Effects of improved early-life conditions on health, welfare, and performance of pigs raised on a conventional farm.

Nowadays, most pigs are raised indoors, on intensive farms providing a poor environment. In these conditions, the risk of the occurrence of damaging behaviours is high, with dramatic consequences for animal health and welfare as well as economic losses for farmers. Early-life conditions may predispose individuals to develop damaging behaviours later in life. In contrast, reinforcing affiliative behaviours between piglets before weaning might help to prevent tail-biting episodes. In this field study, we aimed at improving early-life conditions of piglets on a commercial farm by completely suppressing painful procedures and staggering their exposure to weaning stress factors. The alternative early-life management strategy combined housing in free-farrowing pens with temporary crating of the sow, socialisation during the lactation period with whole-life maintenance of the hierarchical groups, and delayed transfer to the postweaning room after sow removal. Control conditions included birth in farrowing crates, tail docking, absence of socialisation during the lactation period, abrupt weaning with immediate transfer to the postweaning room and mixing with non-littermates. We evaluated the health, welfare, and performance of alternatively raised pigs (n = 80) as compared to controls (n = 75). Visits were made throughout the lifespan of individuals to evaluate their growth and health status. Body and tail lesions were scored as proxy measures of aggressiveness and impaired welfare. Blood and bristle samples were periodically collected to evaluate stress, inflammation and immune competence. While the whole-life performance of pigs was similar among groups, the alternative early-life conditions prevented the growth slowdown usually observed after weaning. In addition, alternatively raised pigs displayed more neutrophils, eosinophils and monocytes the day after weaning, as well as higher C-Reactive Protein levels. One week later, their monocytes displayed greater phagocytic capacity. Altogether, these data suggest an enhanced innate immune competence for alternatively raised pigs around weaning. Piglets reared under alternative conditions also exhibited fewer and less severe body lesions than standard pigs, one week after weaning. In contrast, they showed more tail lesions on days 36 and 66 associated with greater levels of acute phase proteins (C-Reactive Protein and haptoglobin). To conclude, alternative early-life management better prepared piglets for weaning. However, the whole-life maintenance of early-established social groups was not sufficient to prevent the occurrence of damaging behaviours in undocked pigs.

Frequencies of milk protein variants and haplotypes estimated from genotypes of more than 1 million bulls and cows of 12 French cattle breeds.

Due to their major effects on milk composition and cheese-making properties and their putative effects on human health, there is a great deal of interest in bovine milk protein variants. The objectives of this study were to estimate frequencies of milk protein variants and haplotypes in 12 cattle breeds as well as their trends over time to assess the effect of selection on milk traits. Milk protein variants and haplotypes were identified from SNP genotype data from more than 1 million animals from 12 dairy, beef, or dual-purpose cattle breeds that had been genotyped for genomic selection. We examined a total of 15 loci in the genes that encode ß-lactoglobulin (ß-LG) and 3 caseins (αS1-CN, ß-CN, and κ-CN); genotypes were directly called from customized SNP chips (50.6%) or imputed (49.4%). Variants A and B of ß-LG were frequent in the 12 breeds. For the caseins, we found 3 variants for αS1-CN (B, C, and D), 6 for ß-CN (A1, A2, A3, B, C, and I), and 5 for κ-CN (A, B, C, D, and E). For αS1-CN, the B variant was the most frequent in all breeds except Jersey. For ß-CN, the A2 variant was the most abundant in all breeds except Tarentaise, although in Normande animals, the I variant (30.9%) was almost as common as A2 (39.7%). The C variant was very rare except in the Tarentaise sample (4.8%). The most frequent variant for κ-CN was A in 5 breeds (including Holstein), and B in the 7 other breeds. The B variant was present at a particularly high frequency in Jersey (82.6%) and Normande (85.5%) animals. The C and E variants of κ-CN appeared to be particularly frequent in the Tarentaise (12.7%) and Holstein (9%) breeds, respectively. We found 20 haplotype combinations of αS1-ß-κ CN that were present at a frequency >0.1% in at least one breed; however, only 6 to 9 haplotypes were found in any given breed, demonstrating a strong degree of linkage disequilibrium. The most frequent haplotypes were B-A1-A, B-A2-A, B-A2-B, B-I-B, C-A2-A, and C-A2-B. Some alleles were predominantly found in only one haplotype, such as the E and C variants of κ-CN and the I variant of ß-CN, which were mainly found in the B-A1-E, B-A1-C, and B-I-B haplotypes, respectively. We observed changes in the frequency of certain variants over time in several breeds, such as an increase in the frequency of variants A of ß-LG, I of ß-CN, and B of κ-CN. With these results, we update and complete frequency data that were first estimated 30 to 50 yr ago, and, for the first time in these breeds, we assess the effect of selection on milk protein variants.

