Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM

Andersen, Joakim Mark; Barrangou, Rodolphe; Hachem, Maher Abou; Lahtinen, Sampo J.; Goh, Yong Jun; Svensson, Birte; Klaenhammer, Todd R.

doi:10.1371/journal.pone.0044409

Cited by 71 publications

(65 citation statements)

References 56 publications

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“…L. acidophilus NCFM is one of the best-studied gut bacteria, which is ascribed health benefits and used as a commercial probiotic (34)(35)(36)(37). The commercial and physiological relevance of L. acidophilus NCFM has spurred wide interest in the saccharide uptake and catabolism machinery of this organism to identify efficiently utilized glycans with potential as prebiotics (25,38,39). A few intracellular carbohydrate-active enzymes (CAZymes) from L. acidophilus NCFM have been characterized, including those active on maltose (26) and ␣-1,6-linked isomalto-oligosaccharides (24).…”

Section: Discussionmentioning

confidence: 99%

An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus

Møller

Goh

Rasmussen

et al. 2017

Appl Environ Microbiol

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Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 (LaPul13_14), and its role in branched ␣-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for ␣-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of ␣-glucans but was repressed by glucose. The debranching activity is conferred exclusively by LaPul13_14 and is abolished in a mutant strain lacking a functional LaPul13_14 gene. Hydrolysis kinetics of recombinant LaPul13_14 confirmed the preference for short-branched ␣-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest K m reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by ␤-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut.IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched ␣-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of healthbeneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched ␣-glucan oligomers (␣-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for ␣-glucans with short ␣-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.

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Section: Discussionmentioning

confidence: 99%

An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus

Møller

Goh

Rasmussen

et al. 2017

Appl Environ Microbiol

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“…The increased transcription of genes encoding "hypothetical proteins" and an ABC transporter gene may be involved in the phenomenon. A recent analysis of the transcriptional response of Lactobacillus acidophilus to growth on stachyose (a tetrasaccharide) showed increased transcription of ABC transporters (25). Crystal structure studies have shown ABC transporters binding tetrasaccharide with the strongest interactions at the nonreducing end of the carbohydrate (26).…”

Section: Discussionmentioning

confidence: 99%

Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

Lawley

Sims

Tannock

2013

Appl Environ Microbiol

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“…In order to understand the influence of environmental conditions on genome-wide gene expression levels, wholegenome DNA microarrays have often been used to survey the gene expression patterns of strains in the presence and absence of oligosaccharides (2,(5)(6)(7)(8). On the basis of in silico analysis of the Lactobacillus acidophilus NCFM genome sequence, Barrangou et al (2) identified a multiple-sugar metabolism (msm) operon that was involved in the metabolism of FOS.…”

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confidence: 99%

Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

Chen

Zhao

Chen

et al. 2015

Appl Environ Microbiol

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dAlthough fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a ␤-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome-and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. Prebiotics are defined as nondigestible food ingredients that selectively stimulate the growth and activity of beneficial microbial strains residing in the host gastrointestinal tract (GIT) (1). Among the sugars that are qualified as prebiotics, fructooligosaccharides (FOS) are fructose polymers of diverse lengths that can be either derivatives of simple fructose polymers or fructose moieties attached to a sucrose molecule (2). Because of the linkage configuration, FOS are not digested in the upper GIT and have been shown in vivo to beneficially modulate the composition of the intestinal microbiota by preferentially increasing the numbers of bifidobacteria and lactobacilli (3, 4).Despite considerable commercial and research interest in the beneficial effects of FOS, the molecular basis of FOS metabolism by specific members of the intestinal microbiota has only recently been examined. In order to understand the influence of environmental conditions on genome-wide gene expression levels, wholegenome DNA microarrays have often been used to survey the gene expression patterns of strains in the presence and absence of oligosaccharides (2, 5-8). On the basis of in silico analysis of the Lactobacillus acidophilus NCFM genome sequence, Barrangou et al. (2) identified a multiple-sugar metabolism (msm) operon that was involved in the metabolism of FOS. The msm operon encodes an ATP-dependent binding cassette-type transport system and a cytoplasmic ␤-fructosidase, which mediates FOS uptake and intracellular hydrolysis. Moreover, expression of the operon was i...

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Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM

Cited by 71 publications

References 56 publications

An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus

An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus

Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

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