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.

AIM:To determine the composition of both fecal and duodenal mucosa-associated microbiota in irritable bowel syndrome (IBS) patients and healthy subjects using molecular-based techniques. METHODS:Fecal and duodenal mucosa brush samples were obtained from 41 IBS patients and 26 healthy subjects. Fecal samples were analyzed for the composition of the total microbiota using fluorescent in situ hybridization (FISH) and both fecal and duodenal brush samples were analyzed for the composition of bifidobacteria using real-time polymerase chain reaction. RESULTS:The FISH analysis of fecal samples revealed a 2-fold decrease in the level of bifidobacteria (4.2 ± 1.3 vs 8.3 ± 1.9, P < 0.01) in IBS patients compared to healthy subjects, whereas no major differences in other bacterial groups were observed. At the species level, Bifidobacterium catenulatum levels were significantly lower (6 ± 0.6 vs 19 ± 2.5, P < 0.001) in the IBS patients in both fecal and duodenal brush samples than in healthy subjects. CONCLUSION:Decreased bifidobacteria levels in both fecal and duodenal brush samples of IBS patients compared to healthy subjects indicate a role for microbiotic composition in IBS pathophysiology.

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.Recently, we discovered the gene for a relatively unknown type of malate dehydrogenase called malate:quinone oxidoreductase (MQO; (EC 1.1.99.16) [also called "malate dehydrogenase (acceptor)"] (22). Like the NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37), MQO catalyzes the oxidation of malate to oxaloacetate. The enzyme is membrane associated, probably through weak ionic or hydrophobic interactions. Tightly bound flavin adenine dinucleotide serves as a prosthetic group, and quinones instead of NAD are the electron acceptors of the enzyme. The quinones are subsequently oxidized by the electron transfer chain. These properties place MQO, like succinate dehydrogenase (SDH), both in the electron transfer chain and in the citric acid cycle. The existence of MQO as an enzymatic entity was first proven in 1956 (8). MQO activity was since then observed in several bacteria, both gram positive and gram negative (see references cited in reference 22) but not in archaebacteria or eucaryotes. After identification (for the first time) of a DNA sequence encoding an MQO in Corynebacterium glutamicum, it was found t...

In addition to a cytoplasmic, NAD‐dependent malate dehydrogenase ( ), Corynebacterium glutamicum possesses a highly active membrane‐associated malate dehydrogenase (acceptor) ( ). 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.

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