The nickel-dependent urease enzyme is responsible for the hydrolysis of urea to ammonia and carbon dioxide. A number of bacteria produce urease (ureolytic bacteria) and are associated with various infectious diseases and ammonia emissions from agriculture. We report the first comprehensive comparison of the inhibition of urease activity by compounds analysed under the same conditions. Thus, 71 commercially available compounds were screened for their anti-ureolytic properties against both the ureolytic bacterium Klebsiella pneumoniae and purified jack bean urease. Of the tested compounds, 30 showed more than 25% inhibition of the ureolytic activity of Klebsiella pneumoniae or jack bean urease, and among these, carbon disulfide, N-phenylmaleimide, diethylenetriaminepentaacetic acid, sodium pyrrolidinedithiocarbamate, 1,2,4-butanetricarboxylic acid, tannic acid, and gallic acid have not previously been reported to possess anti-ureolytic properties. The diverse effects of metal ion chelators on ureolysis were investigated using a cellular nickel uptake assay. Ethylenediaminetetraacetic acid (EDTA) and dimethylglyoxime (DMG) clearly reduced the nickel import and ureolytic activity of cells, oxalic acid stimulated nickel import but reduced the ureolytic activity of cells, 1,2,4-butanetricarboxylic acid strongly stimulated nickel import and slightly increased the ureolytic activity of cells, while L-cysteine had no effect on nickel import but efficiently reduced the ureolytic activity of cells. Urease (EC 3.5.1.5), a dinickel enzyme, catalyses the hydrolysis of urea to carbonic acid (H 2 CO 3) and ammonia (NH 3) via the formation of carbamic acid (H 2 NCOOH) (Fig. 1) 1. In aqueous solutions, the carbonic acid and NH 3 are in equilibrium with bicarbonate (HCO 3 −) and ammonium (NH 4 +) ions, respectively. Urease is produced by bacteria, fungi, plants, and invertebrates, and its primary structure and active site are surprisingly conserved among different species 2. The active site of urease contains two Ni 2+ ions, which are bridged by a hydroxyl group and a carbamylated lysine. The consequences of urease-driven urea hydrolysis and the accompanying pH increase caused by NH 3 production are widespread and, therefore, are relevant in several aspects. The human pathogenic bacterium Helicobacter pylori (H. pylori), which colonizes the stomach and is linked to diseases such as gastric ulcers, gastritis and stomach cancer 3,4 , produces large amounts of urease and degrades urea to survive the acidic gastric environment 5. In the oral cavity, Streptococcus salivarius produces ammonia from urea hydrolysis in response to low pH, leading to dental plaque and calculus deposition 6. Other ureolytic bacteria, such as Klebsiella pneumoniae (K. pneumoniae) and Proteus mirabilis, are involved in pneumonia, kidney stone formation, and urinary tract infections 7,8. Urease activity is an important pathogenic factor, and ten out of twelve antibiotic-resistant pathogens designated "priority pathogens" by the WHO 9 are, in fact, ureolytic. In ...

Background: Substrate-binding integral membrane proteins of ECF transporters are predicted to undergo reversible rotation during the transport cycle. Results: Capture and release of biotin by a nanodisc-embedded ECF transporter depended on ATP-induced subunit reorientations. Conclusion: ECF transporters mediate vitamin translocation by turning their substrate-specific components within the membrane. Significance: Individual steps of the transport cycle are highlighted by biochemical and biophysical techniques.

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homoor hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotinspecific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [ 3 H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane.

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor-acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor-acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

A., Fach, P., Dorner, B.G. Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry (2016) Analyst, 141 (18), pp. 5281-5297. DOI: 10.1039/C6AN00693KThis is an author manuscript. The definitive version is available at: http://www.pubs.rsc.org/en/journals/journal/an