Structure of the high molecular weight exopolysaccharide produced by Bifidobacterium animalis subsp. lactis IPLA-R1 and sequence analysis of its putative eps cluster

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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...

Section: Resultssupporting

confidence: 73%

“…The strain IPLA-R1 is a derivative of A1dOx, which, after consecutive generations, spontaneously acquired a mucoid phenotype (20). The sequencing of the EPS clusters (32) (Fig. 1A) in the wild-type and the mutant strains allowed the identification of a C to T transition in the gene Balat_1410 at position 266, which causes a codon change.…”

Section: Resultsmentioning

confidence: 99%

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

Hidalgo-Cantabrana

Sánchez

Álvarez‐Martín

et al. 2015

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“…This is the case for the HMW-EPS fraction purified from strain B. animalis subsp. lactis IPLA-R1 (Table 1), whose EPS-unit contains 50% rhamnose (39). This fact has recently been correlated with an overexpression of genes coding for rhamnose biosynthesis precursors located inside the eps cluster of this strain (28).…”

Section: Structure and Genetics Of The Eps-units In Bifidobacteriamentioning

confidence: 89%

“…The cluster was delimited to a fragment of 54.3 kb, including 42 predicted genes flanked by a transposase-encoding gene. Based on homology studies, it was inferred that the genes present in this eps cluster were similar to those detected in LAB-eps clusters (39). Strain B. breve UCC20003 harbors an EPS-encoding cluster of 25.6 kb with 20 predicted genes, which are organized in two adjacent and oppositely oriented gene sets (8 genes in the eps1 operon and 10 genes in the eps2 operon).…”

Section: Structure and Genetics Of The Eps-units In Bifidobacteriamentioning

confidence: 96%

Genomic Overview and Biological Functions of Exopolysaccharide Biosynthesis in Bifidobacterium spp

Hidalgo-Cantabrana

Sánchez

Milani

et al. 2014

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169

162

bFor many years, bacterial exopolysaccharides (EPS) have received considerable scientific attention, mainly due to their contribution to biofilm formation and, above all, because EPS are potential virulence factors. In recent times, interest in EPS research has enjoyed a welcome boost thanks to the discovery of their ability to mediate communication processes with their surrounding environment and to their contribution to host health maintenance. In this review, we provide a fresh perspective on the genetics and activity of these polymers in members of the Bifidobacterium genus, a common gut inhabitant of humans and animals that has been associated with several health-promoting effects. Bifidobacteria can use EPS to protect themselves against the harsh conditions of the gastrointestinal tract, thus improving their persistence in the host. Indeed, the relevant function of EPS for bifidobacteria is underlined by the fact that most genomes sequenced until now contain genes related to EPS biosynthesis. A high interspecies variability in the number of genes and structural organization is denoted among species/subspecies; thus, eps clusters in this genus do not display a consensus genetic architecture. Their different G؉C content compared to that of the whole genome suggests that eps genes have been acquired by horizontal transfer. From the host perspective, EPS-producing bifidobacteria are able to trigger both innate and adaptive immune responses, and they are able to modulate the composition and activity of the gut microbiota. Thus, these polymers seem to be critical in understanding the physiology of bifidobacteria and their interaction with the host.

“…Protein was precipitated via treatment with trichloroacetic acid, and the carbohydrate fraction was precipitated via ethanol treatment as outlined by Marshall et al (54). Monomer compositions were determined by trifluoroacetic acid (TFA) hydrolysis followed by anion-exchange chromatography as described previously (55), with the exception that samples were eluted with aqueous 0.01 M NaOH for 20 min followed by 83% 10 mM NaOH:17% 150 mM NaOH:1 M sodium acetate for 25 min for simultaneous determination of monomers and uronic acids. Dry weights and inorganic contents of the flocs were determined by methods outlined in BS ISO 6496:1999 (56).…”

Section: Methodsmentioning

confidence: 99%

Floc Formation Reduces the pH Stress Experienced by Microorganisms Living in Alkaline Environments

Charles

Rout

Patel

et al. 2017

The survival of microorganisms within a cementitious geological disposal facility for radioactive wastes heavily depends on their ability to survive the calciumdominated, hyperalkaline conditions resulting from the dissolution of the cementitious materials. The results from this study show that the formation of flocs, composed of a complex mixture of extracellular polymeric substances (EPS), provides protection against alkaline pH values up to 13.0. The flocs were dominated by Alishewanella and Dietzia spp., producing a mannose-rich carbohydrate fraction incorporating extracellular DNA, resulting in Ca 2ϩ sequestration. EPS provided a ϳ10-m thick layer around the cells within the center of the flocs, which were capable of growth at pH values of 11.0 and 11.5, maintaining internal pH values of 10.4 and 10.7, respectively. Microorganisms survived at a pH of 12.0, where an internal floc pH of 11.6 was observed, as was a reduced associated biomass. We observed limited floc survival (Ͻ2 weeks) at a pH of 13.0. This study demonstrates that flocs maintain lower internal pHs in response to the hyperalkaline conditions expected to occur within a cementitious geological disposal facility for radioactive wastes and indicates that floc communities within such a facility can survive at pHs up to 12.0. IMPORTANCE The role of extracellular polymeric substances (EPS) in the survival of microorganisms in hyperalkaline conditions is poorly understood. Here, we present the taxonomy, morphology, and chemical characteristics of an EPS-based microbial floc, formed by a consortium isolated from an anthropogenic hyperalkaline site. Short-term (Ͻ2 weeks) survival of the flocs at a pH of 13 was observed, with indefinite survival observed at a pH of 12.0. Measurements from micro-pH electrodes (10-m-diameter tip) demonstrated that flocs maintain lower internal pHs in response to hyperalkaline conditions (pH 11.0, 11.5, and 12.0), demonstrating that floc formation and EPS production are survival strategies under hyperalkaline conditions. The results indicate how microbial communities may survive and propagate within the hyperalkaline environment that is expected to prevail in a cementitious geological disposal facility for radioactive wastes; the results are also relevant to the wider extremophile community.