Mid-infrared spectrometry prediction of the cheese-making properties of raw Montbéliarde milks from herds and cheese dairy vats used for the production of Protected Designation of Origin and Protected Geographical Indication cheeses in Franche-Comté.

Franche-Comté is the primary producing region of Protected Designation of Origin cheeses in France. Normally, mid-infrared (MIR) prediction models for cheese-making property (CMP) traits are developed using individual bovine milks. However, considering the requests of all actors in the dairy sector, the present study aimed to assess the feasibility of MIR spectroscopy to develop CMP equations of Montbéliarde herd and dairy vat milks. For this purpose, 22 CMP traits were analyzed on samples collected in 2016 (half in February-March and half in May-June) from 100 commercial herds and 70 dairy vats (55 cheese dairies) located in Franche-Comté. These characteristics included 11 rennet coagulation traits and 8 lactic acidification traits measured in either soft cheese or pressed cooked cheese conditions and 3 laboratory curd yields. Models of MIR prediction for each of the 22 CMP traits were built using partial least squares regression with external validation by dividing the data set into calibration (70%) and validation (30%) sets. We confirmed that the variability of milk traits depends largely on the production scale and is higher for individual milk than for herd milk and even higher for vat milk. The best prediction models were obtained in herd milk samples for curd yields expressed in dry matter or fresh, with a coefficient of determination (R2) in external validation of 0.78 and 0.77, respectively. As with individual milk, these traits are closely related to the gross composition of the milk and therefore easier to predict by MIR spectroscopy. However, these curd yield traits were poorly predicted (R2 = 0.58) in vat milk samples due to their lower variability. In herd milk samples, prediction models of other CMP traits were poorly accurate except for the ratio of the time to obtain a standard firmness to the rennet coagulation time in soft cheese or pressed cooked cheese conditions, which showed R2 > 0.66 in external validation. Such trait is important in qualifying the behavior of milk during cheese production. Prediction models of other CMP traits for either herd or vat milk samples had poor accuracy, and further work is needed to improve their performance.

Comparison of Bayesian and partial least squares regression methods for mid-infrared prediction of cheese-making properties in Montbéliarde cows.

Assessing the cheese-making properties (CMP) of milks with a rapid and cost-effective method is of particular interest for the Protected Designation of Origin cheese sector. The aims of this study were to evaluate the potential of mid-infrared (MIR) spectra to estimate coagulation and acidification properties, as well as curd yield (CY) traits of Montbéliarde cow milk. Samples from 250 cows were collected in 216 commercial herds in Franche-Comté with the objectives to maximize the genetic diversity as well as the variation in milk composition. All coagulation and CY traits showed high variability (10 to 43%). Reference analyses performed for soft (SC) and pressed cooked (PCC) cheese technology were matched with MIR spectra. Prediction models were built on 446 informative wavelengths not tainted by the water absorbance, using different approaches such as partial least squares (PLS), uninformative variable elimination PLS, random forest PLS, Bayes A, Bayes B, Bayes C, and Bayes RR. We assessed equation performances for a set of 20 CMP traits (coagulation: 5 for SC and 4 for PCC; acidification: 5 for SC and 3 for PCC; laboratory CY: 3) by comparing prediction accuracies based on cross-validation. Overall, variable selection before PLS did not significantly improve the performances of the PLS regression, the prediction differences between Bayesian methods were negligible, and PLS models always outperformed Bayesian models. This was likely a result of the prior use of informative wavelengths of the MIR spectra. The best accuracies were obtained for curd yields expressed in dry matter (CYDM) or fresh (CYFRESH) and for coagulation traits (curd firmness for PCC and SC) using the PLS regression. Prediction models of other CMP traits were moderately to poorly accurate. Whatever the prediction methodology, the best results were always obtained for CY traits, probably because these traits are closely related to milk composition. The CYDM predictions showed coefficient of determination (R2) values up to 0.92 and 0.87, and RSy,x values of 3 and 4% for PLS and Bayes regressions, respectively. Finally, we divided the data set into calibration (2/3) and validation (1/3) sets and developed prediction models in external validation using PLS regression only. In conclusion, we confirmed, in the validation set, an excellent prediction for CYDM [R2 = 0.91, ratio of performance to deviation (RPD) = 3.39] and a very good prediction for CYFRESH (R2 = 0.84, RPD = 2.49), adequate for analytical purposes. We also obtained good results for both PCC and SC curd firmness traits (R2 ≥ 0.70, RPD ≥1.8), which enable quantitative prediction.

