Six genes encoding putative high molecular weight penicillin-binding proteins (Pbp) are present in the genome of the β-lactam-resistant strain Corynebacterium jeikeium K411. In this study, we show that pbp2c, one of these six genes, is present in resistant strains of Corynebacteriaceae but absent from sensitive strains. The molecular study of the pbp2c locus from C. jeikeium and its heterologous expression in Corynebacterium glutamicum allowed us to show that Pbp2c confers high levels of β-lactam resistance to the host and is under the control of a β-lactam-induced regulatory system encoded by two adjacent genes, jk0410 and jk0411. The detection of this inducible resistance may require up to 48 h of incubation, particularly in Corynebacterium amycolatum. Finally, the Pbp2c-expressing strains studied were resistant to all the β-lactam antibiotics tested, including carbapenems, ceftaroline, and ceftobiprole.

Coccidiosis is an important pathology for sheep, causing important economic losses. Medicated so lutions are currently used but increase of coccidian resistance and demand for natural products from consumers is conducting to development of alternative solutions. A trial was conducted on four groups of 30 Lacaune lambs to assess the activity of a mix of plant extracts, commercially named Oilis, on the control of coccidiosis. It was compared to a negative control, to decoquinate (30 mg/kg of concentrate feed) and to another phytogenic product. Animals were followed from weaning (28 days of age) to slaughtering. All evaluated products were incorporated in the concentrate feed that was offered ad libitum, only completed by straw. Growth performances were evaluated by individual weighing every three weeks. Oocysts of coccidia were sampled in faeces and counted using Mac Master Cell at 28, 55, 76 and 97 days of age. Oilis improved growth on global period (p<0.05), particularly compared to decoquinate (+4.9%) and negative control (+8.3%). It enabled also the most important reduction of oocysts excretion at peak (-62.6%).This trial confirms the potency of some natural products as alternative to chemoprevention of coccidiosis in sheep.

Nutritional programming is a very promising strategy for modifying nutrient metabolism to better adapt animals to new diets. Although it is a well-known approach in fish-farmed species, it has never been applied in marine shrimp. A preliminary requirement to perform nutritional programming is to identify the best developmental window for early stimulus. The objective of the present study was to characterize the ontogenesis of the expression of the main genes involved in digestion and metabolism in white shrimp, Litopenaeus vannamei. Four genes involved in digestion, 11 genes in intermediary metabolism (glucose, amino acid and lipid metabolism) and 5 genes in energy metabolism (mitochondria) were studied at 10 different developmental stages (from eggs – 8 h after fertilization - up to post-larval substage 5) using qRT-PCR for measuring mRNA levels. Our data show that almost all of the studied genes present higher mRNA levels during the protozoea substage Z1, which could be explained by the endogenous-exogenous feeding transition in white shrimp. In conclusion, Z1 substage shows a high molecular plasticity for metabolism which becomes the preferential developmental window for future programming experiments in L. vannamei. Our study aimed at characterizing for the first time the ontogenesis of expressions of genes involved in intermediary metabolism (nutrient metabolism). This approach determined developmental windows with high molecular plasticity (Z1 and PL stages). These data will support future studies in nutritional programming for the whiteleg shrimp by determining the timing for early stimulus.

Initial insights of the nutritional programming in shrimp : mapping of the ontogeny metabolic gene expression in whiteleg shrimp (Litopenaeus vannamei). Larvi 2017. 7. Fish and shellfish larviculture symposium

A trial was conducted to determine the effect of 6% hydrolysed wheat gluten (HWG) inclusion in a low-fishmeal diet on growth performance and intestinal microbiota and morphology of Asian seabass (Lates calcarifer). Fish (initial average weight of 36.3g) were allocated into floating cages (eight replicates per diet, 30 fish per cage). They were fed either a fishmeal-based diet (positive, diet code: POS; 36% fishmeal), or a diet based on a mix of animal and plant proteins (negative, diet code: NEG). The fishmeal inclusion rate in this diet was reduced to 6%, with the major protein sources being soybean meal and animal by-products. The third diet (diet code: HWG) was similar to the NEG diet with the exception that 6% HWG was included in replacement of the other proteins. At the end of the 48day trial, fish were sampled for intestinal microbiology and histology. Growth parameters were also assessed. Final body weight, SGR and daily feed intake were not significantly different across diets. However, the feeding rate was significantly affected, with the lowest rate observed in the POS treatment and the highest with the NEG treatment. The FCR was significantly lower for both POS and HWG fed fish than for the NEG fed fish. High throughput sequencing revealed that the majority of reads derived from the mucosa samples belonged to members of Proteobacteria (70.3% of the reads), Cyanobacteria (10.0%) and Firmicutes (7.6%). In the digesta reads were mainly assigned to Proteobacteria (34.5%), Fusobacteria (34.5%), and Firmicutes (22.6%). The alpha diversity did not differ among dietary treatments. Some differences in OTU relative abundances were obtained between diets, however, the overall community was not modified to a large extent by HWG. Histological appraisal revealed that the HWG fed fish exhibited significantly higher posterior intestinal perimeter ratio than that of the POS treatment. Overall, including HWG in a low fishmeal diet positively affects feed efficiency. Concomitantly the absorptive surface area of the posterior intestine was improved while the intestinal microbiota, described comprehensively here for the first time in Asian seabass, was similar to the microbiota of other healthy carnivorous marine fish species. The mechanisms involved in these changes may be related to the high glutamine content and to the high protein digestibility of HWG.

