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Lactic acid bacteria produce γ-aminobutyric acid (GABA) as an acid stress response. GABA is a neurotransmitter that may improve sleep and resilience to mental stress. This study focused on the selection, identification and optimization of a bacterial strain with high GABA production, for development as a probiotic supplement. The scientific literature and an industry database were searched for probiotics and potential GABA producers. In silico screening was conducted to identify genes involved in GABA production. Subsequently, 17 candidates were screened for in vitro GABA production using thin layer chromatography, which identified three candidate probiotic strains Levilactobacillus brevis DSM 20054, Lactococcus lactis DS75843and Bifidobacterium adolescentis DSM 24849 as producing GABA. Two biosensors capable of detecting GABA were developed: 1. a transcription factor-based biosensor characterized by the interaction with the transcriptional regulator GabR was developed in Corynebacterium glutamicum; and 2. a growth factor-based biosensor was built in Escherichia coli, which used auxotrophic complementation by expressing 4-aminobutyrate transaminase (GABA-T) that transfers the GABA amino group to pyruvate, hereby forming alanine. Consequently, the feasibility of developing a workflow based on co-culture with producer strains and a biosensor was tested. The three GABA producers were identified and the biosensors were encapsulated in nanoliter reactors (NLRs) as alginate beads in defined gut-like conditions. The E. coli growth factor-based biosensor was able to detect changes in GABA concentrations in liquid culture and under gut-like conditions. L. brevis and L. lactis were successfully encapsulated in the NLRs and showed growth under miniaturized intestinal conditions., (© 2023. Springer Nature Limited.)

This study evaluated the supplementation of a precision biotic (PB) on the enterohepatic health markers and growth performance of broiler chickens undergoing an enteric challenge. In the first study, three treatments were used: Unchallenged Control (UC); Challenged Control (CC; dietary challenge and 10× dose of coccidia vaccine); and a challenged group supplemented with PB (1.3 kg/ton). In the second study, three treatments were used: control diet, diet supplemented with Avilamycin (10 ppm), and a diet supplemented with PB (0.9 kg/ton). All the birds were exposed to natural challenge composed by dietary formulation and reused litter from a coccidiosis positive flock. In Trial 1, PB decreased ileal histological damage, increased villi length, and the expression of SLC5A8 in ileal tissue versus CC; it reduced ileal expression of IL-1β compared to both UC and CC treatments. PB increased the expression of cell cycling gene markers CCNA2 and CDK2 in the ileum compared to CC. In Trial 2, PB improved the growth performance, intestinal lesion scores and intestinal morphology of broiler chickens. These results indicate that birds supplemented with PB are more resilient to enteric challenges, probably by its action in modulating microbiome metabolic pathways related to nitrogen metabolism and protein utilization.

Fully understanding the dynamic distribution of the gut microbiota in pigs is essential, as gut microorganisms play a fundamental role in physiological processes, immunity, and the metabolism of nutrients by the host. Here, we first summarize the characteristics and the dynamic shifts in the gut microbial community of pigs at different ages based on the results of 63 peer-review publications. Then a meta-analysis based on the sequences from 16 studies with accession numbers in the GenBank database is conducted to verify the characteristics of the gut microbiota in healthy pigs. A dynamic shift is confirmed in the gut microbiota of pigs at different ages and growth phases. In general, Bacteroides , Escherichia, Clostridium , Lactobacillus , Fusobacterium , and Prevotella are dominant in piglets before weaning, then Prevotella and Aneriacter shift to be the predominant genera with Fusobacterium , Lactobacillus , and Miscellaneous as comparative minors in postweaned pigs. A number of 19 bacterial genera, including Bacteroides , Prevotella , and Lactobacillus can be found in more than 90% of pigs and three enterotypes can be identified in all pigs at different ages, suggesting there is a "core" microbiota in the gut of healthy pigs, which can be a potential target for nutrition or health regulation. The "core" members benefit the growth and gut health of the host. These findings help to define an "optimal" gut microbial profile for assessing, or improving, the performance and health status of pigs at different growth stages. IMPORTANCE The ban on feed antibiotics by more and more countries, and the expected ban on ZnO in feed supplementation from 2022 in the EU, urge researchers and pig producers to search for new alternatives. One possible alternative is to use the so-called "next-generation probiotics (NGPs)" derived from gastrointestinal tract. In this paper, we reveal that a total of 19 "core" bacterial genera including Bacteroides , Prevotella , and Lactobacillus etc., can be found in more than 90% of healthy pigs across different ages. These identified genera may probably be the potential candidates of NGPs or the potential target of microflora regulation. Adding substrates preferred by these target microbes will help to increase the abundance of specific symbiotic species and benefit the gut health of pigs. Further research targeting these "core" microbes and the dynamic distribution of microbiota, as well as the related function is of great importance in swine production.

