Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients.

Bacteria are involved in the onset and progression of periodontitis. A promising molecular technique, denaturing gradient gel electrophoresis (DGGE), to study microbial population dynamics in the subgingival pocket is presented. Twenty-three samples were taken from the subgingival pockets of nine patients and six healthy family members. From four periodontitis patients, 12 samples were evaluated before, 1 day after and 3 months after treatment. Part of the 16S rRNA gene of all bacteria was amplified by PCR and separated by DGGE, creating banding patterns representative of the community structure. Shifts in composition and diversity of the microbial population could be determined semiquantitatively, and this showed that treatment resulted in a decrease in the diversity of the population. After 3 months a microbial population 33-47% different from the population before treatment had re-established. Intense bands representing Exiguobacterium aurantiacum were present in 13 out of 25 samples, indicating that this species may play a role in periodontal disease.

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum.

Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Escherichia coli.

Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the beta-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40, respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.

Another unusual type of citric acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase.

The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA.

An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees C instead of 30 degrees C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 x 10(6) cfu micrograms-1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.

Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Corynebacterium glutamicum.

In addition to a cytoplasmic, NAD-dependent malate dehydrogenase (EC 1.1.1.37), Corynebacterium glutamicum possesses a highly active membrane-associated malate dehydrogenase (acceptor) (EC 1.1.99.16). This enzyme also takes part in the citric acid cycle. It oxidizes L-malate to oxaloacetate and donates electrons to ubiquinone-1 and other artificial acceptors or, via the electron transfer chain, to oxygen. NAD is not an acceptor and the natural direct acceptor for the enzyme is most likely a quinone. The enzyme is therefore called malate:quinone oxidoreductase, abbreviated to Mqo. Mqo is a peripheral membrane protein and can be released from the membrane by addition of chelators. The solubilized form was partially purified and characterized biochemically. FAD is probably a tightly but non-covalently bound prosthetic group, and the enzyme is activated by lipids. A C. glutamicum mutant completely lacking Mqo activity was isolated. It grows poorly on several substrates tested. The mutant possesses normal levels of cytoplasmic NAD-dependent malate dehydrogenase. A plasmid containing the gene from C. glutamicum coding for Mqo was isolated by complementation of the Mqo-negative phenotype. It leads to overexpression of Mqo activity in the mutant. The nucleotide sequence of the mqo gene was determined and is the first sequence known for this enzyme. The derived protein sequence is similar to hypothetical proteins from Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis.

Catabolite inactivation of wild-type and mutant maltose transport proteins in Saccharomyces cerevisiae.

The maltose transporter of Saccharomyces cerevisiae is subject to rapid, irreversible inactivation in the presence of glucose. Loss of transport function was paralleled by a decrease in amount of transporter protein and most likely involves endocytosis and degradation of the protein in the vacuole. This (catabolite) inactivation of Mal61p was triggered not only by glucose but also by 2-deoxy-D-glucose, which cannot be metabolized beyond 2-deoxy-D-glucose phosphate. The signal that targets membrane proteins specifically for catabolite inactivation is unknown. To investigate whether or not specific modification of Mal61p triggers the inactivation, putative protein kinase A and C phosphorylation sites were removed, and the transport activities and levels of the mutant proteins upon addition of glucose were followed in time. Three Mal61p mutants, i.e. S295A, T363A, and S487A, exhibited significantly reduced rates of inactivation in the presence of glucose. Likewise, in wild-type Mal61p the rate of inactivation and degradation of the protein paralleled each other in the case of T363A. On the contrary, for the S295A and S487A mutants the rates of protein degradation were slowed down more profoundly than was the loss of transport activity. These observations indicate that (i) some form of modification (e.g. phosphorylation) of the protein precedes breakdown, (ii) the modification inactivates Mal61p, and (iii) the inactivation of Mal61p is not necessarily followed by proteolytic degradation.

Secondary transporters for citrate and the Mg(2+)-citrate complex in Bacillus subtilis are homologous proteins.

Citrate uptake in Bacillus subtilis is mediated by a secondary transporter that transports the complex of citrate and divalent metal ions. The gene coding for the transporter termed CitM was cloned, sequenced, and functionally expressed in Escherichia coli. Translation of the base sequence to the primary sequence revealed a transporter that is not homologous to any known secondary transporter. However, CitM shares 60% sequence identity with the gene product of open reading frame N15CR that is on the genome of B. subtilis and for which no function is known. The hydropathy profiles of the primary sequences of CitM and the unknown gene product are very similar, and secondary structure prediction algorithms predict 12 transmembrane-spanning segments for both proteins. Open reading frame N15CR was cloned and expressed in E. coli and was shown to be a citrate transporter as well. The transporter is termed CitH. A remarkable difference between the two transporters is that citrate uptake by CitM is stimulated by the presence of Mg2+ ions, while citrate uptake by CitH is inhibited by Mg2+. It is concluded that the substrate of CitM is the Mg(2+)-citrate complex and that CitH transports the free citrate anion. Uptake experiments in right-side-out membrane vesicles derived from E. coli cells expressing either CitM or CitH showed that both transporters catalyze electrogenic proton/substrate symport.

