Exploring the association between microbiota and behaviour in suckling piglets.

It is increasingly recognised that the microbes residing in the gastrointestinal tract can influence brain physiology and behaviour, via the microbiota-gut-brain axis. Here, we made a first explorative evaluation at the association between the gut microbiota and behaviour in suckling piglets. 16S microbiota profiling information was obtained from two independent replicate experiments at 2 and 4 weeks of age. Piglets underwent a backtest to assess their personality or coping style at 2 weeks of age, and were subjected to a combined open field and novel object test at 3.5 weeks of age, recording anxiety-related and exploratory behaviour. The number of squeals vocalised during the open field test was associated with microbial groups such as Coprococcus 3 and CAG-873, whereas in the novel object test, explorative behaviour was significantly associated with microbial genera like Atopobium and Prevotella. Overall, this study explores the microbiota-behavioural relation by employing multivariate analysis and exemplifies the importance of individualised analyses when evaluating such relationships.

Using fluorescent promoter-reporters to study sugar utilization control in Bifidobacterium longum NCC 2705.

Bifidobacteria are amongst the first bacteria to colonize the human gastro-intestinal system and have been proposed to play a crucial role in the development of the infant gut since their absence is correlated to the development of diseases later in life. Bifidobacteria have the capacity to metabolize a diverse range of (complex) carbohydrates, reflecting their adaptation to the lower gastro-intestinal tract. Detailed understanding of carbohydrate metabolism regulation in this genus is of prime importance and availability of additional genetic tools easing such studies would be beneficial. To develop a fluorescent protein-based reporter system that can be used in B. longum NCC 2705, we first selected the most promising fluorescent protein out of the seven we tested (i.e., mCherry). This reporter protein was then used to study the carbohydrate mediated activation of PBl1518 and PBl1694, two promoters respectively predicted to be controlled by the transcriptional factors AraQ and AraU, previously suggested to regulate arabinose utilization and proposed to also act as global transcriptional regulators in bifidobacteria. We confirmed that in B. longum NCC 2705 the AraQ controlled promoter (PBl1518) is induced strongly by arabinose and established that the AraU controlled promoter (PBl1694) was mostly induced by the hexoses galactose and fructose. Combining the mCherry reporter system with flow cytometry, we established that NCC 2705 is able to co-metabolize arabinose and glucose while galactose was only consumed after glucose exhaustion, thus illustrating the complexity of different carbohydrate consumption patterns and their specific regulation in this strain.

Early life feeding accelerates gut microbiome maturation and suppresses acute post-weaning stress in piglets.

Early life microbiome perturbations can have important effects on host development, physiology and behaviour. In this longitudinal study, we evaluated the impact of early feeding on gut microbiome colonization in neonatal piglets. Early-fed (EF) piglets had access to a customized fibrous diet from 2 days after birth until weaning in addition to mother's milk, whereas control piglets suckled mother's milk only. Rectal swabs were collected at multiple time points until 6 weeks of age to investigate microbiota development using 16S rRNA gene profiling. The dynamic pre-weaning microbiota colonization was followed by a relatively stable post-weaning microbiota, represented by Prevotella, Roseburia, Faecalibacterium, Ruminococcus, Megasphaera, Catenibacterium and Subdoligranulum. EF piglets showed an accelerated microbiota maturation, characterized by increased microbial diversity, pre-weaning emergence of post-weaning-associated microbes and a more rapid decline of typical pre-weaning microbes. Furthermore, the individual eating behaviour scores of piglets quantitatively correlated with their accelerated microbiome. Importantly, EF piglets displayed a smoother relative weight gain and tended to reach a higher relative weight gain, in addition to reduced diarrhoea scores in the first week post-weaning. Overall, these findings demonstrate the beneficial impact of early feeding on microbiota development as well as pig health and performance during the weaning transition.

Carbohydrate-controlled serine protease inhibitor (serpin) production in Bifidobacterium longum subsp. longum.

The Serine Protease Inhibitor (serpin) protein has been suggested to play a key role in the interaction of bifidobacteria with the host. By inhibiting intestinal serine proteases, it might allow bifidobacteria to reside in specific gut niches. In inflammatory diseases where serine proteases contribute to the innate defense mechanism of the host, serpin may dampen the damaging effects of inflammation. In view of the beneficial roles of this protein, it is important to understand how its production is regulated. Here we demonstrate that Bifidobacterium longum NCC 2705 serpin production is tightly regulated by carbohydrates. Galactose and fructose increase the production of this protein while glucose prevents it, suggesting the involvement of catabolite repression. We identified that di- and oligosaccharides containing galactose (GOS) and fructose (FOS) moieties, including the human milk oligosaccharide Lacto-N-tetraose (LNT), are able to activate serpin production. Moreover, we show that the carbohydrate mediated regulation is conserved within B. longum subsp. longum strains but not in other bifidobacterial taxons harboring the serpin coding gene, highlighting that the serpin regulation circuits are not only species- but also subspecies- specific. Our work demonstrates that environmental conditions can modulate expression of an important effector molecule of B. longum, having potential important implications for probiotic manufacturing and supporting the postulated role of serpin in the ability of bifidobacteria to colonize the intestinal tract.

Impact of early-life feeding on local intestinal microbiota and digestive system development in piglets.

Early-life gut microbial colonisation is known to influence host physiology and development, shaping its phenotype. The developing gastro-intestinal tract of neonatal piglets provides a "window of opportunity" for programming their intestinal microbiota composition and corresponding intestinal development. Here, we investigated the impact of early feeding on jejunum and colon microbiota composition, and intestinal maturation in suckling piglets. From two days of age, early-fed (EF; n = 6 litters) piglets had access to solid feed containing a mixture of fibres till weaning (day29) in addition to sow's milk, whereas the control (CON; n = 6 litters) piglets exclusively fed on sow's milk. Early feeding elicited a significant impact on the colon microbiota, whereas no such effect was seen in the jejunal and ileal microbiota. Quantified eating behavioural scores could significantly explain the variation in microbiota composition of EF piglets and support their classification into good, moderate, and bad eaters. Members of the Lachnospiraceae family, and the genera Eubacterium, Prevotella, and Ruminococcus were quantitatively associated with eating scores. EF piglets were found to have a decreased pH in caecum and colon, which coincided with increased short-chain fatty acid (SCFA) concentrations. Moreover, they also had increased weights and lengths of several intestinal tract segments, as well as a decreased villus-crypt ratio in jejunal mucosa and an increased abundance of proliferative cells in colon mucosa. The approaches in this study indicate that early feeding of a mixed-fibre (pre-weaning) diet changes the microbiota composition, pH, and fermentation products in the distal gut of piglets, while it also alters both macroscopic and microscopic intestinal measurements. These results exemplify the potential of early feeding to modulate intestinal development in young piglets.

Legitimate and Reliable Determination of the Age-Related Intestinal Microbiome in Young Piglets; Rectal Swabs and Fecal Samples Provide Comparable Insights.

A prerequisite for reliable microbiota analysis is having an effective and consistent sampling method. Fecal sampling, commonly used to study the intestinal microbiome, might not be suitable in all situations, especially considering the potential difficulties in obtaining fresh feces from young animals. Indeed, this study shows that the success rate of collecting fecal samples from young piglets (<2 weeks of age) was very low. Therefore, we evaluated rectal swabs as an alternative sample type (to feces) for studying porcine microbiome development and performed a comparative analysis of microbiome composition obtained from fresh fecal samples and rectal swabs in 15 healthy piglets at seven (6 piglets) and 20 (9 piglets) days of age. Three samples (fresh feces, rectal swab before and after defecation) were collected from individual piglets and microbiome composition was assessed by 16S rRNA gene sequencing. The results demonstrated that rectal swabs and fecal samples provide similar microbiome composition profiles, with samples clustering predominantly by individual animal rather than sample type. Furthermore, regardless of the sample type, the biological interpretation with respect to microbiota colonization patterns associated with different ages (7 and 20 days) was found to be comparable. Independent of sample type, we observed age-related changes like increasing microbiota diversity and alterations in relative abundances of the phyla Firmicutes, Bacteroidetes, and Fusobacteria, which was also reflected in consistent family- and genus-level microbiota changes. This study establishes that rectal swabs are a suitable alternative sample type to study the porcine microbiome development in early life, when fecal sampling is challenging.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

Production of aroma compounds in lactic fermentations.

This review describes recent scientific research on the production of aroma compounds by lactic acid bacteria (LAB) in fermented food products. We discuss the various precursor molecules for the formation of aroma compounds in connection with the metabolic pathways involved. The roles of nonmetabolic properties such as cell lysis are also described in relation to aroma formation. Finally, we provide an overview of the literature on methods to steer and control aroma formation by LAB in mixed culture fermentations. We demonstrate that the technological progress made recently in high-throughput analysis methods has been driving the development of new approaches to understand, control, and steer aroma formation in (dairy) fermentation processes. This currently entails proposing new rules for designing stable, high-performance mixed cultures constituting a selection of strains, which in concert and on the basis of their individual predicted gene contents deliver the required functionalities.

Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter.

AIMS: To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST). METHODS AND RESULTS: The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract. CONCLUSIONS: The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.

Transient inflammatory-like state and microbial dysbiosis are pivotal in establishment of mucosal homeostasis during colonisation of germ-free mice.

The gut microbiota is increasingly recognised as a key-player in defining the health status of the gastrointestinal tract. Recently, we demonstrated that colonisation of healthy germfree mice with a conventional microbiota (conventionalisation) elicits temporal and region specific host-microbe communication responses that lead to the establishment of a microbiota-accommodating homeostatic state within 30 days. Here, the microbiota composition profiles, mucosal transcriptomes and plasma-analytes in germ-free and conventionalised C57/BL 6 J mice were assessed to decipher the features of the distinctive and pivotal events occurring four days after initiation of the conventionalisation process. The dominance of the microbial genera Helicobacter, Sphingomonas and Mucispirillum in the gut microbiota coincided with the transient mounting of proinflammatory responses in the mucosa and the transiently elevated levels of specific (inflammatory) cytokines and amines in plasma. The overrepresented microbes have previously been associated with the potential to cause disease under certain conditions, illustrating that conventionalisation proceeds through a transient state that resembles situations associated with dysbiosis. However, no overt mucosal inflammation was observed, suggesting a pivotal role of the overrepresented bacterial groups in priming and maturation of the immune system during the process of conventionalisation. These findings imply that the transiently elevated relative overgrowth of particular microbial genera functions as pivotal adjuvants to elicit the corresponding proinflammatory cascades, which precede the full maturation of the different arms of the immune system following these events and is required to achieve a microbiota-accommodating homeostasis in healthy animals.

The microbiota and the gut-brain axis: insights from the temporal and spatial mucosal alterations during colonisation of the germfree mouse intestine.

The influence of the gut microbiota on the nervous system, brain development and behaviour, in particular during microbial colonisation of the host, has recently been receiving profound interest. Our time-resolved mining of combined data analyses of the ex-germfree mouse intestine during a 30-day course of colonisation with conventional mouse faecal microbiota (conventionalisation), shed light on temporal altered expression of genes of which the products influenced functions of the nervous system. Plasma tryptophan and kynurenine levels reflected high indoleamine dioxygenase activity, which was supported by significant temporal induction of the encoding gene in all gut tissues. However, the majority of genes associated with neuronal development and function were reduced. Colonic substance P elevation in response to conventionalisation was higher only after 30-days. These results support a functional microbiota-neurohumoral relationship during conventionalisation and suggest a delayed neuronal response that is elicited only after the microbiota accommodating homeostasis has been accomplished.

Strain-specific immunomodulatory effects of Lactobacillus plantarum strains on birch-pollen-allergic subjects out of season.

BACKGROUND: Allergic diseases are increasing world-wide, and according to the hygiene hypothesis may be related to a decreased exposure to environmental bacteria. Probiotic bacteria are recognized for their immunomodulating properties, and may benefit allergy patients. In vitro studies reveal immunomodulatory effects that are strain dependent. Differential immunomodulatory in vitro capacities cannot be extrapolated directly to in vivo efficacy. Thus, in vitro screening should preferably be followed by a comparative analysis of the selected immunomodulatory strains in an in vivo setting. OBJECTIVE: We selected five Lactobacillus strains on their IL-10-inducing capacity, and evaluated the immunomodulatory properties in birch-pollen-allergic subjects outside the hayfever season, with a reduction of IL-13 as the primary outcome. METHODS: A double-blind, placebo-controlled parallel study was performed in which 62 subjects with a proven birch-pollen allergy consumed one of five different probiotic yoghurts containing four Lactobacillus plantarum strains and one Lactobacillus casei strain or a placebo yoghurt. Blood samples were collected at the start and after 4 weeks. Several immune parameters were determined in serum and peripheral blood mononuclear cell cultures (PBMC) derived from these subjects. Results A decrease in birch-pollen-specific IgE was found for four probiotic strains. L. casei Shirota reduced the number of CD16(+) /CD56(+) cells in peripheral blood mononuclear cells. For strain L. plantarum CBS125632, the decrease in IgE coincided with significant decreases in IL-5 and IL-13 production by αCD3/αCD28-stimulated PBMC cultures. CONCLUSION AND CLINICAL RELEVANCE: Subjects with seasonal allergy can be used to determine immunomodulatory responses outside the pollen season within a 4-week study period. L. plantarum CBS125632 decreased several immune markers related to allergy, and may have the potential to alleviate the severity of seasonal allergy symptoms.