This study evaluated the effects of vitamin E (Vit E) and selenium (Se) supplementation on mRNA abundance of antioxidant, immune response, and tight junction genes, as well as taxonomic and functional profiles of ileal microbiota of broilers exposed to daily 4-h elevated temperature during d 28 to 35. A total of 640-day-old Cobb male broiler chicks were randomly allocated to 32 floor pens in a 2 × 2 factorial arrangement that included ambient temperature (thermoneutral [TN] or heat stress [HS]) and dietary treatments (basal diet or Vit E + Se). Vit E and organic Se were added to the basal diet at the rate of 250 mg/kg and 1 mg/kg, respectively. Liver and jejunum tissue samples were taken on d 27 (1 bird/pen), d 28 and d 35 (2 birds/pen) from birds for qPCR analysis. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP. Ileal contents were taken on d 27 and d 35 for microbial profiling. Microbiota data were analyzed in QIIME 2 and significance between treatments identified linear discriminant analysis effect size (LEfSe, P < 0.05). Dietary Vit E/Se significantly downregulated the mRNA levels of HSPs in liver and jejunal tissues of the HS-challenged birds both on d 28 and d 35. Moreover, mRNA abundance of TLR2, TNFα, IFNγ, IL-1β, IL-10, and iNOS in the liver were significantly downregulated in birds fed the Vit E/Se diet on d 35. However, dietary treatment had no significant impact on oxidative stress, immunity, and gut integrity related genes analyzed in jejunal tissues on d 28 and d 35, except downregulation of IFNγ on d 35 (P = 0.052). LEfSe analysis revealed that Lachnospiraceae FE2018 and Ruminococcaceae NK4A214 groups was enriched in the Vit E/Se birds on d 35. Moreover, PICRUSt analysis predicted significant functional differences among the treatment groups. In conclusion, dietary supplementation of Vit E/Se mitigated the negative effects of HS potentially via improving antioxidant status, regulating cytokine responses and modifying ileal microbiota and its function., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A dietary glycan-based precision biotic (Glycan PB) was evaluated on the performance, welfare indicators, and litter characteristics of broiler chickens. In Trial 1, the main effects of Glycan PB dose (0, 250 and 500 g/metric ton (MT)) and xylanase supplementation (0 or 100 g/MT) were tested, as was their interaction. In Trial 2, pens located inside a commercial house were used to test the effect of Glycan PB supplementation (500 g/MT) versus a control diet. In Trial 1, Glycan PB supplementation at 250 and 500 g/MT improved feed conversion ratio (FCR) by 7 and 11 points when compared to diets without Glycan PB ( p < 0.001). At 35 d, Glycan PB reduced the pH and ammonia concentration in diets with xylanase. In Trial 1, the supplementation with 500 g of Glycan PB/MT of feed reduced litter scores ( p < 0.05). In both trials, 500 g of Glycan PB/MT of feed increased the proportions of birds without footpad lesions (Trial 1: 72.2% vs. 82.7%; p < 0.001; Trial 2: 14 to 27.3% ( p = 0.05) or gait defects (Trial 1: 96.1% vs. 98.4%; p < 0.001) and decreased the proportion of birds with footpad lesions (Trial 2: 86% vs. 72.7%; p = 0.05).

