Michel Gautier

Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.

Factors Involved in the Inhibition of Growth of Salmonella enteritidis in Liquid Egg White

This study was designed to investigate the growth potential of Salmonella enteritidis in liquid egg white at 30°C and to examine the mechanism of egg white resistance to Salmonella growth. We observed a low and variable growth in whole egg white: Salmonella cell counts rose by 2 log units during the 4 to 6 days of incubation. Treatments to render the egg white components more homogeneous and to facilitate the circulation of nutrients had no effect on the low and variable growth of Salmonella cells. To investigate whether a lack of nutrients or the presence of inhibitory factors could explain this low growth, the growth of various strains at 30°C in egg white filtrate (egg white without protein) was examined. Growth was fast and comparable with growth observed in optimum medium (tryptic soy broth). The addition of 10% egg white to the filtrate decreased the growth of Salmonella enteritidis to the same level observed in egg white, leading us to conclude that inhibitory factors, probably proteins, inhibit the growth of S. enteritidis . To determine the role of the different egg white proteins and to identify which of these inhibit S. enteritidis growth, the effect of each protein added to the filtrate was evaluated. To test the inhibitory potency of three binding proteins, supplementation with their corresponding ligands was also studied. Our study shows that ovotransferrin, or iron deficiency resulting from iron binding to ovotransferrin, was the major protein or mechanism implicated in the inhibition of the growth of S. enteritidis in egg white.

Combinational variation of restriction modification specificities in Lactococcus lactis

Three genes coding for a type I R‐M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R‐M specificity. The presence of these plasmids in IL1403 conferred a new R‐M phenotype on the host, indicating that the plasmid‐encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R‐M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.

Low occurrence of safety hazards in coagulase negative staphylococci isolated from fermented foodstuffs

Some coagulase negative staphylococci (CNS) species play an important role in the fermentation of meat and milk products and are considered as food-grade. However, the increasing clinical significance of CNS and the presence of undesirable and unsafe properties in CNS question their presence or use in food. Our goal was to assess the safety of CNS by developing a diagnostic microarray targeting 268 genes corresponding to safety hazards in a food context i.e. toxins (especially enterotoxins) and determinants of antibiotic resistance and biogenic amine production. Target genes were selected among staphylococci and Gram-positive species that may be in contact with CNS in foodstuffs. The diagnostic microarray was used to screen 129 strains belonging to the 2 dominant species isolated from foodstuffs (S. equorum and S. xylosus) and the 2 main species isolated both in foodstuffs and clinical samples (S. epidermidis and S. saprophyticus). Microarray data were further completed by antibiograms and measurement of biogenic amine production. Safety hazards associated with CNS were mostly limited to the presence of antibiotic resistance. Seventy-one percent of the strains possessed at least one gene encoding antibiotic resistance, while only one strain carried an enterotoxin gene. Most strains did not carry any genes encoding staphylococcal toxins (68%), non-staphylococcal toxins (95%) or decarboxylases involved in biogenic amine production (78%). Food safety hazards were more pronounced in S. epidermidis than in the three other species regardless the food or clinical origin of the strains. Seventy-six percent of the strains carrying genes encoding staphylococcal toxin and 69% of strains carrying 5 or more antibiotic determinants belonged to S. epidermidis species. The dominant antibiotic resistance targeted erythromycin, tetracycline and penicillin and were generally traced back to the presence of tetK and blaZ in the two latest cases. Six percent of the food-related strains produced significant amounts of biogenic amines in vitro without any of the corresponding genes detected, reflecting a lack of knowledge on genetic determinants of such production in staphylococci. This work gives a first picture of safety hazards within four species of CNS frequently isolated from food or clinical environment.

Staphylococcus aureus Virulence Expression Is Impaired by Lactococcus lactis in Mixed Cultures

ABSTRACT Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.

