I shall be released: Assembly platform dynamics during type II secretion.

In this issue of Structure, Dazzoni et al. solve the high-resolution homo- and hetero-dimeric structures of the Klebsiella oxytoca PulL and PulM C-terminal domains and unravel an uncharacterized dynamic interaction interface that is required for correct function of the type II secretion system.

Exopolysaccharides from Klebsiella oxytoca: anti-inflammatory activity

Abstract Exopolysaccharides (EPS) produced by Klebsiella oxytoca are of environmental, pharmaceutical, and medicinal interest. However, studies about the anti-inflammatory activity of EPS produced by this microorganism still remain limited. The aim of this study was to produce, characterize, and evaluate the anti-inflammatory activity of EPS from K. oxytoca in a pleurisy model. Colorimetric analysis revealed that precipitated crude exopolysaccharides (KEPSC) and deproteinated exopolysaccharides (KEPS) present high levels of total carbohydrates (65.57% and 62.82%, respectively). Analyses of uronic acid (7.90% in KEPSC and 6.21% in KEPS) and pyruvic acid (3.01% in KEPSC and 1.68% in KEPS) confirm that the EPS are acidic. Gas chromatography-mass spectrometry analyses demonstrated that the EPS consisted of rhamnose (29.83%), glucose (11.21%), galactose (52.45%), and mannose (6.50%). The treatment of an experimental pleurisy model in rats through subcutaneous administration of 50, 100, 200, and 400 mg/kg of KEPS decreased both the volume of inflammatory exudate and the number of leukocytes recruited to the pleural cavity. The present data showed that EPS production by K. oxytoca using the method described is easy to perform and results in a good yield. In addition, we show that KEPS exhibit anti-inflammatory activity when administered subcutaneously in rats.

Crystallization, structural characterization and kinetic analysis of a GH26 ß-mannanase from Klebsiella oxytoca KUB-CW2-3.

ß-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannan-based polysaccharides at ß-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 ß-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49-72% amino-acid sequence similarity with ß-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Šrevealed an open cleft-shaped active site. The enzyme structure is based on a (ß/α)8-barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to ß-strand 4 and at the end of ß-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency (kcat/Km) towards DP6 (2571.26 min-1 mM-1). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.

Large-Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca*.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 µm Ptm leads to a translocation rate of about one molecule per second and per channel.

Distribution of Ferrihydrite Nanoparticles in the Body and Possibility of Controlling Them in an Isolated Organ by a Permanent Magnetic Field.

We studied the distribution of ferrihydrite nanoparticles isolated from bacteria Klebsiella oxytoca in the whole body in vivo and in a cultured isolated organ (liver). The possibility of controlling these nanoparticles in the body using a magnetic field was assessed. One hour after intravenous injection of ferrihydrite nanoparticles to mice, their accumulation was observed in the liver, lungs, and kidneys. Experiment with cultured isolated rat liver showed that these nanoparticles can be controlled by a magnetic field and the influence of magnetic nanoparticles on the liver over 1 h does not lead to destruction of liver cells associated with the release of the marker enzyme AST. These results show the possibility of using magnetic nanoparticles as a system for controlled drug delivery in the body.

Nanopore Passport Control for Substrate-Specific Translocation.

Membrane protein pores have demonstrated applications in nanobiotechnology and single-molecule chemistry for effective detection of biomolecules. Here, we define the molecular basis of carbohydrate polymers translocation through a substrate-specific bacterial nanopore, CymA, which has a 15-residue N terminus segment inside the pore, restricting its diameter. Using single-channel recordings, we determined the kinetics of cationic cyclic oligosaccharide binding and elucidated the translocation mechanism across the pore in real-time. The cationic cyclic hexasaccharide binds to the densely packed negatively charged residues at the extracellular side of the pore with high affinity, facilitating its entry into the pore driven by the applied voltage. Further, the dissociation rate constant increased with increasing voltages, indicating unidirectional translocation toward the pore exit. Specifically, a larger cationic cyclic octasaccharide rapidly blocked the pore more effectively, resulting in the complete closure of the pore with increasing voltage, implying only strong binding. Further, we show that uncharged oligosaccharides exclusively bind to the extracellular side of the pore and the electroosmotic flow most likely drives their translocation. We propose that CymA favors selective translocation of cyclic hexasaccharide and linear maltooligosaccharides due to an asymmetrical charge pattern and the N terminus that regulates the substrate transport. We suggest that this substrate-specific nanopore with sophisticated geometry will be useful for complex biopolymer characterization.

