of unstained liposomes and differential extraction by urea indicated that the secretin particles were inserted into the liposome membranes. When made in the presence of the detergent Brij-35, PulD and the core domain were synthesized as monomers. Both proteins caused almost immediate growth cessation when synthesized in E. coli without a signal peptide. The small amounts of PulD synthesized before cell death appeared as multimers with characteristics similar to those of the normal outer membrane secretin dodecamers. It was concluded that multimerization and membrane insertion are intrinsic properties of secretin PulD that are independent of a specific membrane environment or membrane-associated factors. The closely related Erwinia chrysanthemi secretin OutD behaved similarly to PulD in all assays, but the more distantly related Neisseria meningitidis secretin PilQ did not form multimers either when made in vitro in the presence of liposomes or when made in E. coli without its signal peptide. This is the first report of the apparently spontaneous in vitro assembly and membrane insertion of a large outer membrane protein complex. Spontaneous multimerization and insertion appear to be restricted to outer membrane proteins closely related to PulD.