Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli.

The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2(4) factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 gl-1 to 1.9 gl-1 (dry weight) and from 10 units ml-1 to 210 units ml-1 within 24 h at 30 degrees C in shake flasks.

Response Surface Models To Describe the Effects of Temperature, pH, and Ethanol Concentration on Growth Kinetics and Fermentation End Products of a Pectinatus sp.

Growth curve data which had been fitted by use of the Gompertz and logistic functions have permitted the development of mathematical models to describe the growth of a Pectinatus sp. by several variables, namely, temperature, pH, and ethanol concentration. The activation energy of this microorganism was lower at 26 to 35(deg)C than at 15 to 22(deg)C. On the basis of the Arrhenius law, growth rate, maximum population density, and cell yield models have been developed by introducing the different activation energy (E(infa)) values. According to the model, optimal conditions were 35(deg)C, pH 6.5, and 0% (vol/vol) ethanol for the growth rate. For cell density and cell yield, optimal conditions were 32(deg)C, pH 6.0, and 1% (vol/vol) ethanol. No growth was observed for ethanol concentrations above 8% and pH values below 4.0. Other equations have also been made to describe the major end products fermented during fermentation by a Pectinatus sp. The synthesis of propionate and acetate is maximal at 28(deg)C at pHs of 5.5 and 6.25, respectively. This model completes the model suggested by Membre and Tholozan (J. Appl. Bacteriol. 77:456-460, 1994), which includes only one variable, i.e., the glucose concentration.

Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron strain 217 can use pectins as a sole carbon source. Preliminary characterization of the pectinolytic enzymes revealed three complementary activities in this strain: a pectin methylesterase (PME), a pectate lyase (PL) and a polygalacturonase (PG), which were all inducible by pectin or polygalacturonate. Use of the lambdoid phage replacement vector lambda EMBL3 allowed a 13.2 kb insert mediating both PL and PME activities to be isolated. Subcloning of two EcoRI fragments in pBR325 led to the separate isolation of the pel and pme genes. They were expressed constitutively in Escherichia coli HB101, as proved by the activities observed even in mineral medium supplemented only with glucose. In addition, the pme gene was expressed in both orientations. These results suggest that each gene represents an individual transcriptional unit. Several properties of the cloned PL were different from those of the original strain: it was mainly associated to the outer membrane, its optimum pH was higher, and its stability at 50 degrees C was lost but partially preserved by CaCl2. In addition, the apparent specific PL activity in the E. coli membrane fraction was about 30-fold higher. On the other hand, most of the properties of the cloned PME were similar to those of the original. Despite an enhanced thermostability, the apparent specific activity of the cloned PME was about 6-fold lower, and was independent of the insert orientation.

Transfer of hybrid plasmids based on the replicon pRRI7 from Escherichia coli to Bacteroides and Prevotella strains.

New shuttle vectors based on a Prevotella ruminicola 9.5 kb cryptic plasmid (pRRI7) inserted within the Escherichia coli vector pKC71, carrying the Ccr/Emr Bacteroides marker, were constructed. These constructs (pKBR23-1 and pKBR23-2) were transferred into Bacteriodes distasonis, Bacteroides thetaiotaomicron, Bacteroides uniformis and into P. ruminicola NCFB 2202 either by conjugal mobilization or by electroporation. Another pRRI7 derivative based on pKC72, pKBR23-3, was smaller (13.1 kb) and non-mobilizable. By electroporation, it was transferred to Bact. distasonis and P. ruminicola. Being derived from pRRI7 which is compatible with the shuttle plasmid pRRI207, the host/vector combination involving P. ruminicola NCFB 2202 and pKBR23-3 offers new possibilities for genetic investigations in rumen anaerobic bacteria after further introduction of a second readily selectable marker within pRRI207 or pKBR23-3.

Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron.

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.

Trophic relationships between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate.