Adaptation of a Bacterial Multidrug Resistance System Revealed by the Structure and Function of AlbA.

To combat the rise of antimicrobial resistance, the discovery of new antibiotics is paramount. Albicidin and cystobactamid are related natural product antibiotics with potent activity against Gram-positive and, crucially, Gram-negative pathogens. AlbA has been reported to neutralize albicidin by binding it with nanomolar affinity. To understand this potential resistance mechanism, we determined structures of AlbA and its complex with albicidin. The structures revealed AlbA to be comprised of two domains, each unexpectedly resembling the multiantibiotic neutralizing protein TipA. Binding of the long albicidin molecule was shared pseudosymmetrically between the two domains. The structure also revealed an unexpected chemical modification of albicidin, which we demonstrate to be promoted by AlbA, and to reduce albicidin potency; we propose a mechanism for this reaction. Overall, our findings suggest that AlbA arose through internal duplication in an ancient TipA-like gene, leading to a new binding scaffold adapted to the sequestration of long-chain antibiotics.

Molecular insights into antibiotic resistance - how a binding protein traps albicidin.

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs.

Biosynthesis of the Klebsiella oxytoca Pathogenicity Factor Tilivalline: Heterologous Expression, in Vitro Biosynthesis, and Inhibitor Development.

Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxytoca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a nonenzymatic indole incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the heterologous system was used to generate novel tilivalline derivatives by supplementation of respective anthranilate and indole precursors. Finally, it could be shown that salicylic and acetylsalicylic acid inhibit the biosynthesis of tilivalline in K. oxytoca liquid culture, presumably by blocking the peptidyl carrier protein ThdA, pointing toward a potential application in combination therapy to prevent or alleviate the symptoms of AAHC.

From the wound to the bench: exoproteome interplay between wound-colonizing Staphylococcus aureus strains and co-existing bacteria.

Wound-colonizing microorganisms can form complex and dynamic polymicrobial communities where pathogens and commensals may co-exist, cooperate or compete with each other. The present study was aimed at identifying possible interactions between different bacteria isolated from the same chronic wound of a patient with the genetic blistering disease epidermolysis bullosa (EB). Specifically, this involved two different isolates of the human pathogen Staphylococcus aureus, and isolates of Bacillus thuringiensis and Klebsiella oxytoca. Particular focus was attributed to interactions of S. aureus with the two other species, because of the high staphylococcal prevalence among chronic wounds. Intriguingly, upon co-cultivation, none of the wound isolates inhibited each other's growth. Since the extracellular proteome of bacterial pathogens is a reservoir of virulence factors, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis and K. oxytoca were characterized by Mass Spectrometry to explore the inherent relationships between these co-exisiting bacteria. This revealed a massive reduction in the number of staphylococcal exoproteins upon co-culturing with K. oxytoca or B. thuringiensis. Interestingly, this decrease was particularly evident for extracellular proteins with a predicted cytoplasmic localization, which were recently implicated in staphylococcal virulence and epidemiology. Furthermore, our exoproteome analysis uncovered potential cooperativity between the two different S. aureus isolates. Altogether, the observed exoproteome variations upon co-culturing are indicative of unprecedented adaptive mechanisms that set limits to the production of secreted staphylococcal virulence factors.

Rapid characterisation of Klebsiella oxytoca isolates from contaminated liquid hand soap using mass spectrometry, FTIR and Raman spectroscopy.