Short communication: Confirmation of candidate causative variants on milk composition and cheesemaking properties in Montbéliarde cows.

In a previous study, we identified candidate causative variants located in 24 functional candidate genes for milk protein and fatty acid composition in Montbéliarde, Normande, and Holstein cows. We designed these variants on the custom part of the EuroG10K BeadChip (Illumina Inc., San Diego, CA), which is routinely used for genomic selection analyses in French dairy cattle. To validate the effects of these candidate variants on milk composition and to estimate their effects on cheesemaking properties, a genome-wide association study was performed on milk protein, fatty acid and mineral composition, as well as on 9 cheesemaking traits (3 laboratory cheese yields, 5 coagulation traits, and milk pH). All the traits were predicted from midinfrared spectra in the Montbéliarde cow population of the Franche-Comté region. A total of 194 candidate variants located in 24 genes and 17 genomic regions were imputed on 19,862 cows with phenotypes and genotyped with either the BovineSNP50 (Illumina Inc.) or the EuroG10K BeadChip. We then tested the effect of each SNP in a mixed linear model including random polygenic effects estimated with a genomic relationship matrix. We confirm here the effects of candidate causative variants located in 17 functional candidate genes on both cheesemaking properties and milk composition traits. In each candidate gene, we identified the most plausible causative variant: 4 are missense in the ALPL, SLC26A4, CSN3, and SCD genes, 7 are located in 5'UTR (AGPAT6), 3' untranslated region (GPT), or upstream (CSN1S1, CSN1S2, PAEP, DGAT1, and PICALM) regions, and 6 are located in introns of the SLC37A1, MGST1, CSN2, BRI3BP, FASN, and ANKH genes.

Genetic parameters for cheese-making properties and milk composition predicted from mid-infrared spectra in a large data set of Montbéliarde cows.

Cheese-making properties of pressed cooked cheeses (PCC) and soft cheeses (SC) were predicted from mid-infrared (MIR) spectra. The traits that were best predicted by MIR spectra (as determined by comparison with reference measurements) were 3 measures of laboratory cheese yield, 5 coagulation traits, and 1 acidification trait for PCC (initial pH; pH0PPC). Coefficients of determination of these traits ranged between 0.54 and 0.89. These 9 traits as well as milk composition traits (fatty acid, protein, mineral, lactose, and citrate content) were then predicted from 1,100,238 MIR spectra from 126,873 primiparous Montbéliarde cows. Using this data set, we estimated the corresponding genetic parameters of these traits by REML procedures. A univariate or bivariate repeatability animal model was used that included the fixed effects of herd × test day × spectrometer, stage of lactation, and year × month of calving as well as the random additive genetic, permanent environmental, and residual effects. Heritability estimates varied between 0.37 and 0.48 for the 9 cheese-making property traits analyzed. Coagulation traits were the ones with the highest heritability (0.42 to 0.48), whereas cheese yields and pH0 PPC had the lowest heritability (0.37 to 0.39). Strong favorable genetic correlations, with absolute values between 0.64 and 0.97, were found between different measures of cheese yield, between coagulation traits, between cheese yields and coagulation traits, and between coagulation traits measured for PCC and SC. In contrast, the genetic correlations between milk pH0 PPC and CY or coagulation traits were weak (-0.08 to 0.09). The genetic relationships between cheese-making property traits and milk composition were moderate to high. In particular, high levels of proteins, fatty acids, Ca, P, and Mg in milk were associated with better cheese yields and improved coagulation. Proteins in milk were strongly genetically correlated with coagulation traits and, to a lesser extent, with cheese yields, whereas fatty acids in milk were more genetically correlated with cheese yields than with coagulation traits. This study, carried out on a large scale in Montbéliarde cows, shows that MIR predictions of cheese yields and milk coagulation properties are sufficiently accurate to be used for genetic analyses. Cheese-making traits, as predicted from MIR spectra, are moderately heritable and could be integrated into breeding objectives without additional phenotyping cost, thus creating an opportunity for efficient improvement via selection.