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and 13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

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.

越来越多的国家禁止抗生素生长促进剂的使用,使得养殖业面临肠道微生物病原过度增殖的风险更高,这些病原会导致肠道功能紊乱、垫料潮湿、死亡率增加和以及生产性能改变.因此,需要寻求一种能控制肠道微生物的替代产品.已经有多种作为添加剂使用的抗菌方案,包括直接抗菌产品,如酸化剂或植物提取物,或间接抗菌产品,如益生菌和益生素.

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219 , encoding a 314 residue NAD + -dependent l -(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA -null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator d -fructose-1,6-bisphosphate (Fru-1,6-P 2 ). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with V max for the reduction of pyruvate being approximately tenfold that of the value for l -lactate oxidation at pH 7.5. No deviation from Michaelis–Menten kinetics was observed in the presence of Fru-1,6-P 2 , while a sigmoidal response (indicative of positive cooperativity) was seen towards l -lactate without Fru-1,6-P 2 . Strikingly, when introduced into an lldD − strain of C. glutamicum , constitutively expressed CgLDH enables the organism to grow on l -lactate as the sole carbon source.

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm2, while non-transformed cells tolerated only up to dose 0.08 J/cm2. It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.

Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding.

The aim of the present study was to check the inter es of forcing the water to feed ratio at 1.7 when rationing rabbits in good sanitary conditions (with out experimental reproduction of Epizootic Rabbit Enteropathy) and in Epizootic Rabbit Enteropathy co nditions. Three hundred thirty six rabbits, housed in cages each containing 8 rabbits, were divided in to 7 groups controlled from weaning at 32 days of age to 67 days. In the control group, rabbits were giv n feed and water ad libitum. In the second group, feed was restricted, but not water (water/fe d ratio >3), and in the last group, feed and water were restricted (water/feed ratio=1.7). In good san itary conditions, the slaughtering weight was highe r for the control group as the compensatory growth wa s not sufficient to compensate the body weight difference due to restrictions. There was no differ ence between the two ways of restriction during the fattening period. Except for the 1 st week (adaptation to restriction) the feed conversi on ratio was improved for groups restricted in feed and/or water . Quantitative feed restriction is interesting in Epizootic Rabbit Enteropathy conditions as it leads to less mortality and morbidity compared to the control group ( ad libitum). There was no significant effect of the type of q uantitative feed restriction (with or without water restriction) on mortality, b ut feed restriction with water ad libitum tended to be more efficient to reduce mortality (from 29.2 to 16 .7). Moreover, this restriction did not generate a difference in slaughtering weight compared to the c ontrol group and produced more meat (+18%).

A wide range of alternative products proposed to feed manufacturers to replace the antibiotic growth promoters in feeds , have been tested between 2000 and 2004 . Commercial diets supplemented with different alternatives ( acidifiers , prebiotics , probiotics , essential oils and vegetal extracts , others ) at different levels from day 1 to day 28 have been compared , alone or in combination , to a negative control and a positive control ( avilamycin 10 ppm ) in 8 experiments on broilers . These results indicate that in the good environmental conditions of an experimental farm , the antibiotic growth promoter and its substitutes can diversely affect the performances in comparison with the negative control , the average effects being rather low . Avilamycin being mostly active against gram-positive bacteria , these observations could be related to differences in the digestive flora of the birds in the different trials . These data confirm that the selection of susbtitutes is difficult to achieve in good sanitary conditions . The use of more discriminating experimental models can be recommanded , either on the field , or by the use of specific models , eventually with microbial challenges , to worse the sanitary conditions and simulate field conditions ; or by a combination of both.

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).

ABSTRACT A “second-generation” production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, dl-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Regulation of gene expression in Corynebacterium glutamicum represents an important issue since this Gram-positive bacterium is a notable industrial amino acid producer. Transcription initiation, beginning by binding of RNA polymerase to the promoter DNA sequence, is one of the main points at which bacterial gene expression is regulated. More than 50 transcriptional promoters have so far been experimentally localized in C. glutamicum. Most of them are assumed to be promoters of vegetative genes recognized by the main sigma factor. Although transcription initiation rate defined by many of these promoters may be affected by transcription factors, which activate or repress their function, the promoter regions share common sequence features, which may be generalized in a consensus sequence. In the consensus C. glutamicum promoter, the prominent feature is a conserved extended −10 region tgngnTA(c/t)aaTgg, while the −35 region is much less conserved. Some commonly utilized heterologous promoters were shown to drive strong gene expression in C. glutamicum. Conversely, some C. glutamicum promoters were found to function in Escherichia coli and in other bacteria. These observations suggest that C. glutamicum promoters functionally conform with the common bacterial promoter scheme, although they differ in some sequence structures.