Genetic Competence Drives Genome Diversity in Bacillus subtilis

This deposit is composed by the main article plus the supplementary materials of the publication.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.Fundação para a Ciência e a Tecnologia grants: (PTDC/EBB-BIO/119006/2010, PEst-OE/EQB/LA0004/2011, SFRH/BPD/89907/2012, SFRH/BD/29397/06); FEDER grant: (LISBOA-01-0145-FEDER-007660).info:eu-repo/semantics/publishedVersio

Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis

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 ΔthiL 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 ΔthiL, 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)

A Gene Encoding a Holin-Like Protein Involved in Spore Morphogenesis and Spore Germination in Bacillus subtilis

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 σ(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 σ(A)-containing RNA polymerase (Eσ(A)), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by Eσ(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

Evaluation of a Precision Biotic on the Growth Performance, Welfare Indicators, Ammonia Output, and Litter Quality of Broiler Chickens

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)

Evaluation of a Novel Precision Biotic on Enterohepatic Health Markers and Growth Performance of Broiler Chickens under Enteric Challenge

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

Development of a workflow for the selection, identification and optimization of lactic acid bacteria with high γ-aminobutyric acid production

Abstract 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