A Single Mutation in the Gene Responsible for the Mucoid Phenotype of Bifidobacterium animalis subsp. lactis Confers Surface and Functional Characteristics

Abstract

Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.T he genus Bifidobacterium includes commensal microorganisms commonly found in the human gut. Some strains, belonging mainly to the species Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, and Bifidobacterium animalis, have been used as probiotics, since there is scientific evidence that links the administration of these bacteria with specific health benefits (1, 2). In particular, B. animalis subsp. lactis has a robust phenotype that allows growth at high numbers in commercial applications under nonanaerobic conditions. Furthermore, strains of this subspecies survive the gastric passage and reach the intestinal tract in a metabolically active state, being also the most common representatives of bifidobacteria in functional food products (3). Because they are more resistant to harsh environmental conditions, strains of B. animalis subsp. lactis have been studied more than those of other members of the genus Bifidobacterium, and clinical studies demonstrated their healthpromoting attributes in some gastrointestinal disorders and allergic processes (4-6).Exopolysaccharides (EPS) are extracellular layers of carbohydrates typically found in microorganisms (7). EPS production has been involved in a variety of physiological processes that have implications for the producer microorganism and the host (8, 9). Specifically, in intestinal bacteria some of the most relevant roles attributed to EPS are the favoring of intestinal colonization and survival (10-12) and immunomodulation (11,13,14).A common set of functional modules, including the synthesis of activated sugar precursors, cytoplasmic assembly of the EPS unit, export...

The Surface-Associated Exopolysaccharide of Bifidobacterium longum 35624 Plays an Essential Role in Dampening Host Proinflammatory Responses and Repressing Local T H 17 Responses

“…In addition, the cytokine response of PBMCs to two isogenic strains of B. animalis subsp. lactis that differ only in their exopolysaccharide-producing phenotype suggest that the mucoid strain could have greater anti-inflammatory activity (31). The data presented here are in agreement with these previous reports and further support the concept that exopolysaccharides from bifidobacteria may elicit immune-modulatory activities.…”

“…A similar phenotype was observed for exopolysaccharide-negative variants of B. breve UCC2003 and Bifidobacterium animalis subsp. lactis (21,31), thereby substantiating the loss of exopolysaccharide production. To ensure that the observed phenotype is directly linked to the inactivation of pgt 624 , the adjacent and cotranscribed genes pgt 624 and BI0343 were cloned together with the presumed promoter sequence in plasmid pBC1.2, after which the resultant construct was introduced into the sEPS neg strain.…”

Ropy exopolysaccharide‐producing Bifidobacterium longum YS108R as a starter culture for fermented milk

“…A mutation in the gene predicted to encode Etk‐like tyrosine kinase in a B. animalis subsp. lactis strain was reported to cause the synthesis of EPS with higher molecular weight and the appearance of a mucoid phenotype (Hidalgo‐Cantabrana et al ., ). In this study, the mutant protein in YS108R was homologous to Etk‐like tyrosine kinases which is predicted to have a large soluble domain and is theoretically responsible for the determination of chain length of saccharide polymer (Woodward et al ., ).…”

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

“…To trigger genome editing, we codelivered a repair template cloned into the self-targeting plasmid containing the CRISPR array to enable the host to overcome Cas3-based targeting and damage. First, we used the p-gtf target to generate a knockout, since the mutants will conveniently display a visibly distinct phenotype due to the altered exopolysaccharide content (51)(52)(53), which can also lead to altered probiotic features, such as adherence, stress resistance, and modulation of the host immune system (54)(55)(56)(57). We designed the repair template to encompass sequences 1 kilobase (kb) upstream and 1 kb downstream of the target protospacer, and cloned into SalI-PvuI-digested pTRK1184 to generate pTRK1185 (SI Appendix, Fig.…”