Learning theorists suggest extinction learning to be a central mechanism in weight loss success; however, empirical studies are scarce. In this pilot study, it was examined whether individual differences in extinction learning predict outcome after weight loss treatment. Overweight and obese individuals first completed a laboratory conditioning task in which individual differences in extinction learning were assessed. Next, they were randomised to one of two weight loss interventions: cue exposure therapy (CET), which is considered the clinical analogue of laboratory extinction, or a control lifestyle intervention. In line with expectations, better extinction learning in the laboratory task was associated with more weight loss at both post-treatment (CET only) and follow-up (both interventions) measurements. In contrast, two other indices of treatment success (reduction in overeating expectancies and ad libitum food intake during a laboratory taste test) showed no associations with pre-treatment extinction learning. It is suggested that extinction learning may be a core mechanism underlying weight loss success, and hence, an important target for new obesity interventions., (© 2020 The Authors. European Eating Disorders Review published by Eating Disorders Association and John Wiley & Sons Ltd.)

Here, we present the complete genome sequence of the Bacillus subtilis strain SP1. This strain is a descendant of the laboratory strain 168. The strain is suitable for biotechnological applications because the prototrophy for tryptophan has been restored. Due to laboratory cultivation, the strain has acquired 24 additional sequence variations., (Copyright © 2020 Richts et al.)

Calorie-reduced diets to combat obesity do work, but they only work when one sticks to the diet and the - relatively small - weight loss usually is short-lived. It is argued that calorie-reduced diets should converge with enduring lifestyle changes: the diet is just the start of a lifelong new eating pattern. Getting people to change their lifestyles forever could increase the amount of lost weight and prevent relapse. However, a real behavior change is difficult, especially when longstanding habits are involved and the change is intended for the rest of life. It is argued here that adherence to a new lifestyle is much easier if sabotaging cognitive processes are tackled. An overview is given of four studies into the effects of exposure to reduce appetitive responding to tempting food cues. A robust effect of exposure on the ad lib intake of exposed foods was found in all studies: participants ate significantly less of exposed foods after exposure compared to control interventions but no generalization to non-exposed foods was found. The reduced food intake after exposure was associated with a violation of overeating expectancies. It is discussed that lifestyle interventions might benefit from techniques that are really able to change longstanding habits. Specifically, the violation of overeating expectancies during exposure seems to be critical for controlled eating and should therefore be part of lifestyle interventions for obesity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Learning models of overeating predict that exposure therapy is effective in reducing food cue reactivity and overeating. This pilot study tested an eight-session exposure therapy aimed at inhibitory learning vs. an active control condition aimed at lifestyle improvement for obesity (treatment-as-usual). Main outcomes are snacking behavior, eating psychopathology, food cue reactivity, and weight loss. Change in overeating expectancies was assessed as mediator for outcomes, and the associations between habituation of eating desires and outcomes were investigated in the exposure condition. Sleep quality was investigated as moderator for outcomes., Methods: 45 overweight women were randomly assigned to the exposure intervention or control condition. The main outcomes, overeating expectancies and sleep quality were re-assessed at post-treatment and three-month follow-up. Habituation of eating desires was measured during exposure sessions., Results: Compared to the control intervention, exposure led to a significantly stronger reduction in snacking behavior of exposed foods, though this effect did not generalize to non-exposed foods, and stronger binge eating frequency. The exposure condition lost significantly more weight at post-treatment and follow-up than the lifestyle condition. Changes of expectancies mediated the effect of condition on kcal consumption of exposed foods, while habituation during exposure was not related to better treatment outcome. Sleep quality did not moderate the effect of condition on treatment outcome., Limitations: Small sample size and limited follow-up period., Conclusions: This short exposure therapy reduced snacking behavior, binge eating and weight more than a lifestyle intervention and is therefore a recommendable intervention for obesity and overeating disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: People with overweight have stronger reactivity (e.g., subjective craving) to food cues than lean people, and this reactivity is positively associated with food intake. Cue reactivity is a learned response that can be reduced with food cue exposure therapy., Objectives: It was hypothesized that participants after food cue exposure therapy would show reduced neural activity in brain regions related to food cue reactivity and increased neural activity in brain regions related to inhibitory-control as compared to participants receiving a control lifestyle intervention., Method: Neural activity of 10 women with overweight (BMI ≥ 27 kg/m 2 ) in response to individually tailored visually presented palatable high-caloric food stimuli was examined before vs. after a cue exposure intervention ( n = 5) or a control lifestyle ( n = 5) intervention. Data were analyzed case-by-case., Results: Neural responses to food stimuli were reduced in food-cue-reactivity-related brain regions after the lifestyle intervention in most participants, and generally not after the cue exposure therapy. Moreover, cue exposure did not lead to increased activity in inhibitory-control-related brain regions. However, decreased neural activity after cue exposure was found in most participants in the lateral occipital complex (LOC), which suggests a decreased visual salience of high-caloric food stimuli., Conclusion: Receiving a cue exposure therapy did not lead to expected neural responses. As cue exposure relies on inhibitory learning mechanisms, differences in contexts (e.g., environments and food types) between the intervention setting and the scanning sessions may explain the general lack of effect of cue-exposure on neural activity., (Copyright © 2020 Franssen, Jansen, Schyns, van den Akker and Roefs.)

