scholarly journals Polymorphisms, Chromosomal Rearrangements, and Mutator Phenotype Development during Experimental Evolution of Lactobacillus rhamnosus GG

2016 ◽  
Vol 82 (13) ◽  
pp. 3783-3792 ◽  
Author(s):  
François P. Douillard ◽  
Angela Ribbera ◽  
Kun Xiao ◽  
Jarmo Ritari ◽  
Pia Rasinkangas ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Chromosomal Rearrangements ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Shearing Stress ◽  
Genomic Integrity ◽  
Mutator Phenotype ◽  
Content Type ◽  
Is Elements ◽  
The Stability

ABSTRACTLactobacillus rhamnosusGG is a lactic acid bacterium widely marketed by the food industry. Its genomic analysis led to the identification of a gene cluster encoding mucus-binding SpaCBA pili, which is located in a genomic island enriched in insertion sequence (IS) elements. In the present study, we analyzed by genome-wide resequencing the genomic integrity ofL. rhamnosusGG in four distinct evolutionary experiments conducted for approximately 1,000 generations under conditions of no stress or salt, bile, and repetitive-shearing stress. Under both stress-free and salt-induced stress conditions, the GG population (excluding the mutator lineage in the stress-free series [see below]) accumulated only a few single nucleotide polymorphisms (SNPs) and no frequent chromosomal rearrangements. In contrast, in the presence of bile salts or repetitive shearing stress, some IS elements were found to be activated, resulting in the deletion of large chromosomal segments that include thespaCBA-srtC1pilus gene cluster. Remarkably, a high number of SNPs were found in three strains obtained after 900 generations of stress-free growth. Detailed analysis showed that these three strains derived from a founder mutant with an altered DNA polymerase subunit that resulted in a mutator phenotype. The present work confirms the stability of the pilus production phenotype inL. rhamnosusGG under stress-free conditions, highlights the possible evolutionary scenarios that may occur when this probiotic strain is extensively cultured, and identifies external factors that affect the chromosomal integrity of GG. The results provide mechanistic insights into the stability of GG in regard to its extensive use in probiotic and other functional food products.IMPORTANCELactobacillus rhamnosusGG is a widely marketed probiotic strain that has been used in numerous clinical studies to assess its health-promoting properties. Hence, the stability of the probiotic functions ofL. rhamnosusGG is of importance, and here we studied the impact of external stresses on the genomic integrity ofL. rhamnosusGG. We studied three different stresses that are relevant for understanding its robustness and integrity under bothex vivoconditions, i.e., industrial manufacturing conditions, andin vivoconditions, i.e., intestinal tract-associated stress. Overall, our findings contribute to predicting the genomic stability ofL. rhamnosusGG and its ecological performance.

scholarly journals Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics

2013 ◽  
Vol 79 (6) ◽  
pp. 1923-1933 ◽  
Author(s):  
François P. Douillard ◽  
Angela Ribbera ◽  
Hanna M. Järvinen ◽  
Ravi Kant ◽  
Taija E. Pietilä ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Transcription Initiation ◽  
Lactobacillus Rhamnosus ◽  
Comparative Genomic ◽  
Phenotypic Analysis ◽  
Carbohydrate Utilization ◽  
Content Type ◽  
Host Signaling ◽  
Toll Like Receptor 2 ◽  
Genome Content

ABSTRACTFourLactobacillusstrains were isolated from marketed probiotic products, includingL. rhamnosusstrains from Vifit (Friesland Campina) and Idoform (Ferrosan) andL. caseistrains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail withL. caseistrain BL23 andL. rhamnosusstrain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization betweenL. caseiandL. rhamnosusstrains, which could be linked to their genotypes. The two isolatedL. rhamnosusstrains had genomes that were virtually identical to that ofL. rhamnosusGG, testifying to their genomic stability and integrity in food products. TheL. caseistrains showed much greater genomic heterogeneity. Remarkably, all strains contained an intactspaCBApilus gene cluster. However, only theL. rhamnosusstrains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of aniso-IS30element upstream of the pilus gene cluster inL. rhamnosusstrains but absent inL. caseistrains had constituted a functional promoter driving pilus gene expression. AllL. rhamnosusstrains triggered an NF-κB response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas theL. caseistrains did not or did so to a much lesser extent. This study demonstrates that the twoL. rhamnosusstrains isolated from probiotic products are virtually identical toL. rhamnosusGG and further highlights the differences between these andL. caseistrains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities.

scholarly journals Lactobacillus rhamnosus GG Genomic and Phenotypic Stability in an Industrial Production Process

