First certified reference materials for molecular fingerprinting of two approved probiotic Bacillus strains.

At present probiotic bacteria are widely used in human and animal nutrition because they beneficially influence the balance of the intestinal flora of the host. Positive effects related to probiotics are various and include enhancement of digestion, strengthening of the immune system and stimulation of vitamin production. Moreover, implementation of probiotics is intended to reduce the use of antibiotics and improve animal growth and feed conversion. To protect human and animal health and to improve consumer confidence, a strict legislation on the use of probiotics exists within the European Union (EU). Official controls by national authorities are performed to ensure verification of compliance with feed and food law. Apart from the risk of using unauthorized strains, mislabelling is a known problem, partly because of the use of phenotyping or genotyping methods with a lack of discriminative power. In addition to official controls, private controls by food and feed producing companies are important in the frame of protection of patented strains and industrial property rights. To support these applications, IRMM has developed certified reference materials (CRMs) consisting of genomic DNA inserts of B. subtilis DSM 5749 and B. licheniformis DSM 5750, two strains that received EU approval. In this study we investigated the use of these CRMs, IRMM-311 and IRMM-312, for the detection and unambiguous discrimination of Bacillus strains by pulsed-field gel electrophoresis (PFGE). Identical fingerprints were obtained for the CRMs and control strains isolated from the feed additive Bioplus 2B. On the other hand a distinction could be made from other not approved B. licheniformis and B. subtilis strains. The reference materials discussed in this study are the first CRMs based on a whole bacterial genome and suitable for PFGE. They offer perspectives for applications in other domains such as analysis of foodborne pathogens in outbreaks or routine analysis.

Exchange, efflux, and substrate binding by cysteine mutants of the lactose permease of Escherichia coli.

In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and reconstituted into proteoliposomes. Lactose equilibrium exchange and efflux activities of mutants with Ser in place of Cys117, Cys176, Cys234, Cys333, Cys353, or Cys355 are essentially the same as wild-type permease. In contrast, mutants in Cys148 and Cys154 exhibit diminished exchange and efflux activities. These mutants in Cys148 and Cys154, except for the C148S mutant, have previously been shown to slow down active transport as well [Van Iwaarden, P.R., Driessen, A. J. M., Menick, D. R., Kaback, H.R., & Konings, W. N. (1991) J. Biol. Chem. 266, 15688-15692]. C148S permease shows monophasic kinetics with a high apparent KM with respect to external lactose in the exchange reaction under nonequilibrium conditions, whereas wild-type permease exhibits biphasic kinetics with both a high and low KM component. Moreover, the absence of the low Km pathway in the C148S permease is correlated with the absence of a high-affinity binding site for p-nitrophenyl alpha-D-galactopyranoside (NPG). Interestingly, the affinity of the permease for NPG appears to increase with the hydrophobicity of the side chain at position 154 (Ser < Cys < Gly < Val). Finally, the presence of a high-affinity binding site for NPG in C154V is consistent with the biphasic exchange kinetics exhibited by this mutant. The results are discussed in the context of a model in which lac permease has two substrate binding sites, a catalytic site and a regulatory site.

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.

Construction of a functional lactose permease devoid of cysteine residues.

By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease). Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced with serine. Remarkably, C-less permease catalyzes lactose accumulation in the presence of a transmembrane proton electrochemical gradient (interior negative and alkaline). Thus, in intact cells and right-side-out membrane vesicles containing comparable amounts of wild-type and Cys-less permease, the mutant protein catalyzes lactose transport at a maximum velocity and to a steady-state level of accumulation of about 35% and 55%, respectively, of wild-type with a similar apparent Km (ca. 0.3 mM). As anticipated, moreover, active lactose transport via C-less permease is completely resistant to inactivation by N-ethylmaleimide. Finally, C-less permease also catalyzes efflux and equilibrium exchange at about 35% of wild-type activity. The results provide definitive evidence that sulfhydryl groups do not play an essential role in the mechanism of lactose/H+ symport. Potential applications of the C-less mutant to studies of static and dynamic aspects of permease structure/function are discussed.

Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli.

lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.

Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus.

In the presence of electrochemical energy, several branched-chain neutral and acidic amino acids were found to accumulate in membrane vesicles of Bacillus stearothermophilus. The membrane vesicles contained a stereo-specific transport system for the acidic amino acids L-glutamate and L-aspartate, which could not translocate their respective amines, L-glutamine and L-asparagine. The transport system was thermostable (Ti = 70 degrees C) and showed highest activities at elevated temperatures (60 to 65 degrees C). The membrane potential or pH gradient could act as the driving force for L-glutamate uptake, which indicated that the transport process of L-glutamate is electrogenic and that protons are involved in the translocation process. The electrogenic character implies that the anionic L-glutamate is cotransported with at least two monovalent cations. To determine the mechanistic stoichiometry of L-glutamate transport and the nature of the cotranslocated cations, the relationship between the components of the proton motive force and the chemical gradient of L-glutamate was investigated at different external pH values in the absence and presence of ionophores. In the presence of either a membrane potential or a pH gradient, the chemical gradient of L-glutamate was equivalent to that specific gradient at different pH values. These results cannot be explained by cotransport of L-glutamate with two protons, assuming thermodynamic equilibrium between the driving force for uptake and the chemical gradient of the substrate. To determine the character of the cotranslocated cations, L-glutamate uptake was monitored with artificial gradients. It was established that either the membrane potential, pH gradient, or chemical gradient of sodium ions could act as the driving force for L-glutamate uptake, which indicated that L-glutamate most likely is cotranslocated in symport with one proton and on sodium ion.