Findings of a few recent studies suggest that obesity is characterized by deficits in appetitive learning about food cues. This could point towards an increased tendency in obese individuals to overgeneralize appetitive responding to stimuli that resemble the conditioned food cue - possibly explaining frequent eating desires and overeating in obesity. The current study aimed to investigate whether obese individuals 1) indeed exhibit appetitive learning deficits and 2) show overgeneralization of conditioned appetitive responses. Obese and matched healthy-weight females (N = 85) completed a differential appetitive conditioning task using food as rewards. First, appetitive responding to a novel stimulus was learned (acquisition phase), after which stimuli with varying resemblance to the food-associated stimulus were presented (generalization phase). Cue-elicited eating expectancies, eating desires, stimulus evaluations, and actual food intake were examined. Results indicated successful acquisition of appetitive responding across all outcome measures. The acquired responses also generalized, indicating that generalization can function as an additional mechanism by which learned food cues can promote food consumption. The data further suggested that overweight and obesity are not characterized by appetitive learning deficits nor by overgeneralization, but that a subgroup of obese individuals (those high in trait anxiety) may be more prone to overgeneralization of appetitive responding., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: This study tested the role of habituation of eating desires and violation of overeating expectancies during food cue exposure in obese women., Method: 52 obese females were randomised into a two-session exposure condition aimed at habituation, a two-session exposure condition aimed at expectancy violation, or a no-treatment control condition. Eating in the absence of hunger of foods included during cue exposure (i.e., exposed foods) and foods not included during cue exposure (i.e., non-exposed foods), and duration of exposure were measured., Results: Both cue exposure conditions ate significantly less of the exposed foods compared to the control condition, though there were no differences between both types of exposure. No differences were found between conditions regarding the eating of non-exposed foods. In addition, the duration of exposure was not different between both cue exposure conditions., Conclusions: While food cue exposure in obese women led to less eating of exposed foods, focusing on either habituation of eating desires or expectancy violation did not matter. It is discussed why exposure works., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Objectives: This study tested whether two sessions of food cue exposure therapy reduced eating in the absence of hunger (EAH), specified for exposed and non-exposed food, in overweight and obese adolescents, and whether habituation of food cue reactivity and reduced CS-US expectancies predicted a decrease in EAH., Methods: 41 overweight adolescents (aged 12-18 years) were randomly assigned to a cue exposure intervention or a lifestyle intervention (control condition). Habituation of food cue reactivity (self-reported desire to eat and salivation) and CS-US expectancy were measured during both sessions, and EAH was measured at the end of session two., Results: Compared to the control condition, the cue exposure condition showed less EAH for the exposed food item as well as for the non-exposed food items. Larger within-session (WSH) and between-session habituation (BSH) of cue reactivity were not related to less EAH, change in CS-US expectancy was unrelated to EAH., Limitations: The study was underpowered, and compliance to homework instructions between sessions was poor, intervention effects might have been larger when participants adhered to daily homework exercises., Conclusions: Food cue exposure was effective to reduce EAH of exposed and non-exposed food items, indicating generalisability of the exposure effect. In line with exposure effects in anxiety disorders, habituation was not found to benefit outcome, though the present data do also not provide evidence that CS-US expectancy violation predicts EAH., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose of Review: This review provides an overview of recent findings relating to the role of Pavlovian conditioning in food cue reactivity, including its application to overeating and weight loss interventions., Recent Findings: Both in the laboratory and in real life, cue-elicited appetitive reactivity (e.g., eating desires) can be easily learned, but (long-term) extinction is more difficult. New findings suggest impaired appetitive learning in obesity, which might be causally related to overeating. The clinical analogue of extinction-cue exposure therapy-effectively reduces cue-elicited cravings and overeating. While its working mechanisms are still unclear, some studies suggest that reducing overeating expectancies is important., Summary: Pavlovian learning theory provides a still undervalued theoretical framework of how cravings and overeating can be learned and how they might be effectively tackled. Future studies should aim to elucidate inter-individual differences in Pavlovian conditioning, study ways to strengthen (long-term) extinction, and investigate the working mechanisms of cue exposure therapy., Competing Interests: Compliance with Ethical StandardsThe authors declare that they have no conflict of interest.All reported studies/experiments with human or animal subjects performed by the authors have been previously published and complied with all applicable ethical standards.

Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Overweight and obese individuals show increased psychological and physiological reactivity to food cues and many of them have difficulties in achieving long-term weight loss. The current study tests whether abnormalities in the learning and extinction of appetitive responses to food cues might be responsible for this. Overweight/obese and healthy weight women completed a differential appetitive conditioning task using food as rewards, while eating expectancies, eating desires, conditioned stimulus evaluations, salivation, and electrodermal responses were assessed during an acquisition and extinction phase. Results suggested reduced discriminative conditioning in the overweight/obese group, as reflected by a worse acquisition of differential eating desires and no successful acquisition of differential evaluative responses. Some evidence was also found for impaired contingency learning in overweight and obese individuals. No group differences in conditioned salivation and skin conductance responses were found and no compelling evidence for differences in extinction was found as well. In sum, the current findings indicate that overweight and obesity may be characterized by reduced appetitive conditioning. It is suggested that this could be causally related to overeating via stronger context conditioning or a tendency towards overgeneralization in overweight and obese individuals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Food cue reactivity is a strong motivation to eat, even in the absence of hunger. Therefore, food cue reactivity might sabotage healthy eating, induce weight gain and impede weight loss or weight maintenance. Food cue reactivity can be learned via Pavlovian appetitive conditioning: It is easily acquired but the extinction of appetitive responding seems to be more challenging. Several properties of extinction make it fragile: extinction does not erase the original learning and extinction is context-dependent. These properties threaten full extinction and increase the risk of full relapse. Extinction procedures are discussed to reduce or prevent the occurrence of rapid reacquisition, spontaneous recovery, renewal and reinstatement after extinction. A translation to food cue exposure treatment is made and suggestions are provided, such as conducting the exposure in relevant contexts, using occasional reinforcement and targeting expectancy violation instead of habituation. A new hypothesis proposed here is that the adding of inhibition training to strengthen inhibition skills that reduce instrumental responding, might be beneficial to improve food cue exposure effects., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The prevalence of overweight and obesity has increased substantially over the last decades. Weight loss attempts in overweight individuals are common, though they seldom result in successful long-term weight loss. One very promising treatment is food cue exposure therapy, during which overweight individuals are repeatedly exposed to food-associated cues (e.g., the sight, smell and taste of high-calorie foods, overeating environments) without eating in order to extinguish cue-elicited appetitive responses to food cues. However, only few studies have tested the effectiveness of cue exposure, especially with regards to weight loss. For exposure treatment of anxiety disorders, it has been proposed that inhibitory learning is critical for exposure to be effective. In this RCT, we translated techniques proposed by Craske et al. (2014) to the appetitive domain and developed a novel cue exposure therapy for overeating aimed at maximizing inhibitory