Environmentally directed mutations and their impact on industrial biotransformation and fermentation processes.

Microbial adaptation plays an important role in the selection of improved strains for biotechnological processes and for the maintenance and stability of the selected production strains. Most of the knowledge about adaptation processes and environmentally directed mutations originates from environmental microbiology and from studies on biological evolution. The increasing information on the molecular mechanisms of adapted mutations and on the development of methods frequently used in environmental and evolutionary microbiology, such as the selection in semi-continuous cultures or chemostats, can be used as input and tools for the improvement of industrial production organisms.

Coordination of a histidine residue of the protein-component S to the cobalt atom in coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co-atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the gz-lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co-atom. A mixture of completely 15N-labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper gz-lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine-splitting of the gz-line, whereas the 15N-labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co-atom. By using a sample of 15N-labelled component S, in which only the histidines were 14N-labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.

Characterization of the coenzyme-B12-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli.

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)

Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans.

Glutaconate coenzyme A-transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N-termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to sigma 70-type promoters from Escherichia coli. Primer-extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)-2-hydroxyglutarate to crotonyl-CoA in the pathway of glutamate fermentation by A. fermentans. The genes gctAB were expressed together in E. coli. Cell-free extracts of a transformant E. coli strain contained glutaconate CoA-transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).

Cloning, sequencing and expression in Escherichia coli of the gene encoding component S of the coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

Adenosylcobalamin (coenzyme B12) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium. The enzyme consists of the two protein components E and S. The gene encoding component S (glmS) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS) gene (80%) and to component S (82%) from the related C. tetanomorphum, respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B12 and component E purified from C. cochlearium. Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells which is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium. This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.

Cloning, sequencing and expression of the gene encoding the coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri in Escherichia coli.

The coenzyme B12 (adenosylcobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri. (a) The mgm gene encoding 2-methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8-kDa protein of 614 amino acids. It shows significant similarity in its C-terminal part to that of other cobamide-dependent enzymes. Probably, this is the coenzyme-binding region. (b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western-blot analysis with rabbit antiserum directed against the native mutase. (c) Cell-free extracts from E. coli carrying the mgm gene showed 2-methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme.

On the role of two different cobalt(II) species in coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri.

Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enzyme liberated the prosthetic groups, cob(II)alamin being oxidized by air to aquocobalamin. HPLC analysis of the released cobamides from several preparations revealed > 90% adenosylcobalamin and < 10% aquocobalamin. Treatment of active 2-methyleneglutarate mutase with 8M urea followed by gelfiltration yielded an inactive enzyme from which 50% of the adenosylcobalamin and up to 70% of the cob(II)alamin was removed. Addition of adenosylcobalamin to the urea-treated enzyme resulted in complete reactivation, but the content of cob(II)alamin was not increased. These data suggest that the oxygen-stable cob(II)alamin is not involved in catalysis. In the presence of the competitive inhibitor itaconate (methylenesuccinate, Ki = 0.7mM), an alteration of the UV/visible spectrum at 470 nm as well as a new line in the EPR spectrum of the enzyme (around g = 2.1) was observed. The results indicate the formation of an unusual, oxygen sensitive Co(II) species during catalysis. The EPR signal of the oxygen-stable cob(II)alamin (gx,y = 2.24) remained unchanged under those conditions.

Alcohol-induced liver injury after jejunoileal bypass operation in rats.

The objective of this study was to investigate whether alcohol administration exerts a synergistic effect on jejunoileal bypass-induced liver dysfunction in rats. Male Wistar rats were subjected to 90% jejunoileal bypass or sham operation. For 10 weeks, subgroups were pair-fed either an alcohol-containing (36% of total calories) liquid diet or a liquid diet where alcohol was replaced isocalorically by starch. Alcohol feeding in rats with jejunoileal bypass increased hepatic triglyceride content about 6-fold as compared with bypassed rats receiving control diet. Neither jejunoileal bypass nor alcohol feeding led to significant changes in hepatic DNA and protein contents. Alcohol feeding increased cytochrome P-450 levels both in operated and in sham-operated rats. The administration of alcohol-containing diet decreased the activity of succinic dehydrogenase, the decrease being distinctly more pronounced in rats with jejunoileal bypass than in the sham-operated controls. Light microscopy revealed no significant morphological alterations in liver sections of rats fed the control diet after jejunoileal bypass or of rats receiving either the alcohol-containing diet or the control diet after sham operation. Alcohol feeding in bypassed rats, however, produced marked diffuse accumulation of fat, and regularly led to other histological abnormalities in the liver. These abnormalities included ballooning of hepatocytes and disarray of the trabecular structure of the liver lobule, hyalin inclusions resembling megamitochondria, single-cell necrosis and focal clustering of necrosis, increased number of mitotic figures, and infiltrates with inflammatory cells. The histological lesions of the liver of bypassed rats receiving alcohol exhibited no obvious zonal distribution. The results demonstrate that alcohol feeding to rats subjected to jejunoileal bypass leads to marked liver injury which mimics, at least in part, that of alcohol-induced liver disease in man. Rats subjected to jejunoileal bypass may, therefore, provide a new model for the study of alcoholic liver disease.