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes.

For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.

The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes fused with proteoliposomes, as model systems for studies on the mechanism and regulation of transport is evaluated.

Transport of citrate catalyzed by the sodium-dependent citrate carrier of Klebsiella pneumoniae is obligatorily coupled to the transport of two sodium ions.

Aerobically grown Escherichia coli GM48 harboring plasmid pKScitS that codes for the sodium-dependent citrate carrier from Klebsiella pneumoniae (CitS) allows initial-rate measurements of citrate uptake in whole cells. The cation stoichiometry and selectivity of CitS was studied using this experimental system. The relationship between the initial rate of uptake of citrate and the Na+ concentration was sigmoidal at pH values between 5 and 7 suggesting a Na+ stoichiometry higher than 1. Rates of uptake increased quadratically in a range of non-saturating Na+ concentrations showing that two Na+ are translocated/catalytic cycle. Symport of Na+ is absolutely required in the range pH 5-7 because no uptake could be detected in the absence of Na+. Protons cannot replace Na+ in the translocation step but the decrease in apparent affinity for Na+ towards lower pH suggests that protons can compete with Na+ for the cation-binding sites. Li+ can replace Na+ in the symport reaction but it takes about a 200-fold higher concentration of Li+ over Na+ to achieve the same rate of uptake, showing that the affinity of CitS for Li+ is much lower than for Na+. Though high Li+ concentrations have an inhibitory effect on citrate uptake, the data suggest that the Li+ stoichiometry is also 2.

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.

Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae.

The gene of the sodium-dependent citrate transport system from Klebsiella pneumoniae (citS) is located on plasmid pES3 (Schwarz, E., and Oesterhelt, D. (1985) EMBO J. 4, 1599-1603) and encodes a 446-amino acid protein. Transport of citrate via this citrate transport protein (CitS) is dependent on the presence of sodium ions and is inhibited by magnesium ions. The delta pH (pH gradient across the membrane) is the major driving force for uptake. It is postulated that, in analogy with the proton-dependent citrate carrier (CitH) of K. pneumoniae (van der Rest, M. E., Abee, T., Molenaar, D., and Konings, W. N. (1990) Eur. J. Biochem. 195, 71-77), only one of the protonated species of citrate is recognized by CitS and that citrate is translocated across the membrane in symport with protons and sodium ions. The hydrophobicity profile of CitS suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments. These segments are not centered around a hydrophilic core as has been suggested for other transport proteins, but the protein is asymmetrical with seven transmembrane segments in front of a large hydrophilic loop and five after this loop. The amino acid sequence is highly similar to a citrate transport system of Lactococcus lactis subsp. lactis var. diacetylactis (CitP) (David, S., van der Rest, M. E., Driessen, A. J. M., Simons, G., and de Vos, W. M. (1990) J. Bacteriol. 172, 5789-5794) and less similar to CitH of K. pneumoniae. We conclude that the citS gene of K. pneumoniae encodes a sodium-dependent citrate transport system that belongs to a novel subclass of transport proteins.

Mechanism and energetics of a citrate-transport system of Klebsiella pneumoniae.

The citrate-transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz, E. & Oesterhelt, D. (1985) EMBO J. 4, 1599-1603] has been subcloned in Escherichia coli DH1. Uptake of citrate in E. coli membrane vesicles via this uptake system is an electrogenic process, although the pH gradient is the main driving force for citrate uptake. The rate of citrate uptake, driven by artificially imposed ion-gradients, is high in the presence of an artificial delta pH and low in the presence of an artificial delta psi. Citrate transport does not depend on the presence of Na+ or Mg2+ as has been observed for other citrate-transport systems. Citrate has three pK values: 3.14, 4.77 and 5.40. Citrate forms a stable complex with Mg2+ with a stability constant of 3.2. Kinetic parameters and calculations of the different citrate (Cit) species at a given pH, indicate that the HCit2- is the species transported and that transport occurs in symport with three protons. This citrate-transport system is thus a unique example of a 3H solute symport system.

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.

DNA sequence of a citrate carrier of Klebsiella pneumoniae.

The citrate transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz E. and D. Oesterhelt (1985) EM BO J. 4, 1599-1603] has been subcloned in Escherichia coli DH1. The DNA sequence of a 1723-base fragment that codes for the citrate carrier has been determined and the gene product has been characterized with the T7 promoter system. The DNA fragment contains an open reading frame of 1332 base pairs and codes for a protein of 444 amino acids. The hydropathy profile suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments centered around a hydrophilic core. The gene for the citrate carrier has 66% similarity with a citrate carrier determinant from a naturally occurring plasmid responsible for secondary transport of citrate across the cytoplasmic membrane of E. coli.