Glucose and citrate are two major carbon sources in fruits or fruit juices such as orange juice. Their metabolism and the microorganisms involved in their degradation were studied by inoculating with an aliquot of fermented orange juice a synthetic model medium containing glucose and citrate. At pH 3.6, their degradation led, first, to the formation of ethanol due to the activity of yeasts fermenting glucose and, eventually, to the formation of acetate resulting from the activity of lactobacilli. The yeast population always outcompeted the lactobacilli even when the fermented orange juice used as inoculum was mixed with fermented beet leaves containing a wider variety of lactic acid bacteria. The evolution of the medium remained similar between pH 3.3 and 5.0. At pH 3.0 or below, the fermentation of citrate was totally inhibited. Saccharomyces cerevisiae and Lactobacillus plantarum were identified as the only dominant microorganisms. The evolution of the model medium with the complex microbial community was successfully reconstituted with a defined coculture of S. cerevisiae and L. plantarum. The study of the fermentation of the defined model medium with a reconstituted microbial community allows us to better understand the behavior not only of fermented orange juice but also of many other fruit fermentations utilized for the production of alcoholic beverages.

Fermentation of citrate by Lactobacillus plantarum in the presence of a yeast under acid conditions.

At pH 3.6, Lactobacillus plantarum is unable to grow on citrate or to ferment it in the absence of another carbon source such as glucose. In a defined medium containing glucose and citrate, with a higher concentration of the former than the latter, as in many fermented alcoholic beverages, L. plantarum will first ferment the sugar. The production of lactate from glucose degradation increases the acidity of the medium and inhibits the fermentation of citrate. In co-culture with Saccharomyces cerevisiae, part of the glucose is fermented by the yeast, partly avoiding the pH drop and the inhibition of citrate fermentation by L. plantarum. Fermentation was still possible at pH values around 3.0.

Reductive carboxylation of propionate to butyrate in methanogenic ecosystems.

During the batch degradation of sodium propionate by the anaerobic sludge from an industrial digestor, we observed a significant amount of butyrate formation. Varying the initial propionate concentrations did not alter the ratio of maximal butyrate accumulation to initial propionate concentration within a large range. By measuring the decrease in the radioactivity of [1-C]butyrate during propionate degradation, we estimated that about 20% of the propionate was converted to butyrate. Labeled butyrate was formed from [1-C]propionate with the same specific radioactivity, suggesting a possible direct pathway from propionate to butyrate. We confirmed this hypothesis by nuclear magnetic resonance studies with [C]propionate. The results showed that [1-C]-, [2-C]-, and [3-C]propionate were converted to [2-C]-, [3-C]-, and [4-C]butyrate, respectively, demonstrating the direct carboxylation on the carboxyl group of propionate without randomization of the other two carbons. In addition, we observed an exchange reaction between C-2 and C-3 of the propionate, indicating that acetogensis may proceed through a randomizing pathway. The physiological significance and importance of various metabolic pathways involved in propionate degradation are discussed, and an unusual pathway of butyrate synthesis is proposed.

Septicaemic Escherichia coli and experimental infection of calves.

Three strains of Escherichia coli with a common surface antigen, 31a, capable of adhering to calf enterocytes in vitro were compared to reference strains of septicaemic E. coli (RVC 330 and vir E. coli). The surface antigen 31a was present in the RVC 330 reference strain. E. coli vir had a surface antigen which was not present in E. coli 31a or E. coli RVC 330. The RVC 330 and vir reference strains also adhered to calf enterocytes in vitro. Oral infection of calves not receiving colostrum with E. coli 31a was generally followed by septicaemia and death in less than 48 h. Post-mortem examination revealed pneumonia and oedema of the kidneys and gall bladder. Oral infection of calves receiving colostrum had no effect, but intravenous inoculation produced arthritis within 15 days. The comparison of these results with those previously described by other workers did not lead to the identification of pathognomonic characteristics, which could be clearly correlated with properties specific to E. coli 31a. It is suggested that, like ColV and vir, antigen 31a may be a virulence marker for certain strains of bovine septicaemic E. coli. Furthermore, the 31a antigen appears to be carried on a plasmid.

[Interaction among anaerobic microbe species in the rumen]. / Interactions entre espèces microbiennes anaérobies dans le rumen.

The rumen is a strictly anaerobic ecosystem colonized by an extremely dense microflora and microfauna. These populations are composed of a grand variety of bacterial and protozoal species. Their cohabitation allows observation of most of the known stages of communal life. The different hydrolytic, fermentative and methanogenic activities of these populations ensure the efficient degradation of cell wall constituent in forages (cellulose, hemicellulose, pectin) ingested by ruminants.

[Anti-K99 vaccination and colostric protection of calves infected experimentally with Escherichia coli K99]. / Vaccination anti-K99 et protection colostrale des veaux infectés expérimentalement avec Escherichia coli K99.

Colostral antibodies of cows vaccinated with E. coli B41 (O101: K99, F41) protect completely B41 experimentally infected calves. In order to know more precisely the role of K99, F41 antibodies in protection, calves receiving the colostrum of B41 vaccinated cows are infected with E. coli B44 (O9: K30: H-K99, F41). Vaccination multiplies K99 antibodies in colostrum by seven. In B44 infected calves, specific K99, F41 antibodies protect only 3 out of 6 calves completely. Additive effect of antibodies against K99 and other surface antigens of enterotoxigenic E. coli seems desirable for a more complete protection.

Homoacetogenic Fermentation of Cellulose by a Coculture of Clostridium thermocellum and Acetogenium kivui.

Interrelationships between methanogens and fermentative or hydrolytic bacteria are well documented; however, such cocultures do not allow a complete fermentation shift to a peculiar metabolite. We describe here a new stable association between Clostridium thermocellum and Acetogenium kivui which converts 1 mol of cellulose (anhydroglucose equivalent) into 2.7 mol of acetate.

Effect of glucose and amino acids on expression of K99 antigen in Escherichia coli.

K99 antigen production by enterotoxigenic Escherichia coli strains of bovine origin was investigated by slide agglutination and in vitro attachment to intestinal villi. Work with two strains (B41 and B44) showed that on minimal medium M2, K99 antigen was not repressed by a high concentration of glucose (2%, w/v). Growth on synthetic or complex medium did not affect K99 antigen detection, which was independent of capsular antigens, and its synthesis was not repressed by Casamino acids or glucose. A survey of 12 strains revealed two groups: in one group K99 antigen production was constitutive on basal medium without glucose, and in the second group K99 antigen was produced only in the presence of glucose. Immunoelectrophoresis patterns, and the results of slide agglutination and attachment tests, were dependent upon K99 type, whereas haemagglutination patterns were not.

Inhibition of K99 antigen synthesis by L-alanine enterotoxigenic Escherichia coli.

The effect of various culture media on K99 antigen production by enterotoxigenic Escherichia coli strains of bovine origin was investigated by slide agglutination tests, enzyme-linked immunosorbent assays and in vitro attachment to intestinal villi. A-Alanine at concentrations higher than 0.7 g 1(-1) was responsible for the inhibition of K99 synthesis observed on media rich in amino acids. The increased inhibitory activity of L-alanine in glucose-rich media after autoclaving suggested the formation of inhibitory products via Maillard's reaction. Of various L-alanine derivatives tested, only those that hydrolysed to L-alanine were inhibitory. L-Alanine analogues were without effect and the addition of 10 mM-cyclic AMP did not overcome the repression of K99 biosynthesis by L-alanine. Enzymes involved in cell wall synthesis such as L-alanine racemase or D-alanyl-D-alanine synthetase were evidently were evidently unaffected by L-alanine.

[Incidence of rotavirus infection and in combined infection of rotavirus and enteropathogenic Escherichia coli in French calves (author's transl)]. / Fréquence du rotavirus et de l' association rotavirus et Escherichia coli K99+ chez des veaux de quelques départements français.

Detection of a rotavirus in faeces of 789 calves developing diarrhoea gave positive results among 48% of the calves. The same investigation extended to 96 apparently healthy animals shows that 12.5% of the faeces contained rotavirus. It appears that all the last ones cannot be considered as healthy controls. Association of rotavirus and E. coli K99+ is found in 5% of sick animals less than 10 days old.