Biodiversity of mannose-specific adhesion in Lactobacillus plantarum revisited: strain-specific domain composition of the mannose-adhesin.

Recently, we have identified the mannose-specific adhesin encoding gene (msa) of Lactobacillus plantarum. In the current study, structure and function of this potentially probiotic effector gene were further investigated, exploring genetic diversity of msa in L. plantarum in relation to mannose adhesion capacity. The results demonstrate that there is considerable variation in quantitative in vitro mannose adhesion capacity, which is paralleled by msa gene sequence variation. The msa genes of different L. plantarum strains encode proteins with variable domain composition. Construction of L. plantarum 299v mutant strains revealed that the msa gene product is the key-protein for mannose adhesion, also in a strain with high mannose adhering capacity. However, no straightforward correlation between adhesion capacity and domain composition of Msa in L. plantarum could be identified. Nevertheless, differences in Msa sequences in combination with variable genetic background of specific bacterial strains appears to determine mannose adhesion capacity and potentially affects probiotic properties. These findings exemplify the strain-specificity of probiotic characteristics and illustrate the need for careful and molecular selection of new candidate probiotics.

A high-throughput cheese manufacturing model for effective cheese starter culture screening.

Cheese making is a process in which enzymatic coagulation of milk is followed by protein separation, carbohydrate removal, and an extended bacterial fermentation. The number of variables in this complex process that influence cheese quality is so large that the developments of new manufacturing protocols are cumbersome. To reduce screening costs, several models have been developed to miniaturize the cheese manufacturing process. However, these models are not able to accommodate the throughputs required for systematic screening programs. Here, we describe a protocol that allows the parallel manufacturing of approximately 600 cheeses in individual cheese vats each with individual process specifications. Protocols for the production of miniaturized Gouda- and Cheddar-type cheeses have been developed. Starting with as little as 1.7 mL of milk, miniature cheeses of about 170 mg can be produced and they closely resemble conventionally produced cheese in terms of acidification profiles, moisture and salt contents, proteolysis, flavor profiles, and microstructure. Flavor profiling of miniature cheeses manufactured with and without mixed-strain adjunct starter cultures allowed the distinguishing of the different cheeses. Moreover, single-strain adjunct starter cultures engineered to overexpress important flavor-related enzymes revealed effects similar to those described in industrial cheese. Benchmarking against industrial cheese produced from the same raw materials established a good correlation between their proteolytic degradation products and their flavor profiles. These miniature cheeses, referred to as microcheeses, open new possibilities to study many aspects of cheese production, which will not only accelerate product development but also allow a more systematic approach to investigate the complex biochemistry and microbiology of cheese making.

In vitro analysis of protection of the enzyme bile salt hydrolase against enteric conditions by whey protein-gum arabic microencapsulation.

The interest in efficient intestinal delivery of health-promoting substances is increasing. However, the delivery of vulnerable substances such as enzymes requires specific attention. The transit through the stomach, where the pH is very low, can be detrimental to the enzymatic activity of the protein to be delivered. Here, we describe the microencapsulation of the model enzyme bile salt hydrolase (Bsh) using whey protein-gum arabic microencapsulates for food-grade and targeted enzyme delivery in the proximal region of the small intestine. Furthermore, the efficacy of enteric coating microencapsulates for site-specific enzyme delivery was compared in vitro with living Lactobacillus plantarum WCFS1 bacteria that endogenously produce the Bsh enzyme. Microencapsulates allowed highly effective protection of the enzyme under gastric conditions. Moreover, Bsh release under intestinal conditions appeared to be very efficient, although in the presence of pancreatin, the Bsh activity decreased in time due to proteolytic degradation. In comparison, L. plantarum appeared to be capable to withstand gastric conditions as well as pancreatin challenge. Delivery using encapsulates and live bacteria each have different (dis)advantages that are discussed. In conclusion, live bacteria and food-grade microencapsulates provide alternatives for dedicated enteric delivery of specific enzymes, and the choice of enzyme to be delivered may determine which mode of delivery is most suitable.

Assessment of real-time RT-PCR for quantification of Lactobacillus plantarum gene expression during stationary phase and nutrient starvation.