Genetic and nongenetic factors contributing to differences in αS-casein phosphorylation isoforms and other major milk proteins.

Relative concentrations of αS-casein (αS-CN) phosphorylation isoforms vary considerably among milk of individual cows. We aimed to explore to what extent genetic and other factors contribute to the variation in relative concentrations of αS-CN phosphorylation isoforms and the phosphorylation degree of αS-CN defined as the proportion of isoforms with higher degrees of phosphorylation. We also investigated the associations of genetic variants of milk proteins and casein haplotypes with relative concentrations of αS-CN phosphorylation isoforms and with the phosphorylation degree of αS-CN in French Montbéliarde cattle from the cheese production area of Franche-Comté. Detailed milk protein composition was determined by liquid chromatography coupled with electrospray ionization mass spectrometry from 531 test-day morning milk samples. Parity, lactation stage, and genetic variation of cows contributed to the phenotypic variation in relative concentrations of individual αS-CN phosphorylation isoforms and in the phosphorylation degree of αS-CN. As lactation progressed, we observed a significant increase for relative concentrations of αS-CN isoforms with higher degrees of phosphorylation (αS1-CN-9P, αS2-CN-13P, and αS2-CN-14P) as well as for the phosphorylation degree of both αS1-CN and αS2-CN. Furthermore, the ß-CN I variant was associated with a greater proportion of isoforms with lower degrees of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P); the ß-CN B variant was associated with a greater proportion of isoforms with higher degrees of phosphorylation (αS1-CN-9P, αS2-CN-12P to αS2-CN-14P). The heritability estimates were low to moderate for relative concentrations of αS2-CN phosphorylation isoforms (0.07 to 0.32), high for relative concentrations of αS1-CN-8P (0.84) and αS1-CN-9P (0.56), and moderate for phosphorylation degrees of αS1-CN (0.37) and αS2-CN (0.23). Future studies investigating relations between the phosphorylation degree of αS-CN and technological properties of milk will be beneficial for the dairy industry.

The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins.

Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese.

Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation.

Cell growth and resistance of Lactococcus lactis subsp. lactis TOMSC161 following freezing, drying and freeze-dried storage are differentially affected by fermentation conditions.

AIMS: To investigate the effects of fermentation parameters on the cell growth and on the resistance to each step of the freeze-drying process of Lactococcus lactis subsp. lactis TOMSC161, a natural cheese isolate, using a response surface methodology. METHODS AND RESULTS: Cells were cultivated at different temperatures (22, 30 and 38°C) and pH (5·6, 6·2 and 6·8) and were harvested at different growth phases (0, 3 and 6 h of stationary phase). Cultivability and acidification activity losses of Lc. lactis were quantified after freezing, drying, 1 and 3 months of storage at 4 and 25°C. Lactococcus lactis was not damaged by freezing but was sensitive to drying and to ambient temperature storage. Moreover, the fermentation temperature and the harvesting time influenced the drying resistance of Lc. lactis. CONCLUSIONS: Lactococcus lactis cells grown in a whey-based medium at 32°C, pH 6·2 and harvested at late stationary phase exhibited both an optimal growth and the highest resistance to freeze-drying and storage. SIGNIFICANCE AND IMPACT OF THE STUDY: A better insight on the individual and interaction effects of fermentation parameters made it possible the freeze-drying and storage preservation of a sensitive strain of technological interest. Evidence on the particularly damaging effect of the drying step and the high-temperature storage is presented.

Digestibility energy and amino acids of canola meal from two species (Brassica juncea and Brassica napus) fed to distal ileum cannulated grower pigs.

