Martin Bastian Pedersen

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.

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.

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.

Task Distribution between Acetate and Acetoin Pathways to Prolong Growth in Lactococcus lactis under Respiration Conditions.

Lactococcus lactis is used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications. ABSTRACT Lactococcus lactis is the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme under aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation, in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter-reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures. First, the acetate pathway contributes to biomass yield in respiration without affecting medium pH. Second, the acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways will lead to fine-tuning respiration growth, with improved yield and stability. IMPORTANCE Lactococcus lactis is used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications.