The lactic acid bacteria (LAB) are essential for food fermentations and their impact on gut physiology and health is under active exploration. In addition to their well-studied fermentation metabolism, many species belonging to this heterogeneous group are genetically equipped for respiration metabolism. In LAB, respiration is activated by exogenous heme, and for some species, heme and menaquinone. Respiration metabolism increases growth yield and improves fitness. In this review, we aim to present the basics of respiration metabolism in LAB, its genetic requirements, and the dramatic physiological changes it engenders. We address the question of how LAB acquired the genetic equipment for respiration. We present at length how respiration can be used advantageously in an industrial setting, both in the context of food-related technologies and in novel potential applications.

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 hemeresponsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.

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We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F 1 domain of the membrane-bound (F 1 F 0 ) H ؉ -ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F 1 -ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F 1 -ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.Lactic acid bacteria are used extensively in the dairy industry, where the production of lactic acid is important for texture, flavor, and preservation purposes. In addition, lactic acid bacteria are also used for industrial lactate production, which has numerous applications, such as cosmetics, cleaning agents, and biodegradable polylactic acid polymers. From an industrial point of view there is great interest in improving the performance of these organisms with respect to both the rate and the yield of lactate production.In spite of the importance of glycolysis for fermentation purposes, it is still not known what controls the glycolytic flux in microbial bioreactors. It has been suggested that the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a high level of control (estimated to be 90% of the control) over the glycolytic flux in nonproliferating cells of Lactococcus lactis (33). However, it has recently been shown that GAPDH has no control over the glycolytic flux in steadily growing L. lactis cells (Solem, Koebmann, and Jensen, unpublished data). The control over the glycolytic flux exerted by lactate dehydrogenase was also reported to be close to zero (2).According to metabolic control theory (16, 25), flux control can reside in any of the steps in a system; i.e., it can reside in the numerous processes that consume the ATP generated in glycolysis (8,17). Indeed, we have recently shown that at least 75% of the control over glycolysis in aerobic Escherichia coli cultures occurs in the ATP-consuming reactions (26). This result was obtained by overexpression of genes encoding part of the F 1 unit of the (F 1 F 0 ) H ϩ -ATPase, which resulted in uncoupling of glycolysis from biomass production and a 70% increase in the glycolytic flux.In this paper we show that expression of genes encoding...

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.

A series of mutant strains of Lactococcus lactis were constructed with lactate dehydrogenase (LDH) activities ranging from below 1% to 133% of the wild-type activity level. The mutants with 59% to 133% of lactate dehydrogenase activity had growth rates similar to the wild-type and showed a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C J F1 LDH ¼ 2 1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate dehydrogenase, only 70% more than needed to catalyze the lactate flux in the wild-type cells.Keywords: lactic acid bacteria; metabolic control analysis; gene expression; fermentation.Lactococcus lactis plays an important role in dairy fermentations, mainly in the production of cheeses. In these fermentation processes, lactose is present at high concentrations (50 g·L 21 ) and is converted through glycolysis to lactic acid, with minor amounts of other compounds being produced in addition (homolactic fermentation). The resulting low pH contributes to the texture and flavor of cheeses and inhibits the growth of other bacterial species. Under conditions where sugar becomes limiting for growth of Lactococcus lactis, the metabolism shifts to mixed-acid products, i.e. formate, acetate and ethanol along with smaller amounts of lactate [1,2].Work has been performed in the past to study the mechanisms involved in the shift between the two different fermentation modes in L. lactis. In the presence of excess sugar, the concentration of fructose 1,6-bisphosphate, the triose-phosphates, pyruvate, and the NADH/NAD þ ratio are high, whereas the concentration of phosphoenolpyruvate and inorganic phosphate are relative low [3 -6]. In contrast, when sugar is limiting the concentration of these metabolites and cofactors are reversed to the opposite, high or low level. Particularly, the level of fructose-1,6-bisphosphate, which is known to activate both pyruvate kinase and lactate dehydrogenase, has been suggested to play a key role in the regulation of the fermentation mode [1].Work has also been performed to determine the factors that control the flux through glycolysis by applying metabolic control analysis [7,8]. Based on inhibitor titration, Poolman et al. [9] suggested that glyceraldehyde 3-phosphate dehydrogenase had a large amount of control ove...

Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacterium animalis 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 (␤-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.Prebiotics are defined as food components that confer a health benefit on the host through modulation of the microbiota (17). Among different kinds of prebiotics, special focus has been given to nondigestible oligosaccharides (NDO), which are the most abundant nutrients in the lower gastrointestinal tract (GIT), the ecological niche of bifidobacteria. The majority of the members of the genus Bifidobacterium are capable of degrading NDO to monosaccharides, which in turn are converted into intermediates of the D-fructose-6-phosphate (F6P) shunt (also known as the bifid shunt), the central carbohydrate catabolic pathway characteristic of bifidobacteria.Xylo-oligosaccharides (XOS) are NDO that have received increasing attention as potential prebiotic candidates (18). XOS are sugar oligomers composed of a ␤-1,4-linked xylopyranosyl backbone that are obtained by either chemical or, more commonly, enzymatic hydrolysis of xylan polysaccharides extracted from plant cell wall. The bifidogenic effect of XOS was demonstrated both by in vitro studies (22) and by small-scale in vivo human studies (2). Some intestinal bacterial strains are able to grow on XOS, yet numerous studies have demonstrated that the ability to utilize these oligosaccharides varies considerably among these bacteria (3,25,29). Moreover, a recent semicontinuous, anaerobic colon simulator study demonstrated that growth on XOS can also result in decreased levels of pathogenic strains, an ...

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.The consumption of bifid bacteria to restore a healthy gut microflora was first suggested in 1906 (8). Since then, the use of probiotic bacteria to confer health benefits has developed into a global industry in which probiotic bacteria are delivered to consumers in functional foods, infant formulas, and dietary supplements. A number of species, primarily of the genera Bifidobacterium and Lactobacillus, are employed. It is generally believed that the ability of a strain to provide a health benefit is a strain-specific property (see, for example, reference 6). Research on strains with documented probiotic capabilities is therefore of particular scientific interest.Bifidobacterium animalis subsp. lactis strain BB-12 has been commercially available for more than 25 years and is the subject of numerous independent clinical trials. We are aware of more than 200 scientific publications involving BB-12 (see, for example, reference 5), making this one of the most thoroughly studied probiotic strains available. We have determined the complete genome sequence of BB-12 by using cells from the inoculation material employed for commercial production of this strain.Genomic DNA was shotgun cloned into plasmid and cosmid vectors and sequenced by Integrated Genomics (Chicago, IL) using Sanger methodology. This resulted in 11-fold coverage of the genome and, initially, 56 contigs. Initial alignment of the contigs was done by comparison to the published genome sequence of B. longum NCC2705 (7). Even though NCC2705 shows little homology to BB-12 at the DNA sequence level, several large clusters of genes were found to have the same organization, allowing alignment and closing of several gaps. The remaining gaps were closed by combinatorial PCR using 70 primers designed from sequences at the ends of the original contigs and by additional DNA sequencing following primer walking. This approach resulted in a single circular contig containing 1,942,198 bp. No plasmids are present in BB-12.An optical map of the BB-12 chromosome digested by NotI was produced by OpGen (Madison, WI) and analyzed using the OpGen MapSolver package. There are 193 NotI sites in the genome sequence, and the optical map confirmed the correctness of the assembly. The overall chromosomal structure is the same as that determined for the type strain DSM 10140 (1), whereas strain AD011 (4) has apparently undergone a number of genome rearrangements, yielding several differences in the in silico-predicted locations of N...