Berend Poolman

The proteolytic systems of lactic acid bacteria

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

Overcoming barriers to membrane protein structure determination.

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ∼30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines.

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their α-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis,Escherichia coli, Staphylococcus aureus, andLactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

Multidrug resistance in Lactococcus lactis : Evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane

Lactococcus lactis possesses an ATP‐dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P‐glycoprotein. One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds. It has been suggested that P‐glycoprotein removes drugs directly from the membrane. Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF‐AM and cationic diphenylhexatriene (DPH) derivatives from the membrane. The non‐fluorescent probe BCECF‐AM can be converted intracellularly into its fluorescent derivative, BCECF, by non‐specific esterase activities. The development of fluorescence was decreased upon energization of the cells. These and kinetic studies showed that BCECF‐AM is actively extruded from the membrane before it can be hydrolysed intracellularly. The increase in fluorescence intensity due to the distribution of TMA‐DPH into the phospholipid bilayer is a biphasic process. This behaviour reflects the fast entry of TMA‐DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane. The initial rate of TMA‐DPH extrusion correlates with the amount of probe associated with the inner leaflet. Taken together, these results demonstrate that the lactococcal MDR transporter functions as a ‘hydrophobic vacuum cleaner’, expelling drugs from the inner leaflet of the lipid bilayer. Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters.

Lateral diffusion of membrane proteins

We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a alpha-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per microm(2) of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 microm(-2)), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of approximately 25,000 microm(-2)) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed.

Bioenergetics and Solute Transport in Lactococci

During the last few years the studies about the physiology and bioenergetics of lactic acid bacteria during growth and starvation have evolved from a descriptive level to an analysis of the molecular events in the regulation of various processes. Considerable progress has been made in the understanding of the modes of metabolic energy generation, the mechanism of homeostasis of the internal pH, and the mechanism and regulatory processes of transport systems for sugars, amino acids, peptides, and ions. Detailed studies of these transport processes have been performed in cytoplasmic membrane vesicles of these organisms in which a foreign proton pump has been introduced to generate a high proton motive force.

How do membrane proteins sense water stress

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmo‐protectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift‐activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein–lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift‐activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.

Membrane reconstitution of ABC transporters and assays of translocator function.

In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (Kd) for ligand binding, number of binding-sites, transport affinities (Km), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4–5 d.

The Lactococcal lmrP Gene Encodes a Proton Motive Force- dependent Drug Transporter*

To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombination using DNA fragments of the flanking region of the gene. The L. lactis (ΔlmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton antiporter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N.(1994) J. Bacteriol. 176, 6957-6964).

S-carvone as a natural potato sprout inhibiting, fungistatic and bacteristatic compound

S-Carvone, a common monoterpene found in caraway (Carum carvi L.), inhibits the sprouting of potatoes very efficiently at continuous low head space concentrations. The length growth of potato sprouts was inhibited within 2 days following exposure to S-carvone. Sprouts were able to convert S-carvone into more reduced compounds. In addition, growth of the plant-pathogenic fungi Fusarium solani and Fusarium sulphureum was found to be inhibited by S-carvone at concentrations between 1 and 3 mM. At sub-inhibitory concentration, both F. solani and F. sulphureum converted S-carvone into more reduced compounds which were shown to be less toxic as compared to S-carvone. Finally, the growth rate of Streptococcus thermophilus, Lactococcus lactis and Escherichia coli decreased at S-carvone concentrations above 1 mM. This decrease correlated with a dissipation of the proton motive force generated in these cells.