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Marianne Stage ◽  
Anita Wichmann ◽  
Mette Jørgensen ◽  
Natalia Ivonne Vera-Jimenéz ◽  
Malue Wielje ◽  
...  
Keyword(s):  
Production Process ◽  
Gene Cluster ◽  
Industrial Production ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Properties ◽  
Phenotypic Stability ◽  
Content Type ◽  
Freeze Dried ◽  
Probiotic Strains

ABSTRACT Lactobacillus rhamnosus GG is one of the most widely marketed and studied probiotic strains. In L. rhamnosus GG, the spaCBA-srtC1 gene cluster encodes pili, which are important for some of the probiotic properties of the strain. A previous study showed that the DNA sequence of the spaCBA-srtC1 gene cluster was not present in some L. rhamnosus GG variants isolated from liquid dairy products. To examine the stability of the L. rhamnosus GG genome in an industrial production process, we sequenced the genome of samples of L. rhamnosus GG (DSM 33156) collected at specific steps of the industrial production process, including the culture collection stock, intermediate fermentations, and final freeze-dried products. We found that the L. rhamnosus GG genome sequence was unchanged throughout the production process. Consequently, the spaCBA-srtC1 gene locus was intact and fully conserved in all 31 samples examined. In addition, different production batches of L. rhamnosus GG exhibited consistent phenotypes, including the presence of pili in final freeze-dried products, and consistent characteristics in in vitro assays of probiotic properties. Our data show that L. rhamnosus GG is highly stable in this industrial production process. IMPORTANCE Lactobacillus rhamnosus GG is one of the best-studied probiotic strains. One of the well-characterized features of the strain is the pili encoded by the spaCBA-srtC1 gene cluster. These pili are involved in persistence in the gastrointestinal tract and are important for the probiotic properties of L. rhamnosus GG. Previous studies demonstrated that the L. rhamnosus GG genome can be unstable under certain conditions and can lose the spaCBA-srtC1 gene cluster. Since in vitro studies have shown that the loss of the spaCBA-srtC1 gene cluster decreases certain L. rhamnosus GG probiotic properties, we assessed both the genomic stability and phenotypic properties of L. rhamnosus GG throughout an industrial production process. We found that neither genomic nor phenotypic changes occurred in the samples. Therefore, we demonstrate that L. rhamnosus GG retains the spaCBA-srtC1 cluster and exhibits excellent genomic and phenotypic stability in the specific industrial process examined here.

scholarly journals Evaluation of Cereals and Pseudocereals Suitability for the Development of New Probiotic Foods

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Monika Kocková ◽  
Monika Dilongová ◽  
Eva Hybenová ◽  
L’ubomír Valík
Keyword(s):  
Organic Acids ◽  
Ph Value ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Colony Form ◽  
Cell Counts ◽  
The Stability ◽  
And Storage

The aim of the present work was to evaluate suitability of cereals and pseudocereals for the development of new probiotic foods and to evaluate the stability of cereal and pseudocereal porridges fermented by probiotic strainLactobacillus rhamnosusGG. Ten samples of cereals and pseudocereals obtained from Slovak mill house and markets were used in this work. A mixture of each cereal and pseudocereal samples with water (10% w/v) was inoculated after sterilization with coequal number ofLactobacillus rhamnosusGG, to obtain approximately 5-6 log colony form units per gram of suspensions. Fermentation was led at 37°C during 10 hours. Fermented suspensions were stored for 21 days at 5°C. Monitoring of cell counts, pH value, and concentration of organic acids during fermentation and storage was done.

scholarly journals Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

2014 ◽  
Vol 81 (4) ◽  
pp. 1387-1396 ◽  
Author(s):  
Milica Zivkovic ◽  
Marija Miljkovic ◽  
Patricia Ruas-Madiedo ◽  
Ivana Strahinic ◽  
Maja Tolinacki ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gene Cluster ◽  
Probiotic Strain ◽  
Gene Clusters ◽  
Size Exclusion ◽  
High Molecular Weight Fraction ◽  
Transposase Gene ◽  
Content Type ◽  
Lactobacillus Paraplantarum

ABSTRACTLactobacillus paraplantarumBGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of theepsgene cluster, a putative transposase gene was identified, followed by an additionalrfbgene cluster containing therfbACBDgenes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in theepsand the other in therfbgene cluster, as confirmed by size exclusion chromatography analysis. Therefore, bothepsandrfbgenes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strainL. paraplantarumBGCG11.