Microbiological monitoring of consumer products and the efficiency of early warning systems and outbreak investigations depend on the rapid identification and strain characterisation of pathogens posing risks to the health and safety of consumers. This study evaluates the potential of three rapid analytical techniques for identification and subtyping of bacterial isolates obtained from a liquid hand soap product, which has been recalled and reported through the EU RAPEX system due to its severe bacterial contamination. Ten isolates recovered from two bottles of the product were identified as Klebsiella oxytoca and subtyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS), near-infrared Fourier transform (NIR FT) Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Comparison of the classification results obtained by these phenotype-based techniques with outcomes of the DNA-based methods pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis of whole-genome sequencing (WGS) data revealed a high level of concordance. In conclusion, a set of analytical techniques might be useful for rapid, reliable and cost-effective microbial typing to ensure safe consumer products and allow source tracking.

Role of Electroosmosis in the Permeation of Neutral Molecules: CymA and Cyclodextrin as an Example.

To quantify the flow of small uncharged molecules into and across nanopores, one often uses ion currents. The respective ion-current fluctuations caused by the presence of the analyte make it possible to draw some conclusions about the direction and magnitude of the analyte flow. However, often this flow appears to be asymmetric with respect to the applied voltage. As a possible reason for this asymmetry, we identified the electroosmotic flow (EOF), which is the water transport associated with ions driven by the external transmembrane voltage. As an example, we quantify the contribution of the EOF through a nanopore by investigating the permeation of α-cyclodextrin through CymA, a cyclodextrin-specific channel from Klebsiella oxytoca. To understand the results from electrophysiology on a molecular level, all-atom molecular dynamics simulations are used to detail the effect of the EOF on substrate entry to and exit from a CymA channel in which the N-terminus has been deleted. The combined experimental and computational results strongly suggest that one needs to account for the significant contribution of the EOF when analyzing the penetration of cyclodextrins through the CymA pore. This example study at the same time points to the more general finding that the EOF needs to be considered in translocation studies of neutral molecules and, at least in many cases, should be able to help in discriminating between translocation and binding events.

Bacteria cell properties and grain size impact on bacteria transport and deposition in porous media.

The simultaneous role of bacteria cell properties and porous media grain size on bacteria transport and deposition behavior was investigated in this study. Transport column experiments and numerical HYDRUS-1D simulations of three bacteria with different cell properties (Escherichia coli, Klebsiella oxytoca, and Rhodococcus rhodochrous) were carried out on two sandy media with different grain sizes, under saturated steady state flow conditions. Each bacterium was characterized by cell size and shape, cell motility, electrophoretic mobility, zeta potential, hydrophobicity and potential of interaction with the sand surface. Cell characteristics affected bacteria transport behavior in the fine sand, but similar bacteria breakthroughs and retardation factors observed in the coarse sand, indicated that bacteria transport was more depended on grain size than on bacteria cell properties. Retention decreased with increasing hydrophobicity and increased with increasing electrophoretic mobility of bacteria for both sand. The increasing sand grain size resulted in a decrease of bacteria retention, except for the motile E. coli, indicating that retention of this strain was more dependent on cell motility than on the sand grain size. Bacteria deposition coefficients obtained from numerical simulations of the retention profiles indicated that straining was an important mechanism affecting bacteria deposition of E. coli and Klebsiella sp., in the fine sand, but the attachment had the same importance as straining for R. rhodochrous. The results obtained in the coarse sand did not permit to discriminate the predominant mechanism of bacteria deposition and the relative implication of bacteria cell properties of this process.

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.

Aqueous biphasic treatment of some nitrocompounds with hydrogen in the presence of a biogenerated Pd-polysaccharide.

A strain of Klebsiella oxytoca BAS-10, known to produce a specific exopolysaccharide (EPS), when grown aerobically in static mode in the presence of Pd(NO3)2, generated the species Pd-EPS that was used as catalyst precursor in the aqueous biphasic treatment of some nitrocompounds with hydrogen. Nitrobenzene was hydrogenated to aniline with almost quantitative yields and the catalyst, embedded in the aqueous phase, was used with success and with near the same efficiency in three recycling experiments. In the case of 1-iodo-4-nitrobenzene only nitrobenzene was obtained while the unsaturated nitro compound ß-methyl-ß-nitrostyrene afforded both the corresponding oxime and the saturated nitro derivative.

Characterisation of biosynthesised silver nanoparticles by scanning electrochemical microscopy (SECM) and voltammetry.