learning. The current RCT tested the effectiveness of this 8-session cue exposure intervention relative to a control intervention in 45 overweight adult (aged 18-60) females at post-treatment and 3-month follow-up, of which 39 participants completed the study. Weight loss, eating psychopathology, food cue reactivity, and snacking behaviour were studied as main treatment outcomes, and mediators and moderators of treatment effects were studied. The presented study design represents an innovative effort to provide valuable clinical recommendations for the treatment of overeating and obesity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present study investigated whether a single-session of food cue exposure for overweight women would decrease 'if CS then US' expectancies, cue reactivity and eating in the absence of hunger (EAH). EAH was measured in a behavioural paradigm that enabled to also investigate whether the cue exposure effects were specific for exposed foods or would generalise to food items that were not present during exposure. Overweight women were randomly assigned to either the cue exposure intervention or a control intervention that focused on body image. In line with the hypotheses, results showed that cue exposure induced a significant decrease in 'if CS then US' expectancies, in contrast to the control intervention. It was also found that, compared to the control intervention, desires to eat initially increased during cue exposure while gradual extinction was observed towards the end of the intervention. No extinction of increased salivation responses was found. Regarding EAH, the intake of the exposed food item was significantly less in the exposure condition than in the control condition, whereas total caloric food intake was not different between conditions, indicating that cue exposure was effective in reducing intake but did not generalise to the intake of other food items., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Obesity prevalence among children is high and knowledge on cognitive factors that contribute to children's reactivity to the "obesogenic" food environment could help to design effective treatment and prevention campaigns. Empirical studies in adults suggest that attention bias for food could be a risk factor for overeating. Accordingly, the current study tested if children with obesity have an elevated attention bias for food when compared to healthy-weight children. Another aim was to explore whether attention biases for food predicted weight-change after 3 and 6 months in obese children., Method: Obese children (n = 34) were recruited from an intervention program and tested prior to the start of this intervention. Healthy-weight children (n = 36) were recruited from local schools. First, attention biases for food were compared between children with obesity (n = 30) and matched healthy-weight children (n = 30). Second, regression analyses were conducted to test if food-related attention biases predicted weight changes after 3 and 6 months in children with obesity following a weight loss lifestyle intervention., Results: Results showed that obese children did not differ from healthy-weight children in their attention bias to food. Yet automatically directing attention toward food (i.e., initial orientation bias) was related to a reduced weight loss (R² = .14, p = .032) after 6 months in children with obesity., Discussion: High palatable food is a salient stimulus for all children, irrespective of their weight status. However, automatically directing attention to food cues might facilitate further weight gain in children with obesity., ((c) 2015 APA, all rights reserved).)