Genome-Wide Analysis of Ruminant Staphylococcus aureus Reveals Diversification of the Core Genome

Staphylococcus aureus causes disease in humans and a wide array of animals. Of note, S. aureus mastitis of ruminants, including cows, sheep, and goats, results in major economic losses worldwide. Extensive variation in genome content exists among S. aureus pathogenic clones. However, the genomic variation among S. aureus strains infecting different animal species has not been well examined. To investigate variation in the genome content of human and ruminant S. aureus, we carried out whole-genome PCR scanning (WGPS), comparative genomic hybridizations (CGH), and the directed DNA sequence analysis of strains of human, bovine, ovine, and caprine origin. Extensive variation in genome content was discovered, including host- and ruminant-specific genetic loci. Ovine and caprine strains were genetically allied, whereas bovine strains were heterogeneous in gene content. As expected, mobile genetic elements such as pathogenicity islands and bacteriophages contributed to the variation in genome content between strains. However, differences specific for ruminant strains were restricted to regions of the conserved core genome, which contained allelic variation in genes encoding proteins of known and unknown function. Many of these proteins are predicted to be exported and could play a role in host-pathogen interactions. The genomic regions of difference identified by the whole-genome approaches adopted in the current study represent excellent targets for studies of the molecular basis of S. aureus host adaptation.

Lactobacillus helveticus: Strain Typing and Genome Size Estimation by Pulsed Field Gel Electrophoresis

Abstract. Genomic DNAs of 22 strains of Lactobacillus helveticus of various geographical origins were analyzed by pulsed-field gel electrophoresis. Two endonucleases, SmaI and SgrAI, of the 19 tested produced DNA fragments useful for strain comparison. With the endonuclease SmaI, a characteristic restriction pattern was identified for 18 of the 22 strains. The percentage of similarity (Dice coefficient) between the profiles varied between 26% and 100%, and clustering was accomplished by using the unweighted pair group method with arithmetic averages (UPGMA). For the strains showing identical profiles, the high genomic similarity was confirmed when the endonuclease SgrAI was used instead of SmaI. From summation of SmaI and SgrAI fragments from three L. helveticus strains (CNRZ 241, CNRZ 303, and CIP 57.15), the genomic length was estimated at ca. 1.85–2.0 Mb.

Isolation and characterization of a psychrotolerant toxin producer, Bacillus weihenstephanensis, in liquid egg products.

A psychrotolerant bacteria of the Bacillus cereus group was found responsible for the spoilage of whole liquid egg products. By sequencing a 16S rRNA region and performing a PCR amplification of specific 16S rRNA and cspA signatures, a Bacillus weihenstephanensis was identified. Characterization of this strain shows its ability to grow in defined medium as well as in whole liquid egg at refrigerated temperatures. The strain isolated possesses genes encoding for hemolysin BL, nonhemolytic enterotoxin, and B. cereus enterotoxins and produces enterotoxins with cytotoxic activity in whole liquid egg, even at refrigerated temperatures. The isolate exhibits a clear ability to stick and form biofilms on stainless steel, the most common material used in egg breaking factories, as well as on model hydrophilic (glass) and hydrophobic (polytetrafluoroethylene) materials. These findings show the necessity to monitor for Bacillus contamination in egg products that are often used in the composition of particularly susceptible finished products such as cream, dessert, dairy, meat, and seafood.

Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii

We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.

Effect of dry heating on the microbiological quality, functional properties, and natural bacteriostatic ability of egg white after reconstitution.

Spray-dried egg white (powder) is widely used in the food industry because of its variety of functional properties and its practical advantages. Moreover, egg white powder is generally considered safe because it can withstand high temperatures that allow for the destruction of all pathogens, especially Salmonella. In France, two types of treatments are used to improve the functional properties (whipping and gelling) of dried egg white: standard storage at 67 degrees C for about 15 days and storage at 75 to 80 degrees C for 15 days. The objective of this study was to investigate the effects of two dry-heating treatments (storage at 67 and 75 degrees C for 15 days) on the subsequent ability of egg white to resist Salmonella growth after reconstitution. The impact on the endogenous microflora of the powder and on its functional properties was also considered. Both dry-heating treatments were efficient in destroying a large number of Salmonella. Dry heating at 75 degrees C affected the bacteriostatic ability of reconstituted egg white to a greater extent than did dry heating at 67 degrees C. This loss of bacteriostatic ability could be attributable to the thermal denaturation of ovotransferrin, resulting in a reduction in its activity as an iron chelator. However, dry heating at 75 degrees C resulted in improved functional properties. Ultimately, no complete compromise between better functional quality and the preservation of the bacteriostatic ability of egg white after reconstitution is possible. Our results underline the importance of the use of hygienic conditions with egg white powder, especially with powder subjected to high-temperature treatments.