Horn N.Wegmann U.Dertli E.Mulholland F.Collins S.R.A.Waldron K.W.Bongaerts R.J.Mayer M.J.Narbad A.20.04.20192019-04-2020.04.20192019-04-2020131932-6203https://dx.doi.org/10.1371/journal.pone.0059957https://hdl.handle.net/20.500.12403/863As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ?epsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions. © 2013 Horn et al.eninfo:eu-repo/semantics/openAccessexopolysaccharidephosphotransferaseprotein tyrosine kinaseunclassified drugundecaprenyl phosphate galactose phosphotransferaseamino acid substitutionarticlebacterial colonizationbacterial genebacterial strainbacterium adherencebacterium isolationcarbohydrate analysiscarbohydrate synthesiscell structurecontrolled studyDNA sequenceenzyme synthesisexopolysaccharide C geneexopolysaccharide E geneexopolysaccharide genegenegene clustergene expressiongene functiongene mutationhost pathogen interactionhumanhuman cellLactobacillus johnsoniimutational analysisnonhumannucleotide sequencephenotypeprotein transporttwo dimensional gel electrophoresiswild typeAmino Acid SequenceBacterial AdhesionBacterial ProteinsCarbohydratesCell MembraneColony Count, MicrobialGene DeletionGenes, BacterialGenetic Complementation TestHT29 CellsHumansLactobacillusMolecular Sequence DataMolecular WeightMultigene FamilyMutationPhenotypePolysaccharides, BacterialProteomicsSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationBacteria (microorganisms)Lactobacillus johnsoniiexopolysaccharidephosphotransferaseprotein tyrosine kinaseunclassified drugundecaprenyl phosphate galactose phosphotransferaseamino acid substitutionarticlebacterial colonizationbacterial genebacterial strainbacterium adherencebacterium isolationcarbohydrate analysiscarbohydrate synthesiscell structurecontrolled studyDNA sequenceenzyme synthesisexopolysaccharide C geneexopolysaccharide E geneexopolysaccharide genegenegene clustergene expressiongene functiongene mutationhost pathogen interactionhumanhuman cellLactobacillus johnsoniimutational analysisnonhumannucleotide sequencephenotypeprotein transporttwo dimensional gel electrophoresiswild typeAmino Acid SequenceBacterial AdhesionBacterial ProteinsCarbohydratesCell MembraneColony Count, MicrobialGene DeletionGenes, BacterialGenetic Complementation TestHT29 CellsHumansLactobacillusMolecular Sequence DataMolecular WeightMultigene FamilyMutationPhenotypePolysaccharides, BacterialProteomicsSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationBacteria (microorganisms)Lactobacillus johnsoniiArticle8310.1371/journal.pone.0059957235441142-s2.0-84875446710Q1WOS:000317480700073Q1