Yellow-seeded Brassica juncea is a novel canola species targeted to grow in the southern Canadian prairies where thermotolerance, disease resistance, and adaptation to dry agronomic conditions are required. The support of its cultivation needs nutritional evaluation of its coproduct. The B. juncea canola meal (CM) contains less fiber than conventional, dark-seeded Brassica napus CM but also slightly less Lys. In a 6 × 6 Latin square, 6 distal ileum cannulated pigs (47 kg BW) were fed 6 diets to determine the apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of CP and AA, AID and apparent total tract digestibility (ATTD) of energy, and VFA content in digesta and feces. Pigs were fed 6 diets: basal [46% wheat (Triticum aestivum) and corn (Zea mays) starch], 4 diets with 46% wheat and either B. juncea or B. napus CM at 25 or 50%, and a N-free diet based on corn starch. The B. juncea CM had higher (P < 0.05) ATTD of energy than B. napus CM (68.6 vs. 60.3%) likely due to its lower fiber content. Ileal total VFA was lower (P < 0.001) in pigs fed B. juncea than B. napus CM diets. In pigs fed B. juncea CM, the molar ratio in digesta was lower (P < 0.001) for acetate and butyrate whereas the propionate ratio was lower (P < 0.001) in feces than in pigs fed B. napus CM diets. The CM species did not affect the AID of energy, SID of AA, and feces VFA content. The DE value was higher (P < 0.05) and content of SID Lys was lower (P < 0.05) for B. juncea than B. napus CM. In conclusion, availability of B. juncea CM, a coproduct of a canola species grown in Canadian prairie land, will increase flexibility in swine feed formulation.

Assessment of the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11 during growth by flow cytometry.

AIMS: The aim of this study was to improve knowledge about the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11, a chain-forming bacterium, during growth, and to evaluate whether flow cytometry (FCM) combined with fluorescent probes can assess these different physiological states. METHODS AND RESULTS: Cellular viability was assessed using double labelling with carboxyfluorescein diacetate and propidium iodide. FCM makes it possible to discriminate between three cell populations: viable cells, dead cells and cells in an intermediate physiological state. During exponential and stationary phases, the cells in the intermediate physiological state were culturable, whereas this population was no longer culturable at the end of the stationary phase. CONCLUSIONS, AND IMPACT OF THE STUDY: We introduced a new parameter, the ratio of the means of the fluorescence cytometric index to discriminate between viable culturable and viable nonculturable cells. Finally, this work confirms the relevance of FCM combined with two fluorescent stains to evaluate the physiological states of L. lactis SK11 cells during their growth and to distinguish viable cells from viable but not culturable cells.

In situ gene expression in cheese matrices: application to a set of enterococcal genes.

Transcriptional approaches are increasingly used to compare the behaviour of pathogenic and non-pathogenic bacteria in different culture conditions. The purpose of this study was to apply these methods in cheese to better characterize food and clinical Enterococcus faecalis isolates during cheese processing. Because of the complex biochemical composition of the cheese matrix, e.g. the presence of casein and fat, we developed an efficient method to recover total RNA from bacteria in a semi-hard cheese model. To validate the RNA extraction method, we analysed expression of 7 genes from two E. faecalis strains (one clinical and one food isolate) in both cheese and culture medium by semi-quantitative RT-PCR. We then used PCR-based DNA macro-arrays to compare expression of 154 genes from two E. faecalis strains in both cheese and culture medium. The food strain isolated from cheese is transcriptionally active in cheese, as reflected by the higher transcript levels of various genes. Conversely, overall transcript levels of the V583 clinical isolate were lower in cheese, suggesting that the food strain may be more adapted to a dairy environment than the clinical strain. The method described here constitutes a very promising tool for future transcriptomic studies in cheese matrices. Global profiling in foods may prove to be a valid criterion for differentiating food from clinical isolates.

Bacterial biodiversity of traditional Zabady fermented milk.

The aim of this work was to identify the bacterial biodiversity of traditional Zabady fermented milk using PCR-temporal temperature gel electrophoresis (PCR-TTGE) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Most of the identified bacterial species in Zabady samples belonged to lactic acid bacteria (LAB), e.g., Streptococcus thermophilus, Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis, Leuconostoc citreum, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus johnsonii. Using the culture-dependent and independent methods, the streptococcal and lactococcal groups appeared to be the major bacterial species in Zabady fermented milk, whereas the lactobacilli were the minor ones. The main dominant species was St. thermophilus followed by Lc. garvieae. Other molecular tools, e.g., species-specific PCR assay and cloning and sequencing strategy were used to confirm the TTGE and DGGE results. Lc. garvieae, Lc. raffinolactis, Ln. citreum, and Lb. johnsonii were identified for the first time in this type of Egyptian fermented milk.