Effect of ammonia on plasma and cerebrospinal fluid amino acids in dogs with and without portacaval anastomoses.

To elucidate the possible connection between ammonia-induced changes of plasma and cerebrospinal fluid (CSF) amino acid levels and the development of hepatic encephalopathy in dogs, beagle dogs were given an ammonium acetate infusion both before and following portacaval shunt (PCS). During ammonia-induced coma and after recovery in the dogs prior to PCS the plasma and CSF concentrations of most amino acids were decreased. Following PCS the plasma and CSF concentrations of the aromatic amino acids (AAA), phenylalanine and tyrosine, increased and the levels of the branched chain amino acids (BCAA), valine, leucine, and isoleucine, decreased during ammonia-induced coma. The CSF/plasma molar ratio for the AAA exhibited a marked increase after recovery as compared to the value during coma in the Eck-fistula dogs. With respect to the AAA, no correlation was observed between signs of neurologic impairment in the animals and the following parameters: glutamine and methionine levels of CSF, and the plasma molar ratio (Formula: see text). The data obtained do not support the hypothesis that high concentrations of phenylalanine and tyrosine in the brain may be primarily responsible for altered neurotransmission leading to the development of hepatic encephalopathy.

[Need for randomizing microvascular surgical experiments for testing new prosthesis material, with special reference to personal surgical errors from a morphological viewpoint]. / Uber die Notwendigkeit der Randomisierung mikrovaskulärer chirurgischer Experimente bei der Uberprüfung eines neuen Prothesenmaterials unter Berücksichtigung eigener chirurgischer Fehler aus morphologischer Sicht.

In the examination of material involving two microvascular prostheses of different nature the need is discussed of randomising experimental series to achieve a self-critical control of operative dexterity in microsurgical techniques and to exclude eventual errors arising and influencing the results. A comparison was made of two polyurethane prostheses, one porous (n = 15), and the other covered internally with a smooth silicon layer (n = 15). They were implanted using microsurgical techniques into the infrarenal portion of the abdominal aorta in rats. The main criterion for successful implantation was a patency over a prolonged period of observation lasting a maximum of 407 days. The porous prosthesis showed the best results. Most frequent complications were early thrombosis and technical faults demonstrated by histology. Therefore, the distribution through prospective randomisation of good and bad results based on technical errors enhances the significance of material analysis.

The use of microvascular graft as an arterial substitute in the abdominal aorta of the rat.

The purpose of this study was to compare by means of microsurgical techniques a porous with an interiorly smooth siliconized vascular graft. Compatibility and patency of the graft were examined in a long term follow-up. A microvascular prosthesis with a particular fibrous texture was used. The fibres consisted of polyurethane (PUR), a new prosthetic material. The lumen of the prosthesis measured 1.6 mm; the outer diameter was 2.4 mm. In group A an in- and exteriorly microporous graft was applied; in group B the inner surface of the prosthesis was smoothed by a silicon layer. 30 SPF-Wistar rats were randomized in group A (n = 15) and B (n = 15), and both prosthetic materials were implanted as a substitute for an infrarenal part of the abdominal aorta. Monofile suture material (MirafilR--DR 5) size 10:0 was used for the anastomosis. The mean pressure of the aorta amounted to 108 +/- 30 mm Hg. After 4 +/- 2 months the total survival rate was 77% (n = 23): in group A, 80% (n = 12); in group B, 73% (n = 11). Cause of death was early thrombosis due to technical mistakes. The following results were obtained by angiography: highest patency--group A, 8; group B, 1; constriction--group A, 6; group B, 9; occlusion--group A, 1; group B, 4; total--group A, 15; group B, 14; 1 angiography technically failed. Arterial substitution in the abdominal aorta of the rat by PUR-grafts is possible and is a suitable model for further experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

[Idiopathic hepatic and choledochal cyst--diagnosis and surgical treatment]. / Die idiopathische Hepatikus- und Choledochuszyste--Diagnostik und chirurgische Behandlung.

Special uncertainty in diagnosis and operative therapy of infrequent hepatic and/or choledochal cyst is discussed by an own case report and recent literature. Indication for operation persists always. Best operative treatment is total excision of the cyst with Roux-en-Y hepatico-(choledocho-) jejunostomy.

[Frequency and significance of infections due to Yersinia enterocolitica in "acute appendicitis" (author's transl)]. / Häufigkeit und Bedeutung von Yersinia-enterocolitica-Infektionen bei "akuter Appendizitis".

In 352 patients who were hospitalized with symptoms of an acute appendicitis, Yersinia infection were determined in 18.2% of the cases by cultural and serological methods. Infections due to Y. enterocolitica (Y. e.) serovar 0:3 were approximately 6 times more frequent than those due to Y. e. serovar 0:9. Yersinia pseudotuberculosis (Y. pstbc.) could only be isolated in one patient from a mesenterial lymph node. In another case Yersinia serovar 0:6 could be isolated as well as Y. e. serovar 0:3. The majority of the infections were found in the age group 9-12 years. The incidence was highest in the summer months June-August.

