Frédéric Borges

Oligomeric States of the Detergent-solubilized Human Serum Paraoxonase (PON1)

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C12E8 micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.

Identification of Streptococcus thermophilus CNRZ368 Genes Involved in Defense against Superoxide Stress

ABSTRACT To better understand the defense mechanism of Streptococcus thermophilus against superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt, ossF, and ossG) from those whose action may be generalized to other stressful conditions (mreD, rodA, pbp2b, cpsX, and iscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1 in two loci close to each other. More precisely, these two loci are homologous to the sufD and iscU genes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called the suf region, was further characterized in S. thermophilus CNRZ368 by sequencing and by construction of ΔsufD and iscU97 nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.

Carnobacterium maltaromaticum: identification, isolation tools, ecology and technological aspects in dairy products.

Carnobacterium species constitute a genus of Lactic Acid Bacteria (LAB) present in different ecological niches. The aim of this article is to summarize the knowledge about Carnobacterium maltaromaticum species at different microbiological levels such as taxonomy, isolation and identification, ecology, technological aspects and safety in dairy products. Works published during the last decade concerning C. maltaromaticum have shown that this non-starter LAB (NSLAB) could present major interests in dairy product technology. Four reasons can be mentioned: i) it can grow in milk during the ripening period with no competition with starter LAB, ii) this species synthesizes different flavouring compounds e.g., 3-methylbutanal, iii) it can inhibit the growth of foodborne pathogens as Listeria monocytogenes due to its ability to produce bacteriocins, iv) it has never been reported to be involved in human diseases as no cases of human infection have been directly linked to the consumption of dairy products containing this species.

Bacterial diversity of Darfiyeh, a Lebanese artisanal raw goat's milk cheese.

In order to contribute to the preservation of the Lebanese dairy heritage, the aim of this study was to characterize the Darfiyeh cheese, a traditional variety made from raw goats milk and ripened in goats skin. Three independent batches of Darfiyeh production were analyzed after 20, 40 and 60 days of ripening. Mesophilic lactobacilli, thermophilic coccal-shaped lactic acid bacteria (LAB) and thermophilic lactobacilli were enumerated. In order to explore the Darfiyeh natural ecosystem, a combination of phenotypical and molecular approaches was applied. The latter included Polymerase Chain Reaction-temporal temperature gel electrophoresis (PCR-TTGE), classical PCR and quantitative PCR. These methods revealed the presence of Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus malodoratus, group D Streptococcus sp., Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus haemolyticus, Escherichia coli, Clostridium sp./Eubacterium tenue. Real-time PCR enabled quantification of E. faecium, with a detection of 10(7)-10(9) cfu g(-1) of product. The present molecular approaches combined with phenotypic method allowed describing the complex natural ecosystem of Darfiyeh, giving useful information for the preservation of Lebanese artisanal dairy products.

Lactic acid bacteria in dairy food: surface characterization and interactions with food matrix components.

This review gives an overview of the importance of interactions occurring in dairy matrices between Lactic Acid Bacteria and milk components. Dairy products are important sources of biological active compounds of particular relevance to human health. These compounds include immunoglobulins, whey proteins and peptides, polar lipids, and lactic acid bacteria including probiotics. A better understanding of interactions between bioactive components and their delivery matrix may successfully improve their transport to their target site of action. Pioneering research on probiotic lactic acid bacteria has mainly focused on their host effects. However, very little is known about their interaction with dairy ingredients. Such knowledge could contribute to designing new and more efficient dairy food, and to better understand relationships between milk constituents. The purpose of this review is first to provide an overview of the current knowledge about the biomolecules produced on bacterial surface and the composition of the dairy matter. In order to understand how bacteria interact with dairy molecules, adhesion mechanisms are subsequently reviewed with a special focus on the environmental conditions affecting bacterial adhesion. Methods dedicated to investigate the bacterial surface and to decipher interactions between bacteria and abiotic dairy components are also detailed. Finally, relevant industrial implications of these interactions are presented and discussed.