Molecular fingerprinting of dairy microbial ecosystems by use of temporal temperature and denaturing gradient gel electrophoresis.

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of approximately 150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.

Positive co-regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator.

Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co-dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine-sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15 kDa protein on SDS-PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai-fix regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp half-sites separated by 13 bp, and centred at -70 and -127 from the fix transcription start site. They were found to overlap the two low-affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co-operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai-fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP-CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N-terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.

Comparative study of cadmium transfer in ewe and cow milks during rennet and lactic curds preparation.

Cadmium transfer from whole milk to cream, rennet, or lactic curds was studied before and following a repeated oral cadmium administration to three lactating ewes and one cow. Before cadmium administration, the cadmium levels in milk were around 0.4 microg/L in ewes and less than 0.2 microg/L in cow. Throughout cadmium administration the mean cadmium levels in milk were 3.3+/-1.4 microg/L in ewes and 2.5+/-1 microg/L in cow. During cadmium administration, 86% of cadmium in ewe milk was dispersed in the skimmed milk and 17% in the cream, whereas only 72% was dispersed in the cow skimmed milk and 27% in the cow cream. Most of milk cadmium was associated with casein fractions. About 70% of milk cadmium was transferred to the rennet or lactic curds of ewe. The remaining cadmium present in whole milk, about 9%, was transferred to the rennet or lactic curd whey. In cow, the proportion of cadmium associated with rennet or lactic curds, rennet curd whey, and lactic curd whey was, respectively, 60%, 56%, 14% and 12% of total milk cadmium. The fraction of total cadmium transferred from milk to its milk products, whatever the species, ranged from 94% to 103%. The factor of concentration of cadmium from whole milk to milk products ranged from three to six. We suggest that the excretion of cadmium into milk is mainly achieved via the milk casein secretion. This is, to our knowledge, the first in vivo study where the cadmium transfer from milk to its milk products after repeated cadmium oral administration to ewe and cow has been studied.

Regulation of the carnitine pathway in Escherichia coli: investigation of the cai-fix divergent promoter region.

The divergent structural operons caiTABCDE and fixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopy lacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an fnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at -41.5 of the cai transcriptional start site, plays a direct role in the sole cai activation. In contrast, mutation in CRP site 2, positioned at -69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at -126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

Proteolytic specificity of chymosin on caprine alpha s1-caseins A and F.

From hydrolysis experiments carried out on alpha s1-caseins A and F at pH 5.2 in the presence of 30 g NaCl/l, i.e. the conditions encountered in many young goats' cheeses, it was found that minima of 19 and 9 bonds were sensitive to chymosin in variants A and F respectively. Variant A was hydrolysed faster than variant F and the proteolytic pattern (reversed-phase HPLC and polyacrylamide agarose gel electrophoresis) differed between the variants. Hydrolysates from both variants had a number of cleavage sites in common (Leu20-Leu21, Phe23-Ala24 and Phe32-Arg33 in both variants, Leu101-Lys102 and Leu64-Lys65, Leu120-His121 and Leu83-His84, Leu142-Ala143 and Leu105-Ala106, Leu149-Phe150 and Leu112-Phe113, Leu156-Asp157 and Leu119-Asp120, Trp164-Tyr165 and Trp127-Tyr128 in variants A and F respectively), while other bonds were split only in variant A (Leu16-Asn17, Glu18-Asn19, Phe28-Pro29, Ile44-Gly45, Tyr80-Ile81, Gln82-Lys83, Tyr91-Leu92, Tyr94-Leu95, Leu109-Glu110 and Phe179-Ser180). Major cleavage sites appeared to be at Phe23-Val24, Leu142-Ala143 and Trp164-Tyr165 for variant A, and Phe23-Val24 and Leu64-Lys65 for variant F. Cleavage site Phe23-Val24 could be the origin of the first breakdown product from goat alpha s1-caseins A and F visible in polyacrylamide agarose gel electrophoresis.

Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli.

Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene. The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon. This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries. In vivo overexpression consistently led to the synthesis of a 16-kDa protein. The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine. Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon. It was preceded by a cyclic AMP receptor protein motif centered at position -41.5. Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine. This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism. Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway. Expression of a caiF-lacZ operon fusion was subject to FNR regulator-mediated anaerobic induction and cyclic AMP receptor protein activation. The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors. Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism.