Specific and non-specific immunity of piglets from sows fed diets containing specific fatty acids in field conditions.

The transfer of passive immunity from sows to piglets is important and it is the first immune protection of the new born piglet. Improving sows immunity by adding immuno-stimulating product in sows diet can positively affect colostrum composition and transfer of immune molecules to piglets. The aim of the current study is to evaluate the benefit of a different solution, made of specific fatty acids from marine origin that have been used in human medicine for decades, for sows and piglets. Two trials were conducted in commercial farm, involving 240 sows at different periods of the year. Sows were divided in a control group, without supplementation, and a test group, supplemented with the feed additive from the 90th day of gestation to weaning. Sows body condition, piglets viability and growth were recorded. Milk immunoglobulin content was measured, as well as Aujeszky antibodies in sows and piglets blood as marker of specific immunity, and blood bactericidal activity, complement activity and lysozyme as markers of non specific immunity. No effect of the product was observed on piglets zootechnical criteria and specific immunity parameters but significant improvement of piglet non specific immunity, was observed. No difference was observed neither in the piglets blood PRRSV and PCV2 antibodies and viruses nor in Aujeszky antibodies. Blood complement activity seems to be an accurate indicator of immuno-stimulating additive efficiency. Giving alkyl-glycerol fatty acids to sows in late gestation and lactation can improve the passive immunity transfer to piglets.

Corynebacterium glutamicum superoxide dismutase is a manganese-strict non-cambialistic enzyme in vitro.

Superoxide dismutase (SOD) of Corynebacterium glutamicum was purified and characterized. The enzyme had a native molecular weight of about 80kDa, whereas a monomer with molecular weight of 24kDa was found on SDS-PAGE suggesting it to be homotetramer. The native SOD activity stained gel revealed a unique cytosolic enzyme. Supplementing growth media with manganese increased the specific activity significantly, while adding iron did not result in significant difference. No growth perturbation was observed with the supplemented media. In vitro metal removal and replacement studies revealed conservation of about 85% of the specific activity by substitution with manganese, while substitution with copper, iron, nickel or zinc did not restore any significant specific activity. Manganese was identified by atomic absorption spectrometer, while no signals corresponding to fixing other metallic elements were detected. Thus, C. glutamicum SOD could be considered a strict (non-cambialistic) manganese superoxide dismutase (MnSOD).

Postprandial kinetics of some biotic and abiotic characteristics of the gastric ecosystem of horses fed a pelleted concentrate meal.

Our knowledge of the microflora of the stomach of the horse is still limited, although some data indicate its important role in nutrition. The objective of this experiment was to investigate the microbial and biochemical profiles in the stomach of the horse and to quantify the disappearance of dietary starch. Total anaerobic bacteria, lactate-utilizing bacteria, lactobacilli, and streptococci were determined, and biochemical characteristics (pH, and DM, D- and L-lactate, D-glucose, NH3, and VFA concentrations) were measured in chyme collected from 4 horses by naso-gastric intubation aided by endoscopy, at 30 min before and 60, 120, and 210 min after the meal. The total anaerobic population exhibited a linear increase (5.54 to 6.98 log10 cfu/mL; P = 0.018) within the first postprandial hour and reached 8.32 log10 cfu/mL at 210 min after the meal. The concentrations of lactobacilli, streptococci, and lactate-utilizing bacteria in the stomach contents were 5.52, 4.82, and 6.95 log10 cfu/mL, respectively. Lactate concentration increased linearly from 0.25 mmol/L before the meal to 7.98 mmol/L at the last collection point (P = 0.013). This increase was mostly due to L-lactate accumulation. The VFA concentration increased linearly (P = 0.002) during the postprandial period from 1.96 to 8.17 mmol/L. Acetate represented, on average, 78 mol/100 mol of total VFA. The average concentration of NH3 in the stomach content was 2.48 mmol/L. Dietary starch disappearance did not respond during the post-prandial period and was not consistent with previous findings. These in vivo data provide complementary information on the postprandial microbial and biochemical kinetics in the stomachs of horses and confirm its abundant microbial colonization.

Cloning of the sodA gene from Corynebacterium melassecola and role of superoxide dismutase in cellular viability.

The sodA gene encoding the Corynebacterium melassecola manganese-cofactored superoxide dismutase (SOD) has been cloned in Escherichia coli and sequenced. The gene is transcribed monocistronically; the predicted polypeptide is 200 amino acids long and associates in a homotetrameric, manganese-dependent form, able to complement an SOD-deficient E. coli mutant. A second open reading frame, coding for a putative 217-amino-acid protein with high homology to peptide methionine sulfoxide reductases from various origins, has been identified immediately upstream of sodA in the opposite transcription orientation. The sodA gene was inactivated by insertion of an integrative vector carrying a kanamycin resistance gene. The growth rate of the SOD-deficient integrant was only slightly affected in BHI rich medium as well as in BMCG chemically defined medium, but was strongly affected by the presence of the redox-cycling agent paraquat. The SOD deficiency had, on the other hand, a deleterious effect on viability as soon as the culture entered the stationary phase of growth in BHI medium. Surprisingly, SOD deficiency was able to rescue the dramatic loss of viability observed for the wild-type strain in BMCG synthetic medium when glucose was not the limiting growth factor.

Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism.

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (Delta malE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.

Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation.

To give clues about the respective importance of phosphoenol-pyruvate carboxylase (PEPc) and pyruvate carboxylase (Pc) in Corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, PEPc activity was genetically amplified and Pc activity was suppressed by biotin limitation in the culture medium. In absence of Pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. Whereas PEPc amplification in excess of biotin (4 mg/L) only slightly modified the cell kinetics, under biotin limiting conditions this amplification strongly improved the glutamate production (4 microg/L). When Pc was absent, PEPc activity was sufficient to allow up to 70% of the maximal glutamate production rate and seemed to have an important anaplerotic role, especially at the beginning of the production phase. In contrast, Pc was predominant during the remainder of the glutamate fermentation.

Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose.

Growth of Corynebacterium glutamicum on fructose was significantly less than that obtained on glucose, despite similar rates of substrate uptake. This was in part due to the production of overflow metabolites (dihydroxyacetone and lactate) but also to the increased production of CO2 during growth on fructose. These differences in carbon-metabolite accumulation are indicative of a different pattern of carbon-flux distribution through the central metabolic pathways. Growth on glucose has been previously shown to involve a high flux (> 50% of total glucose consumption) via the pentose pathway to generate anabolic reducing equivalents. NMR analysis of carbon-isotope distribution patterns of the glutamate pool after growth on 1-13C- or 6-13C-enriched fructose indicates that the contribution of the pentose pathway is significantly diminished during exponential growth on fructose with glycolysis being the predominant pathway (80% of total fructose consumption). The increased flux through glycolysis during growth on fructose is associated with an increased NADH/NAD+ ratio susceptible to inhibit both glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenase, and provoking the overflow of metabolites derived from the substrates of these two enzymes. The biomass yield observed experimentally is higher than can be estimated from the apparent quantity of NADPH associated with the pentose pathway and the flux through isocitrate dehydrogenase, suggesting an additional reaction yielding NADPH. This may involve a modified tricarboxylic acid cycle involving malic enzyme, expressed to significantly higher levels during growth on fructose than on glucose, and a pyruvate carboxylating anaplerotic enzyme.

Growth Rate-Dependent Modulation of Carbon Flux through Central Metabolism and the Kinetic Consequences for Glucose-Limited Chemostat Cultures of Corynebacterium glutamicum.

The physiological behavior of Corynebacterium glutamicum in glucose-limited chemostat cultures was examined from both growth kinetics and enzymatic viewpoints. Metabolic fluxes within the central metabolism were calculated from growth kinetics and analyzed in relation to specific enzyme activities. At high growth rates, incomplete glucose removal was observed, and this was attributed to rate-limiting capacity of the phosphotransferase system transporter and the probable contribution of a low-affinity permease uptake mechanism. The improved biomass yield observed at high growth rates was related to a shift in the profile of anaplerotic carboxylation reactions, with pyruvate carboxylase replacing malic enzyme. Phosphoenolpyruvate carboxylase, an activity often assumed to be the major anaplerotic reaction during growth of C. glutamicum on glucose, was present at only low levels and is unlikely to contribute significantly to tricarboxylic acid cycle fuelling other than at low growth rates.

13C-NMR studies of Corynebacterium melassecola metabolic pathways.

Coryneform bacteria are widely used to produce amino acids, in particularly glutamic acid, by fermentation. To study the metabolic fate of glucose as the carbon source, we developed a method to analyze intracellular extracts by NMR and HPLC. The intracellular metabolites represent the metabolic state of the cells. Glutamic acid was the major metabolic intermediate found in the extracts and its 13C isotopic enrichment reflected that of pyruvic acid. Thus, it was possible to determine the respective contributions of the two major glucose catabolic pathways during the exponential growth phase; glycolysis (55%) and the pentose phosphate pathway (45%). Absolute glutamate 13C enrichments resulting from the incorporation of [1-13C]glucose were determined to quantify the contribution of several metabolic pathways such as anaplerotic pathways (61%; phosphoenolpyruvate carboxylase, pyruvate carboxylase, malic enzyme), a single turn (32%) or multiple turns of the Krebs cycle and the glyoxylate shunt, to oxaloacetate synthesis. A previously described model was adapted to C. melassecola for these calculations. The Krebs cycle was active, whereas the glyoxylate shunt was inactive in exponentially growing cells of C. melassecola with glucose as the sole carbon source. The contributions of anaplerotic enzymes and pyruvate dehydrogenase to replenishing the Krebs' cycle were determined to be 38% and 62%, respectively.

