Marie-Christine Champomier-Vergès

The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K

Lactobacillus sakei is a psychrotrophic lactic acid bacterium found naturally on fresh meat and fish. This microorganism is widely used in the manufacture of fermented meats and has biotechnological potential in biopreservation and food safety. We have explored the 1,884,661-base-pair (bp) circular chromosome of strain 23K encoding 1,883 predicted genes. Genome sequencing revealed a specialized metabolic repertoire, including purine nucleoside scavenging that may contribute to an ability to successfully compete on raw meat products. Many genes appear responsible for robustness during the rigors of food processing – particularly resilience against changing redox and oxygen levels. Genes potentially responsible for biofilm formation and cellular aggregation that may assist the organism to colonize meat surfaces were also identified. This genome project is an initial step for investigating new biotechnological approaches to meat and fish processing and for exploring fundamental aspects of bacterial adaptation to these specific environments.

Proteomic Analysis of Global Changes in Protein Expression during Bile Salt Exposure of Bifidobacterium longum NCIMB 8809

Adaptation to and tolerance of bile stress are among the main limiting factors to ensure survival of bifidobacteria in the intestinal environment of humans. The effect of bile salts on protein expression patterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with or without bile extract allowed us to identify 34 different proteins whose expression was regulated. The majority of these proteins were induced after both a minor (0.6 g liter(-1)) and a major (1.2 g liter(-1)) exposure to bile. These include general stress response chaperones, proteins involved in transcription and translation and in the metabolism of amino acids and nucleotides, and several enzymes of glycolysis and pyruvate catabolism. Remarkably, xylulose 5-phosphate/fructose 6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacterial shunt, was found to be upregulated, and the activity on fructose 6-phosphate was significantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of metabolic end products (acetate and lactate) were also detected. These results suggest that bile salts, to which bifidobacteria are naturally exposed, induce a complex physiological response rather than a single event in which proteins from many different functional categories take part. This study has extended our understanding of the molecular mechanism underlying the capacity of intestinal bifidobacteria to tolerate bile.

Low-pH Adaptation and the Acid Tolerance Response of Bifidobacterium longum Biotype longum

ABSTRACT Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum biotype longum NCIMB 8809 and its acid-pH-resistant mutant B. longum biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pH-resistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in B. longum biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.

Adaptation and Response of Bifidobacterium animalis subsp. lactis to Bile: a Proteomic and Physiological Approach

ABSTRACT Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cells physiology.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Lactic acid bacteria and proteomics: current knowledge and perspectives.

Lactic acid bacteria (LAB) are widely used in the agro-food industry. Some of the LAB also participate in the natural flora in humans and animals. We review here proteomic studies concerning LAB. Two methods of research can be distinguished. In the first one, a systematic mapping of proteins is attempted, which will be useful for taxonomy and to function assignment of proteins. The second one focuses particularly on proteins whose synthesis is induced by various environmental situations or stresses. However, both approaches are complementary and will give new insights for the use of bacteria in industry, in human health and in the struggle against bacterial pathogens. Interest in LAB is growing, showing thus an increasing concern of their rational use and one can foresee in the near future an increasing use of proteomics as well as genomics.

Lactobacillus sakei: recent developments and future prospects.

Lactobacillus sakei is one of the most important bacterial species involved in meat preservation and meat fermentation. In the last fifteen years, numerous studies have focused on this species due to its important role in food microbiology. The present paper reviews current knowledge of this emerging species in the fields of taxonomy, phylogeny and physiology, and metabolism. Recent developments in genetic tools and molecular genetics will also be emphasized to evaluate future prospects.

Lactate Racemization as a Rescue Pathway for Supplying d-Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon inactivation or inhibition of the LdhD enzyme.

Intraspecies Genomic Diversity and Natural Population Structure of the Meat-Borne Lactic Acid Bacterium Lactobacillus sakei

ABSTRACT Lactobacillus sakei is a food-borne bacterium naturally found in meat and fish products. A study was performed to examine the intraspecies diversity among 73 isolates sourced from laboratory collections in several different countries. Pulsed-field gel electrophoresis analysis demonstrated a 25% variation in genome size between isolates, ranging from 1,815 kb to 2,310 kb. The relatedness between isolates was then determined using a PCR-based method that detects the possession of 60 chromosomal genes belonging to the flexible gene pool. Ten different strain clusters were identified that had noticeable differences in their average genome size reflecting the natural population structure. The results show that many different genotypes may be isolated from similar types of meat products, suggesting a complex ecological habitat in which intraspecies diversity may be required for successful adaptation. Finally, proteomic analysis revealed a slight difference between the migration patterns of highly abundant GapA isoforms of the two prevailing L. sakei subspecies (sakei and carnosus). This analysis was used to affiliate the genotypic clusters with the corresponding subspecies. These findings reveal for the first time the extent of intraspecies genomic diversity in L. sakei. Consequently, identification of molecular subtypes may in the future prove valuable for a better understanding of microbial ecosystems in food products.

Evidence for Involvement of at Least Six Proteins in Adaptation of Lactobacillus sakei to Cold Temperatures and Addition of NaCl

ABSTRACT Lactobacillus sakei is a lactic acid bacterium widely represented in the natural flora of fresh meat. The aim of this study was to analyze the differences in protein expression during environmental changes encountered during technological processes in which L. sakei is involved in order to gain insight into the ability of this species to grow and survive in such environments. Using two-dimensional electrophoresis, we observed significant variation of a set of 21 proteins in cells grown at 4°C or in the presence of 4% NaCl. Six proteins could be identified by determination of their N-terminal sequences, and the corresponding gene clusters were studied. Two proteins belong to carbon metabolic pathways, and four can be clustered as general stress proteins. A phenotype was observed at low temperature for five of the six mutants constructed for these genes. The survival of four mutants during stationary phase at 4°C was affected, and surprisingly, one mutant showed enhanced survival during stationary phase at low temperatures.