The CHAP domain of Cse functions as an endopeptidase that acts at mature septa to promote Streptococcus thermophilus cell separation

Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall‐binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N‐terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis and S. thermophilus. Additionally, RP‐HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus.

Intraspecific Variability of the Terminal Inverted Repeats of the Linear Chromosome of Streptomyces ambofaciens

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.

Antibacterial activity of class IIa bacteriocin Cbn BM1 depends on the physiological state of the target bacteria.

Carnobacteriocin BM1 (Cbn BM1) is a class IIa bacteriocin produced by Carnobacterium maltaromaticum CP5 isolated from a French mold ripened cheese. Numerous studies highlight variations in numerous parameters, such as bacterial membrane composition and potential, according to physiological changes. In this work, the mechanism of action of an oxidized form of Cbn BM1 was studied on C. maltaromaticum DSM20730 in log and stationary growth phases. Membrane integrity assessment and high resolution imaging by atomic force microscopy confirmed the link between physiological state and bacterial sensitivity to Cbn BM1. Indeed, these approaches enable visualizing morphological damage of C. maltaromaticum DSM20730 only in an active dividing state. To specifically address the interaction between peptide and bacterial membrane, fluorescence anisotropy measurements were conducted. Results revealed strong modifications in membrane fluidity by Cbn BM1 only for C. maltaromaticum DSM20730 in log growth phase. In a similar way, the Δψ component, but not the ΔpH component of the proton-motive force, was perturbed only for bacteria in log growth phase. These results clearly show that a class IIa bacteriocin antimicrobial mechanism of action can be modulated by the physiological state of its target bacteria.

Characterisation of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368

During industrial processes, the dairy organism Streptococcus thermophilus is exposed to stress conditions. Its ability to survive and grow in an aerobic environment indicates that it must possess defensive mechanisms against reactive oxygen species. To identify the genes involved in oxidative stress defence, a collection of mutants was generated by random insertional mutagenesis and screened for menadione sensitivity and resistance. Results obtained for resistant clones allowed the identification of eight loci. The insertions affected genes whose homologues in other bacteria were previously identified as being involved in stress response (deoB, gst) or transcription regulation (rggC) and five ORFs of unknown function. The tolerance of the eight mutants to air-exposure, methyl viologen and H2O2 was studied. Real-time quantitative PCR was used to analyse the transcript level of mutated genes and revealed that most were down-regulated during oxidative stress.

Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA 28

Carnobacterium maltaromaticum strains are widely found in food including fish, meat and some dairy products. Producing a malty/chocolate like aroma due to 3-methylbutanal from the catabolism of leucine is a general characteristic of this species. In this study, we investigated metabolic routes responsible for the biosynthesis of this flavor compound from the catabolism of leucine in C. maltaromaticum LMA 28, a strain isolated from mold ripened soft cheese. Depending on the lactic acid bacterium, leucine can be converted into 3-methylbutanal following two possible metabolic pathways: either directly by α-ketoacid decarboxylase (KADC) pathway or indirectly by α-ketoacid dehydrogenase (KADH) pathway. Both KADC (41.0±3.0 nmol/mg protein/min) and KADH (1.43±0.62 nmol/mg protein/min) activities were detected and determined in vitro in C. maltaromaticum LMA 28. C. maltaromaticum LMA 28 slightly reduced the production of 3-methylbutanal from leucine in the presence of a specific inhibitor of KADH enzyme complex, i.e. sodium meta-arsenite, suggesting that both pathways were involved in vivo in leucine catabolism. Moreover the presence of genes encoding aminotransferase, glutamate dehydrogenase, α-ketoacid decarboxylase, α-ketoacid dehydrogenase and aldehyde dehydrogenase was confirmed. C. maltaromaticum is then the first lactic acid bacterium in which presence of both metabolic routes responsible for the biosynthesis of 3-methylbutanal from leucine catabolism was confirmed in vitro and in vivo as well.