[Frequence of K99 antigen and antibioresistance in Escherichia coli from calves in France (author's transl)]. / Fréquence de l'antigène K99 antibiorésistance chez Escherichia coli d'origine bovine en France.

During the winter of 1979-1980, an epizootiological study of diarrhoeic calves revealed the presence of K99+ E. coli among 8.2 p. cent of clinically healthy calves and in 18.9 p. cent of diseases calves. Some calves which seemed healthy on the day of sampling possibly became diarrhoeic on the following days. In diarrhoeic calves, K99+ E. coli were mainly found during the early life, i.e. in 33.7 p. cent of calves less than 4 days old. These results were obtained with 147 healthy calves and 1053 diarrhoeic calves. They confirmed previous results obtained with more limited numbers of animals. Moreover, K99+ E. coli were found in all breeding systems. Antibiotics resistance among the isolated E. coli were very high, especially in K99+ strains. The results proved the interest of E. coli K99 diagnosis in liquid diarrhoea of very young calves.

Specific protection by colostrum from cows vaccinated with the K 99 antigen in newborn calves experimentally infected with E. coli Ent+ K99+.

We have measured the protective effect of colostrum of vaccinated cows with the K99 antigen comparatively with the colostrum of non vaccinated cows, in calves receiving at birth 2 liters of colostrum and just after 5 times 10(10) to 1 times 10(11) E. coli Ent+, K99+. The K99 antigen was prepared from E coli B41 (0101:K99:H-) cultured in a fermentor. Cows were vaccinated subcutaneously with 100 mg or 300 mg (wet weight) in emulsion in Freund uncomplete adjuvant 45 and 15 days before parturition. Of 4 calves receiving a colostrum of non vaccinated cows 4 had a diarrhoea, 3 became dehydrated and 2 died. Of 6 calves receiving colostrum of vaccinated cows, 5 were healthy and the 6th calf had a mild diarrhoea for few hours and became again healthy. Control and protected calves excreted E. coli K99+, 24 HOURS AFTER INFECTION. The protection is probably due to K99 antibodies which inhibit adhesion of E. coli K99+ to the intestinal epithelium.

[Diarrhea of the newborn animal: nature and mechanism of action of enteropathogenic Escherichia coli in the calf and piglet]. / Diarrhée du nouveau-né: propriétés et mécanismes d'action des Escherichia coli entéropathogènes chez le veau et le porcelet.

The present knowledge of the enteropathogenic characteristics of Escherichia coli (adhesion and toxinogenesis) is reviewed in the calf, and compared with piglet data. A pathogenic model of E. coli diarrhoea in the calf is proposed, taking into account the electromyographic, bacteriologic and physiologic data.

The experimental production of diarrhoea in colostrum deprived axenic and gnotoxenic calves with enteropathogenic Escherichia coli, rotavirus, coronavirus and in a combined infection of rotavirus and E. coli.

We attempted to produce diarrhoea experimentally in the newborn calf by orally injecting 17 colostrum-deprived calves with two serotypes of Escherichia coli Ent+ K99+, a rotavirus and a coronavirus. With E. coli alone, a dose of 2 x 10(8) bacteria administered 24 hours after birth causes a mild attack of diarrhoea, whereas 1 x 10(10) bacteria leads to dehydration and death. An inoculation of rotavirus is followed by diarrhoea which always contains large quantities of rotavirus. These animals were anorectic for a time, but none was dehydrated or died. With coronavirus, there were large quantities of watery diarrhoea, which led to dehydration and death. The inoculation of rotavirus, not lethal in itself, followed by a similarly non lethal inoculation of E. coli in doses of 3 x 10(8) to 2 x 10(9) led to dehydration and death. The authors conclude that dehydration and death of the animal can caused by large doses of E. coli or coronavirus or by two non-lethal doses of rotavirus and E. coli administered one after the other.