AIMS: In this study, we evaluated the impact of different real-time reverse-transcription PCR (RT-PCR) data normalization methods on the interpretation of stationary-phase and nutrient-starved Lactobacillus plantarum WCFS1 gene expression levels. METHODS AND RESULTS: Lactobacillus plantarum WCFS1 culture characteristics and housekeeping gene transcripts were measured during stationary phase in standard growth medium and during extreme nutrient starvation. These conditions differentially affected L. plantarum viability and RNA/DNA ratios. Real-time RT-PCR gene expression data were normalized according to three different methods: (i) total RNA amounts added to the reactions; (ii) the comparative 2(-Delta Delta Ct) method using recA as a reference; and (iii) the geNorm approach based on the average expression values of several housekeeping genes. Each of these methods revealed differences in the abundance of housekeeping gene transcripts between L. plantarum in the exponential phase of growth and in stationary phase or undergoing nutrient starvation. CONCLUSIONS: Real-time RT-PCR data analysis with a normalization factor comprised of several of the most stably expressed housekeeping genes best accounted for the expected activity levels of the cells contained in the different cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The relative normalization of real-time RT-PCR data using multiple housekeeping reference genes should be useful for the quantification of bacterial gene expression levels in nonoptimal growth conditions in situ.

Mutational analysis of the Lactococcus lactis NIZO B40 exopolysaccharide (EPS) gene cluster: EPS biosynthesis correlates with unphosphorylated EpsB.

AIMS: To determine the role of the EpsA, EpsB, and EpsC proteins encoded at the 5'-end of the exopolysaccharide (EPS) gene cluster in regulation of EPS production in Lactococcus lactis. METHODS AND RESULTS: Deletion and paralog-replacement mutants of epsABCD were used to determine the function of EpsA, EpsB and EpsC in EPS production and polymer chain length determination in L. lactis. EpsA and EpsB appeared to be essential for EPS biosynthesis in L. lactis, while deletion of the phosphatase (EpsC) only had a minor effect on the EPS production level. Determination of the phosphorylation state of EpsB and analysis of a C-terminally truncated EpsB variant indicate that EPS biosynthesis in L. lactis is driven by a nonphosphorylated form of EpsB. CONCLUSIONS: The data presented here show that in L. lactis, EPS production is under control of a phosphoregulatory system and that EPS biosynthesis correlates with an unphosphorylated EpsB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides molecular understanding of polysaccharide production in L. lactis that could eventually enable novel approaches to control EPS production by lactic acid bacteria during industrial fermentation processes.

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecal samples after passage through the gastrointestinal tract of mice.

AIM: This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. METHODS AND RESULTS: Nine Lact. plantarum ivi gene replacement mutants were constructed, focussing on ivi genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation. The in vitro growth characteristics of the mutants appeared identical to those observed for the wild-type strain, which agrees with the recombination-based in vivo expression technology suggestion that these genes are not transcribed in the laboratory. Quantitative PCR experiments demonstrated differences in the relative population dynamics of the Lact. plantarum ivi mutants in faecal samples after passage through the GI tract of mice. CONCLUSIONS: The in situ competition experiments revealed a 100- to 1000-fold reduction of the relative abundance of three of the ivi gene mutants, harbouring deletions of genes predicted to encode a copper transporter, an orphan IIC cellobiose PTS and a cell wall anchored extracellular protein. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments clearly establish that the proteins encoded by these three genes play a key role in Lact. plantarum performance during passage of the GI tract.

DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum.

AIMS: The purpose of this study was to determine the global transcriptional response in a food-associated lactic acid bacterium during bile stress. METHODS AND RESULTS: Clone-based DNA micro-arrays were employed to describe the global transcriptional response of Lactobacillus plantarum WCFS1 towards 0.1% porcine bile. Comparison of differential transcript profiles obtained during growth of Lact. plantarum on plates with and without bile revealed 28 and 62 putative genes, of which the expression was at least 2.5-fold up- or down-regulated by bile, respectively. Approximately, 50% of these genes appeared genetically linked, and 12 bile-responsive gene clusters were identified. Seven of the identified bile-responsive genes and gene clusters encode typical stress-related functions, including glutathione reductase and glutamate decarboxylase, involved in oxidative and acid stress, respectively. Moreover, 14 bile-responsive genes and gene clusters were identified that encode proteins that are located in the cell envelope, including the dlt operon and the F1F0 ATPase. CONCLUSIONS: The identification of a relatively high number of genes encoding cell envelope functions indicates a major impact of bile acids on the integrity and/or functionality of the cytoplasmic membrane and cell wall. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here provide valuable clues towards the defence mechanisms that play a role during bile stress in Lact. plantarum.

Metabolic engineering of acetaldehyde production by Streptococcus thermophilus.

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.