Christel Garrigues

Streptococcus thermophilus Core Genome: Comparative Genome Hybridization Study of 47 Strains

ABSTRACT A DNA microarray platform based on 2,200 genes from publicly available sequences was designed for Streptococcus thermophilus. We determined how single-nucleotide polymorphisms in the 65- to 75-mer oligonucleotide probe sequences affect the hybridization signals. The microarrays were then used for comparative genome hybridization (CGH) of 47 dairy S. thermophilus strains. An analysis of the exopolysaccharide genes in each strain confirmed previous findings that this class of genes is indeed highly variable. A phylogenetic tree based on the CGH data showed similar distances for most strains, indicating frequent recombination or gene transfer within S. thermophilus. By comparing genome sizes estimated from the microarrays and pulsed-field gel electrophoresis, the amount of unknown DNA in each strain was estimated. A core genome comprised of 1,271 genes detected in all 47 strains was identified. Likewise, a set of noncore genes detected in only some strains was identified. The concept of an industrial core genome is proposed. This is comprised of the genes in the core genome plus genes that are necessary in an applied industrial context.

Impact of Aeration and Heme-Activated Respiration on Lactococcus lactis Gene Expression: Identification of a Heme-Responsive Operon

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O(2) and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.

Using heme as an energy boost for lactic acid bacteria

Lactic acid bacteria (LAB) are a phylogenetically diverse group named for their main attribute in food fermentations, that is, production of lactic acid. However, several LAB are genetically equipped for aerobic respiration metabolism when provided with exogenous sources of heme (and menaquinones for some species). Respiration metabolism is energetically favorable and leads to less oxidative and acid stress during growth. As a consequence, the growth and survival of several LAB can be dramatically improved under respiration-permissive conditions. Respiration metabolism already has industrial applications for the production of dairy starter cultures. In view of the growth and survival advantages conferred by respiration, and the availability of heme and menaquinones in natural environments, we recommend that respiration be accepted as a part of the natural lifestyle of numerous LAB.

Bile-Inducible Efflux Transporter from Bifidobacterium longum NCC2705, Conferring Bile Resistance

ABSTRACT Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.

Complete Genome Sequence of Bifidobacterium animalis subsp. lactis BB-12, a Widely Consumed Probiotic Strain

Bifidobacterium animalis subsp. lactis BB-12 is a commercially available probiotic strain used throughout the world in a variety of functional foods and dietary supplements. The benefits of BB-12 have been documented in a number of independent clinical trials. Determination of the complete genome sequence reveals a single circular chromosome of 1,942,198 bp with 1,642 predicted protein-encoding genes, 4 rRNA operons, and 52 tRNA genes. Knowledge of this sequence will lead to insight into the specific features which give this strain its probiotic properties.

Combined Transcriptome and Proteome Analysis of Bifidobacterium animalis subsp. lactis BB-12 Grown on Xylo-Oligosaccharides and a Model of Their Utilization†

ABSTRACT Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacteriumanimalis subsp. lactis strains in particular. To elucidate the metabolism of XOS in the well-documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either XOS or glucose. The analyses show that 9 of the 10 genes that encode proteins predicted to play a role in XOS catabolism (i.e., XOS-degrading and -metabolizing enzymes, transport proteins, and a regulatory protein) were induced by XOS at the transcriptional level, and the proteins encoded by three of these (β-d-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides) to bind XOS on the cell surface and transport them into the cell. XOS are then degraded intracellularly through the action of xylanases and xylosidases to d-xylose, which is subsequently metabolized by the d-fructose-6-P shunt. The findings obtained in this study may have implications for the design of a synbiotic application containing BB-12 and the XOS used in the present study.

Pangenomics--an avenue to improved industrial starter cultures and probiotics.

With the dramatic reductions in the cost and time involved in DNA sequencing, a new approach to characterisation of bacteria is emerging. It is based on a comparison of complete genome sequences of a number of members of the same species (pangenomics). Pangenomics opens an array of new opportunities for understanding and improving industrial starter cultures and probiotics. These include understanding the formation of texture and flavour in dairy products, understanding the functionality of probiotics as well as providing information that can be used for strain screening, strain improvement, safety assessments and process improvements.

Responses of Lactic Acid Bacteria to Oxidative Stress

Lactic acid bacteria (LAB) include those designated as generally recognized as safe (LABGRAS), used in dairy industries, and opportunistic pathogens like most of the streptococceae. They are usually classified as strict fermentative bacteria producing mainly lactic acid as the end product of carbohydrate catabolism and they are oxygen-sensitive. Oxygen, in conjunction with the reducing environment, can generate highly toxic byproducts: superoxide (O2.−), hydrogen peroxide (H2O2), and hydroxyl radical (HO.). These species damage macromolecules like enzymes, leading to growth arrest or mortality in LAB. However, in the last decade, a basic functional oxygen-dependent respiratory chain has been identified in several LAB, suggesting that they might be better adapted to an oxygen environment than we thought previously. Interestingly, LAB are defective in their capacity to synthesize heme (and quinone in some LAB), both essential cofactors in respiratory chains. This chapter focuses on recent studies of oxygen toxicity, the respiratory metabolism in LAB, exemplified by Lactococcus lactis, and the signaling pathway associated with oxidative stress responses.

Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production.

Binding Interactions Between α-glucans from Lactobacillus reuteri and Milk Proteins Characterised by Surface Plasmon Resonance

Interactions between milk proteins and α-glucans at pH 4.0–5.5 were investigated by use of surface plasmon resonance. The α-glucans were synthesised with glucansucrase enzymes from Lactobacillus reuteri strains ATCC-55730, 180, ML1 and 121. Variations in the molecular characteristics of the α-glucans, such as molecular weight, linkage type and degree of branching, influenced the interactions with native and denatured β-lactoglobulin and κ-casein. The highest overall binding levels were reached with α-(1,4) compared to α-(1,3) linked glucans. Glucans with many α-(1,6) linkages demonstrated the highest binding levels to κ-casein, whereas the interaction with native β-lactoglobulin was suppressed by α-(1,6) linkages. Glucans with a higher degree of branching generally displayed lower protein binding levels whereas a higher molecular weight resulted in increased binding to κ-casein. The interactions with κ-casein were not pH dependent, whereas binding to denatured β-lactoglobulin was highest at pH 4.0 and binding to native β-lactoglobulin was optimal at pH 4.5–5.0. This study shows that molecular weight, linkage type and degree of branching of α-glucans highly influence the binding interactions with milk proteins.