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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Intestinal microbiota may play a role in the pathophysiology of irritable bowel syndrome (IBS). In this case-control study, mucosa-associated small intestinal and faecal microbiota of IBS patients and healthy subjects were analysed using molecular-based methods. Duodenal mucosal brush and faecal samples were collected from 37 IBS patients and 20 healthy subjects. The bacterial 16S rRNA gene was amplified and analysed using PCR denaturing gradient gel electrophoresis (DGGE). Pooled average DGGE profiles of all IBS patients and all healthy subjects from both sampling sites were generated and fingerprints of both groups were compared. The DGGE band fragments which were confined to one group were further characterized by sequence analysis. Quantitative real-time PCR (q-PCR) was used to quantify the disease-associated microbiota. Averaged DGGE profiles of both groups were identical for 78.2 % in the small intestinal samples and for 86.25 % in the faecal samples. Cloning and sequencing of the specific bands isolated from small intestinal and faecal DGGE patterns of IBS patients showed that 45.8 % of the clones belonged to the genus Pseudomonas, of which Pseudomonas aeruginosa was the predominant species. q-PCR analysis revealed higher levels (P,0.001) of P. aeruginosa in the small intestine of IBS patients (8.3 %±0.950) than in the small intestine of healthy subjects (0.1 %±0.069). P. aeruginosa was also significantly (P,0.001) more abundant (2.34 %±0.31) in faeces of IBS patients than in faeces of healthy subjects (0.003 %± 0.0027). This study shows that P. aeruginosa is detected more frequently and at higher levels in IBS patients than in healthy subjects, suggesting its potential role in the pathophysiology of IBS. INTRODUCTIONIrritable bowel syndrome (IBS) is a gastrointestinal disorder of unknown aetiology characterized by abdominal pain and change in bowel habit. Alteration in faecal microbiota composition and abnormal colonic fermentation imply that gastrointestinal microbiota may play a role in the pathogenesis of IBS (Spiller, 2007;Quigley, 2007). In 7-30 % of IBS patients, acute gastrointestinal infection has been proposed as trigger of the onset of IBS symptoms, and the inflammatory response to the infection may cause persistent sensory-motor dysfunction (Madden & Hunter, 2002;Rodríguez & Ruigó mez, 1999;Parry et al., 2003;Collins et al., 2001). Moreover, antibiotic therapy and probiotic supplementation have been shown to reduce IBS symptoms in a subset of IBS patients, by either eradication of small intestinal bacterial overgrowth or modulation of the composition of the microbiota (Pimentel et al., 2000;Nobaek et al., 2000;Niedzielin et al., 2001;O'Mahony et al., 2005). Antibiotic therapy may also be a risk factor for developing IBS symptoms due to changes in bowel microbiota and colonization of pathogenic bacteria (Maxwell et al., 2002). It is well recognized that the microbial community is significantly altered in IBS (King et al., 1998;Posserud et al., 2007). Conventional culturing methods have shown th...

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:quinoneoxidoreductase (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 ␤-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.In Escherichia coli, several enzymes or pathways are able to convert malate to oxaloacetate. The NAD-dependent (cytoplasmic) malate dehydrogenase (MDH; EC 1.1.1.37) has always been considered to be the principal malate-oxidizing enzyme in the citric acid cycle (tricarboxylic acid [TCA] cycle) of this organism (9). Recently, a malate:quinone-oxidoreductase (MQO; EC 1.1.99.16) which is an essential enzyme in the TCA cycle of Corynebacterium glutamicum (reference 23 and accompanying paper) was described. MQO is a flavin adenine dinucleotide (FAD)-and lipid-dependent peripheral membrane protein catalyzing the oxidation of L-malate to oxaloacetate. The electrons are donated to the electron transfer chain at the level of quinones. This reaction is essentially irreversible (17). In C. glutamicum, MQO and the cytoplasmic NAD-dependent malate dehydrogenase, which is capable of reversible oxidation of malate to oxaloacetate, are active at the sam...

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 controversy with regard to the presence of malate dehydrogenase (MDH) in Helicobacter pylori became apparent when the genomic sequences of two strains of this organism were published (2, 29). Whereas biochemical measurements indicated that MDH activity (EC 1.1.1.37) was present in this organism, no open reading frame (ORF) for a possible MDH could be found in the genomic sequences (14,17,19,24). In some organisms genes encoding MDH are more similar to genes for L-lactate dehydrogenases (6). However, such ORFs were also lacking in H. pylori, excluding the possibility that an mdh gene had been erroneously annotated as an ldh gene. H. pylori does have an ORF (dld) for a lactate dehydrogenase, but this is a membrane-bound D-lactate dehydrogenase.The presence or absence of an MDH in H. pylori has implications for its central metabolism. Considerable confusion exists as to whether H. pylori possesses a complete citric acid cycle and whether this cycle can operate oxidatively or functions only in a branched mode. Physiological studies of lactate and pyruvate oxidation by well-aerated cells indicated that some oxidative citric acid cycle activity might be present (5). However, the original annotation of the genome indicated three omissions in the list of ORFs comprising a typical citric acid cycle (29). The omissions were ␣-ketoglutarate dehydrogenase, succinyl-coenzyme A (CoA) ligase, and MDH. Earlier biochemical studies showed that instead of ␣-ketoglutarate dehydrogenase H. pylori possesses ␣-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3) (18). Furthermore, a succinylCoA:acetoacetyl-CoA transferase could convert succinyl-CoA to succi...