Silver nanoparticles (AgNPs) were biosynthesised by a Klebsiella oxytoca strain BAS-10, which, during its growth, is known to produce a branched exopolysaccharide (EPS). Klebsiella oxytoca cultures, treated with AgNO3 and grown under either aerobic or anaerobic conditions, produced silver nanoparticles embedded in EPS (AgNPs-EPS) containing different amounts of Ag(0) and Ag(I) forms. The average size of the AgNPs-EPS was determined by transmission electron microscopy, while the relative abundance of Ag(0)- or Ag(I)-containing AgNPs-EPS was established by scanning electrochemical microscopy (SECM). Moreover, the release of silver(I) species from the various types of AgNPs-EPS was investigated by combining SECM with anodic stripping voltammetry. These measurements allowed obtaining information on the kinetic of silver ions release from AgNPs-EPS and their concentration profiles at the substrate/water interface.

Structural similarity of secretins from type II and type III secretion systems.

Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.

Metabolic changes in Klebsiella oxytoca in response to low oxidoreduction potential, as revealed by comparative proteomic profiling integrated with flux balance analysis.

Oxidoreduction potential (ORP) is an important physiological parameter for biochemical production in anaerobic or microaerobic processes. However, the effect of ORP on cellular physiology remains largely unknown, which hampers the design of engineering strategies targeting proteins associated with ORP response. Here we characterized the effect of altering ORP in a 1,3-propanediol producer, Klebsiella oxytoca, by comparative proteomic profiling combined with flux balance analysis. Decreasing the extracellular ORP from -150 to -240 mV retarded cell growth and enhanced 1,3-propanediol production. Comparative proteomic analysis identified 61 differentially expressed proteins, mainly involved in carbohydrate catabolism, cellular constituent biosynthesis, and reductive stress response. A hypothetical oxidoreductase (HOR) that catalyzes 1,3-propanediol production was markedly upregulated, while proteins involved in biomass precursor synthesis were downregulated. As revealed by subsequent flux balance analysis, low ORP induced a metabolic shift from glycerol oxidation to reduction and rebalancing of redox and energy metabolism. From the integrated protein expression profiles and flux distributions, we can construct a rational analytic framework that elucidates how (facultative) anaerobes respond to extracellular ORP changes.

Independent domain assembly in a trapped folding intermediate of multimeric outer membrane secretins.

The outer membrane portal of the Klebsiella oxytoca type II secretion system, PulD, is a prototype of a family of proteins, the secretins, which are essential components of many bacterial secretion and pilus assembly machines. PulD is a homododecamer with a periplasmic vestibule and an outer chamber on either side of a membrane-spanning region that is poorly resolved by electron microscopy. Membrane insertion involves the formation of a dodecameric membrane-embedded intermediate. Here, we describe an amino acid substitution in PulD that blocks its assembly at this intermediate "prepore" stage. Electron microscopy indicated that the prepore has an apparently normal periplasmic vestibule but a poorly organized outer chamber. A peptide loop around this amino acid appears to be important for the formation/stability of the fully folded complex. A similar assembly intermediate results from creation of the same amino acid substitution in the Pseudomonas aeruginosa secretin XcpQ.

Structural insights into the broadened substrate profile of the extended-spectrum ß-lactamase OXY-1-1 from Klebsiella oxytoca.

Klebsiella oxytoca is a pathogen that causes serious infections in hospital patients. It shows resistance to many clinically used ß-lactam antibiotics by producing chromosomally encoded OXY-family ß-lactamases. Here, the crystal structure of an OXY-family ß-lactamase, OXY-1-1, determined at 1.93 Šresolution is reported. The structure shows that the OXY-1-1 ß-lactamase has a typical class A ß-lactamase fold and exhibits greater similarity to CTX-M-type ß-lactamases than to TEM-family or SHV-family ß-lactamases. It is also shown that the enzyme provides more space around the active cavity for the R(1) and R(2) substituents of ß-lactam antibiotics. The half-positive/half-negative distribution of surface electrostatic potential in the substrate-binding pocket indicates the preferred properties of substrates or inhibitors of the enzyme. The results reported here provide a structural basis for the broadened substrate profile of the OXY-family ß-lactamases.