Bacillus subtilis is a Gram-positive, rod-shaped, spore-forming bacterium. We present the genome sequence of an undomesticated strain, BSP1, isolated from poultry. The sequence of the BSP1 genome supports the view that B. subtilis has a biphasic lifestyle, cycling between the soil and the animal gastrointestinal tract, and it provides molecular-level insight into the adaptation of B. subtilis to life under laboratory conditions.

The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric beta-glucuronidase enzyme from Escherichia coli.

The physiological function for thiaminase II, a thiamin-degrading enzyme, has eluded investigators for more than 50 years. Here, we demonstrate that this enzyme is involved in the regeneration of the thiamin pyrimidine rather than in thiamin degradation, and we identify a new pathway involved in the salvage of base-degraded forms of thiamin. This pathway is widely distributed among bacteria, archaea and eukaryotes. In this pathway, thiamin hydrolysis products such as N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine (formylaminopyrimidine; 15) are transported into the cell using the ThiXYZ transport system, deformylated by the ylmB-encoded amidohydrolase and hydrolyzed to 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP; 6)-an intermediate on the de novo thiamin biosynthetic pathway. To our knowledge this is the first example of a thiamin salvage pathway involving thiamin analogs generated by degradation of one of the heterocyclic rings of the cofactor.

In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this DeltathiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the DeltathiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).

We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a sigma(A)-type promoter bearing the TG dinucleotide motif of "extended" -10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified sigma(A)-containing RNA polymerase (Esigma(A)), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by Esigma(A) during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.

The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.

During endospore formation in Bacillus subtilis, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing sigmaK. GerE binds to a site on one of these promoters, cotX, that overlaps its -35 region. We tested the model that GerE interacts with sigmaK at the cotX promoter by seeking amino acid substitutions in sigmaK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the sigmaK-dependent, GerE-independent promoter gerE. We identified two amino acid substitutions in sigmaK, E216K and H225Y, that decrease cotX promoter utilization but do not affect gerE promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in sigmaK on transcription in vitro. We found that these substitutions specifically reduced utilization of the cotX promoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent cotX promoter activity, that the histidine at position 225 of sigmaK may interact with GerE at the cotX promoter, and that this interaction may facilitate the initial binding of sigmaK RNA polymerase to the cotX promoter. We also found that the alanine substitutions at positions 216 and 225 of sigmaK had no effect on utilization of the GerE-dependent promoter cotD, which contains GerE binding sites that do not overlap with its -35 region.

Spo0A is a DNA binding protein in Bacillus subtilis required for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the sigmaA subunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in sigmaA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in sigmaA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the sigmaA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIA promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, sigmaH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in sigmaA-dependent promoter activation.

To study the transcriptional apparatus and the mechanisms that control gene expression in cyanobacteria, the RNA polymerase was purified from the filamentous Calothrix sp. PCC 7601 and used in in vitro transcription assays. Conditions required for specific transcription initiation to occur were analyzed with the eleven Calothrix PCC 7601 genes for which the 5' ends have been mapped. Most of the transcripts directly obtained did not have the expected size, providing a test for looking at specific transcription factors. Addition of RcaA, a protein that binds to the promoter region of the phycobiliprotein cpeBA operon, restored accurate initiation of transcription in the in vitro system for three phycobiliprotein promoters. RcaA thus is a transcription factor that allows to mimick in vivo transcription. In parallel, the functional properties of the Escherichia coli and cyanobacterial RNA polymerases were compared. The enteric enzyme could not precisely initiate transcription at the promoter of a phycobiliprotein gene and, reciprocally, the cyanobacterial RNA polymerase could initiate transcription at PlacUV5, but not from wild-type Plac promoters. The different behaviours of the enzymes are discussed in the light of the structural differences that exist between subunits of the RNA polymerases.

Although in cyanobacteria many genes have been shown to be transcriptionally controlled by specific stimuli, little is known about promoter structure and the form of RNA polymerase that recognizes individual promoters. RNA polymerase holoenzyme has been purified from Calothrix sp. PCC 7601. Its polypeptide composition resembles that of the plant chloroplast enzymes. To study transcription in cyanobacteria further, we have analysed the promoter-recognition properties of the purified enzyme. In vitro transcription was assayed with the promoter of the phycocyanin gene (cpc1) that is expressed whatever the incident light conditions. Transcription initiation at the same start point as in vivo was obtained with the Calothrix sp. PCC 7601 purified enzyme and the Escherichia coli core enzyme supplemented with a Calothrix sp. PCC 7601 sigma factor, but not with the E. coli holoenzyme.