[Piperacillin: pharmacokinetic studies on the biliary and renal excretion (author's transl)]. / Piperacillin: Kinetische Studien über biliäre und renale Ausscheidung.

9 patients (6 female, 3 male) with either cholelithiasis or choledocholithiasis submitted to cholecystectomy were studied during the postoperative period. Sodium 6-[D-(--)-alpha-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-alpha-phenyl-ace tamido]penicillinate (piperacillin) was administered in single i.v. doses of 2 and 4 g on the 4th and 6th postoperative days. Serum samples were taken at intervals of 20 min over a period of 3 h post injection and bile was collected via T-tube drainage in 20-min periods also for 180 min. The total urine outputs were collected for the periods 0--6 h, 6--12 h and 12--24 h after administration of the drug. The maximum concentration of piperacillin in the bile was observed 60--120 min post administration. The values in the bile were found to exceed the corresponding serum concentrations 30--40fold. Within 3 h 9.6 (+/- 6.5)% and 13.4 (+/- 5.0)% were eliminated in the bile after 2 and 4 g injections, respectively. The percentage of the administered drug excreted in the urine within 24 h was 56.3% (2 g i.v.) and 66.0% (4 g i.v.).

[Epicillin concentrations in bile and serum after parenteral application (author's transl)]. / Epicillin-Konzentrationen in Galle und Serum nach parenteraler Gabe.

11 patients with cholelithiasis and choledocholithiasis, respectively, were cholecystectomized with subsequent inspection of the common bile duct. The drainage of the bile alpha-amino-3,6-dihydrobenzylpenicillin (epicillin, Spectacillin) were administered i.v. and the antibiotic concentrations in the bile and the serum determined. Samples were taken at intervals of 20 min over a period of 3 h. The maximum concentration in the bile was observed 60 min following administration of the antibiotic. The values in the bile were found to exceed the corresponding serum concentrations 6--14 fold. By the end of the observation period the concentrations of epicillin in the bile as well as in the serum were still higher than the minimum inhibitory concentrations for most of the gram-positive and gram-negative organisms.

Hemodynamic parameters and blood gas analyses in the normal and the cirrhotic rat.

Thioacetamide-fed rats developed cirrhosis with portal hypertension (P portal=23.0 +/- 5.2 cm H2O, controls: 14.4 +/- 1.0 cm H2O). The PO2 of liver tissue was markedly reduced in cirrhosis (PO2=7.6 +/- 3.4 torr, controls 22.3 +/- 5.8 torr), and the aortal pH was significantly lower as well. No correlation was found between portal hypertension, development of large--nodular cirrhosis, and ascites.

Glucagon therapy in acute pancreatitis. Report of a double-blind trial.

The results of a double-blind trial of glucagon in 69 patients with acute pancreatitis are reported. In a subgroup of 59 patients statistical analysis showed no significant differences between the glucagon-treated (n = 29; 2 X 5 mg protamine-zinc glucagon intramuscularly per day) and the placebo-treated (n = 30) subjects for the following data: duration of pain left spontaneously and induced by palpation, amounts of analgesics and antispasmodics required by the patients, duration of hospital stay, amylase activities in serum and 24 hour urine collections. Mortality rates did not differ significantly between the glucagon-treated and the placebo-treated subjects in the total group of 69 patients and in the two subgroups of patients who were treated conservatively (n = 59) and those who underwent laparotomy because of severe peritonitis (n = 10). From the results of this study it is concluded that favourable effects of glucagon upon the course of acute pancreatitis--if they do exist--are not significant.

[Improved control of the pressure during use of the Sengstaken-Blakemore tube]. / Verbesserte Druckkontrolle bei der Anwendung der Sengstaken-Blakemore-Sonde

For the control of acute hemorrhage from esophageal varices by balloon tamponade a proper application of the Sengstaken-Blakemore tube is necessary to arrest bleeding and to minimize complications. A simple modification of the pressure measurement used up to now is described with other suitable applications. By this modified technique a correct pressure can be kept in the esophageal balloon spontaneously; its ease in handling in intensive care makes for better results in emergency management of balloon-tamponade in treatment of acute bleeding from esophageal varices.

[Current status of liver transplantation. Clinical and experimental studies. Microsurgery]. / Der gegenwärtige Stand der Lebertransplantation. Klinik-Experiment-Mikrochirurgie.

Special problems of liver transplantation were discussed on results and experiences in clinical and experimental transplantation during the last two decades. Heterotopic auxiliary transplantation in animals especially in rats by means of microsurgical techniques were pointed out. Although more than 200 orthotopic transplantations had been carried out, this procedure is still in clinical trial. Heterotopic auxiliary liver transplantation had been done almost exclusively in research with disappointing results in the early phase and different results later on. In the last years the indication for orthotopic liver transplantation was reduced to benign liver diseases with severe follow-up, originally a field for heterotopic auxiliary transplantation. In future many problems, e.g. on preservation and blood supply, regeneration and rejection or biochemical function of liver grafts must be studied in clinic and research.