scholarly journals Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

2009 ◽  
Vol 75 (11) ◽  
pp. 3554-3563 ◽  
Author(s):  
Sarah Lebeer ◽  
Tine L. A. Verhoeven ◽  
Grégory Francius ◽  
Geert Schoofs ◽  
Ivo Lambrichts ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Single Molecule ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Extracellular Polysaccharides ◽  
Gene Encoding ◽  
Single Molecule Force Spectroscopy ◽  
Monomer Composition ◽  
Composition Determination ◽  
Surface Polysaccharides

ABSTRACT Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.

scholarly journals Genomic Characterization of Non-Mucus-Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis

2014 ◽  
Vol 80 (22) ◽  
pp. 7001-7009 ◽  
Author(s):  
Pia Rasinkangas ◽  
Justus Reunanen ◽  
François P. Douillard ◽  
Jarmo Ritari ◽  
Virva Uotinen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Lactobacillus Rhamnosus ◽  
Illumina Miseq ◽  
Class Iii ◽  
Dot Blot ◽  
Content Type ◽  
Intestinal Mucus ◽  
Pilus Biogenesis ◽  
Dot Blot Analysis ◽  
The Impact

ABSTRACTLactobacillus rhamnosusGG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed thatL. rhamnosusGG harbors a genomic island including thespaCBA-srtC1gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis inL. rhamnosusGG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied toL. rhamnosusGG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in thesrtC1gene; class II, with a deletion containing thespaCBA-srtC1gene cluster; and class III, with mutations in thespaAgene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficientsrtC1gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.

scholarly journals Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG

2011 ◽  
Vol 77 (13) ◽  
pp. 4465-4472 ◽  
Author(s):  
Ingemar von Ossowski ◽  
Reetta Satokari ◽  
Justus Reunanen ◽  
Sarah Lebeer ◽  
Sigrid C. J. De Keersmaecker ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Functional Characterization ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Surface Proteins ◽  
Adhesive Interaction ◽  
Content Type ◽  
Intestinal Mucus ◽  
Mucosal Surfaces

ABSTRACTIn spite of the wealth of clinical evidence supporting the health benefits ofLactobacillus rhamnosusGG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover,L. rhamnosusGG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) inL. rhamnosusGG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction betweenL. rhamnosusGG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean thatL. rhamnosusGG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic.

scholarly journals A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

2014 ◽  
Vol 81 (5) ◽  
pp. 1708-1714 ◽  
Author(s):  
Min-Sik Kim ◽  
Ae Ran Choi ◽  
Seong Hyuk Lee ◽  
Hae-Chang Jung ◽  
Seung Seob Bae ◽  
...  
Keyword(s):  
Production Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Transcriptional Regulator ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

scholarly journals Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

2014 ◽  
Vol 58 (10) ◽  
pp. 6185-6196 ◽  
Author(s):  
Marius Spohn ◽  
Norbert Kirchner ◽  
Andreas Kulik ◽  
Angelika Jochim ◽  
Felix Wolf ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
High Performance ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Gene Clusters ◽  
Von Willebrand Disease ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

Fusion based learning approach for predicting concrete pouring productivity based on construction and supply parameters

2016 ◽  
Vol 16 (2) ◽  
pp. 185-202 ◽  
Author(s):  
Mojtaba Maghrebi ◽  
Ali Shamsoddini ◽  
S. Travis Waller
Keyword(s):  
Supply Chain ◽  
Production Rate ◽  
Learning Method ◽  
Data Set ◽  
Content Type ◽  
Field Database ◽  
The Stability ◽  
Stable Learning ◽  
Ready Mixed Concrete ◽  
Practical Implications

Purpose The purpose of this paper is to predict the concrete pouring production rate by considering both construction and supply parameters, and by using a more stable learning method. Design/methodology/approach Unlike similar approaches, this paper considers not only construction site parameters, but also supply chain parameters. Machine learner fusion-regression (MLF-R) is used to predict the production rate of concrete pouring tasks. Findings MLF-R is used on a field database including 2,600 deliveries to 507 different locations. The proposed data set and the results are compared with ANN-Gaussian, ANN-Sigmoid and Adaboost.R2 (ANN-Gaussian). The results show better performance of MLF-R obtaining the least root mean square error (RMSE) compared with other methods. Moreover, the RMSEs derived from the predictions by MLF-R in some trials had the least standard deviation, indicating the stability of this approach among similar used approaches. Practical implications The size of the database used in this study is much larger than the size of databases used in previous studies. It helps authors draw their conclusions more confidently and introduce more generalised models that can be used in the ready-mixed concrete industry. Originality/value Introducing a more stable learning method for predicting the concrete pouring production rate helps not only construction parameters, but also traffic and supply chain parameters.

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