Gene replacement, integration, and amplification at the gdhA locus of Corynebacterium glutamicum.

Gene replacement and integration in a Corynebacterium glutamicum ATCC 21086 derivative were achieved by transformation with a nonreplicative plasmid that contains the C. glutamicum ATCC 17965 gdhA gene modified by the insertion of an aphIII cartridge. We isolated rare derivatives of the integrative transformants that have higher levels of expression of the integrated plasmid genes than the parent. Different types of such amplified clones were distinguished according to their antibiotic resistance levels, enzyme specific activities, and physical structures. All amplified clones share a structural DNA motif confined to the chromosomal gdhA locus: a variable number (up to 10) of tandem copies of a unit that includes the selected gene and one flanking repeat. A given clone contains subpopulations that differ in the number of repeats of this unit.

'Integron'-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria.

A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli gltA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called 'integron', that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.

Cloning and structure of the pyrE gene of Lactobacillus plantarum CCM 1904.

The pyrE gene of Lactobacillus plantarum CCM 1904, coding for the orotate phosphoribosyl transferase involved in the pyrimidine biosynthetic pathway, was cloned in Escherichia coli and sequenced. The predicted polypeptide sequence extending over 212 amino acids (MW 22,690) was compared to those of E. coli and to those of lower eukaryotes (Saccharomyces cerevisiae, Podospora anserina, Sordaria macrospora, Dictyostelium discoideum). Important conserved stretches were revealed, implying that these proteins are closely related.

Interspecies electro-transformation in Corynebacteria.

Plasmid DNA was efficiently electro-transformed into intact cells of nine Corynebacteria strains belonging to Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum and Corynebacterium melassecola. Relationships were explored between transformation efficiency and parameters such as electric field strength and pulse length, DNA concentration, physiological state and concentration of the cells. In optimal conditions, more than 10(7) transformants per microgram of DNA could be obtained. Electro-transformation with plasmid DNA isolated from different sources indicates that DNA modification may play a role in transformation efficiency.

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904.

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa.

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.

Structure of the Saccharomyces cerevisiae URA4 gene encoding dihydroorotase.

The URA4 gene of Saccharomyces cerevisiae, coding for the third enzyme of the pyrimidine pathway, has been cloned through phenotypic complementation of a ura4 mutant of S. cerevisiae. Subcloning of an original 9 kb DNA fragment, carrying the yeast URA4 gene, allowed us to localize the gene on a 2 kb ClaI--BamHI fragment. The sequence of the URA4 structural gene and surrounding DNA was determined by the dideoxynucleotide chain termination method. The URA4 gene encodes a dihydroorotase subunit of calculated molecular weight 40,600. S1 nuclease mapping indicated that transcription of URA4 is initiated at four major start sites located at positions -41, -30, -22 and -18. A set of potentially significant sequences was identified in the 5' OH non-coding region of the gene. The deduced amino acid sequence of dihydroorotase was examined and compared with homologous amino acid sequences of Salmonella typhimurium, Escherichia coli and Drosophila melanogaster. S. cerevisiae dihydroorotase shows 40% homology with the S. typhimurium and E. coli enzymes and 23% homology with the D. melanogaster enzyme. A potential active site has been predicted for dihydroorotase from these comparisons.

Yeast regulatory gene PPR1. I. Nucleotide sequence, restriction map and codon usage.

The PPR1 gene of Saccharomyces cerevisiae controls the transcription of two unlinked structural genes URA1 and URA3. The primary structure of this eukaryotic regulatory gene and its flanking regions has been established by the dideoxynucleotide chain termination method. Our data show an open reading frame of 2712 nucleotides, corresponding to 904 amino acid residues. The 3' untranslated messenger RNA region presents consensus yeast termination and polyadenylation sequences. The pattern of codon usage in the gene is clearly random. This result is discussed in relation to protein abundance and is compared with the codon usage in 20 yeast structural and regulatory genes and with that found for Escherichia coli genes.

Complete sequence of a eukaryotic regulatory gene.

Dihydroorotase, the third enzymatic activity of the pyrimidine pathway, is encoded in Saccharomyces cerevisiae by a single gene URA4, which is induced at the transcriptional level by accumulation of ureidosuccinic acid. A regulatory gene PPR2 (pyrimidine pathway regulatory 2) acting specifically on this step, has been characterized, cloned and sequenced. The main open reading frame is 384 nucleotides long and potentially codes for a basic protein, favoring a molecular mechanism involving direct binding of a regulatory protein to DNA. The short length of the PPR2 polypeptide chain and the presence of seven cysteine residues suggest that the active form of the protein is an oligomer assembled through disulphide bonds. An uninducible allele has been cloned and sequenced. The mutation corresponds to an A leads to T transversion changing a lysine triplet into an ochre codon. The uninducible phenotype of this mutant is completely suppressed by an ochre suppressor, strengthening the hypothesis that PPR2 acts on URA4 transcription through the synthesis of a regulatory protein.