REVIEW ARTICLE published: 26 September 2012 doi: 10.3389/fmicb.2012.00340 Metabolism of oligosaccharides and starch in lactobacilli: a review

Michael G. Gänzle* and Rainer Follador †

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

Edited by: Oligosaccharides, compounds that are composed of 2–10 monosaccharide residues, are Kostas Koutsoumanis, Aristotle major carbohydrate sources in habitats populated by lactobacilli. Moreover, oligosaccharide University, Greece metabolism is essential for ecological fitness of lactobacilli. Disaccharide metabolism by Reviewed by: Eva Van Derlinden, Katholieke lactobacilli is well understood; however, few data on the metabolism of higher oligosac- Universiteit Leuven, Belgium charides are available. Research on the ecology of intestinal microbiota as well as the com- Alexandra Lianou, Aristotle University mercial application of prebiotics has shifted the interest from (digestible) disaccharides to of Thessaloniki, Greece (indigestible) higher oligosaccharides.This review provides an overview on oligosaccharide *Correspondence: metabolism in lactobacilli. Emphasis is placed on maltodextrins, isomalto-oligosaccharides, Michael G. Gänzle, Department of Agricultural, Food and Nutritional fructo-oligosaccharides, galacto-oligosaccharides, and raffinose-family oligosaccharides. Science, University of Alberta, 4-10 Starch is also considered. Metabolism is discussed on the basis of metabolic studies Ag/For Centre, Edmonton, AB, related to oligosaccharide metabolism, information on the cellular location and substrate Canada T6G 2P5. specificity of carbohydrate transport systems, glycosyl hydrolases and phosphorylases, e-mail: [email protected] and the presence of metabolic genes in genomes of 38 strains of lactobacilli. Metabolic †Present address: Rainer Follador, Microsynth AG, pathways for disaccharide metabolism often also enable the metabolism of tri- and tetrasac- Balgach, Switzerland. charides. However, with the exception of amylase and levansucrase, metabolic enzymes for oligosaccharide conversion are intracellular and oligosaccharide metabolism is limited by transport. This general restriction to intracellular glycosyl hydrolases differentiates lac- tobacilli from other bacteria that adapted to intestinal habitats, particularly Bifidobacterium spp.

Keywords: Lactobacillus, metabolism, isomalto-oligosaccharides, starch, fructo-oligosaccharides, galacto-oligosaccharides, raffinose-family oligosaccharides, prebiotic

INTRODUCTION use of the term “oligosaccharides” in the current scientific lit- Lactobacilli have complex nutritional requirements for fer- erature, however, differs from this definition in a number of mentable carbohydrates, amino acids, nucleic acids and other cases. For example, the term “fructo-oligosaccharides” is gen- substrates, and derive metabolic energy from homofermentative erally used to include β-(1 → 2) linked fructo-oligosaccharides, or heterofermentative carbohydrate fermentation (Hammes and excluding the digestible disaccharide sucrose; the term “galacto- Hertel, 2006). Habitats of lactobacilli are nutrient-rich and often oligosaccharides” generally includes the (indigestible) β-(1 → 3 or acidic and include plants, milk and meat, and mucosal surfaces 6) linked disaccharides and α-galactosyllactose but excludes the of humans and animals (Hammes and Hertel, 2006). Intestinal disaccharide lactose, which is also indigestible in a majority of microbiota are characterized by a high proportion of lactobacilli humans, and melibiose; the term“isomalto-oligosaccharides”gen- particularly in animals harboring non-secretory epithelia in the erally includes the digestible disaccharide isomaltose (Roberfroid upper intestinal tract, including the crop of poultry, the pars et al., 1998; MacFarlane et al., 2008; Seibel and Buchholz, 2010). esophagus of swine, and the forestomach or rodents and mem- This paper will use the IUPAC definition of oligosaccharides to bers of the Equidae family (Walter, 2008). Lactobacilli are also include the corresponding disaccharides. dominant in fermentation microbiota of a majority of food fer- Oligosaccharide metabolism is essential for ecological fitness mentations, and are applied as probiotic cultures to benefit host of lactobacilli in most of their food-related and intestinal habi- health (Hammes and Hertel, 2006). Owing to their association tats (de Vos and Vaughan, 1994; Bron et al., 2004; Gänzle et al., with humans, food animals, and food as well as their economic 2007; Walter, 2008; Tannock et al., 2012). Oligosaccharides are the importance, they have been studied for more than a century and major carbohydrate sources in cereals, milk, fruits, and the upper the physiology and genetics of their monosaccharide metabolism intestine of animals. The metabolism of mono- and disaccharides is well understood (Orla-Jensen, 1919; Kandler, 1983; de Vos and is well understood; however, few data are available on the metab- Vaughan, 1994; Axelsson, 2004; Makarova et al., 2006; Gänzle et al., olism of higher oligosaccharides which are equally abundant in 2007). many habitats. Moreover, interest in the intestinal microbial ecol- Oligosaccharides are defined as compounds that are composed ogy as well as the widespread commercial application of prebiotic of few (2–10) monosaccharide residues (Anonymous, 1982). The oligosaccharide preparations has shifted the research interest from

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(digestible) disaccharides to (indigestible) higher oligosaccharides milk, meat, cereal fermentations, vegetable fermentations, and (Barrangou et al., 2003; Kaplan and Hutkins, 2003; Walter, 2008; intestinal habitats. Data were obtained from NCBI GenBank Seibel and Buchholz, 2010). However, the sound description of the (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) on 9 September 2011 metabolism of higher oligosaccharides is challenging. First, the (Table 1). For each species and its associated plasmids, coding in silico assignment of the specificity of carbohydrate transport sequences were extracted, translated, and a BLAST database was systems or glycosyl hydrolases is unreliable (see e.g., Thompson built using “makeblastdb” of the NCBI Standalone BLAST+ soft- et al., 2008; Francl et al., 2010) and often results in question- ware package (version 2.2.25; Camacho et al., 2009). The query able assignments of gene function. Second, few of the relevant sequences were searched in each database using “blastp” of the higher oligosaccharides are available in purified form for use as BLAST+ software package with the standard settings and the best substrate. However, the determination of the growth of lactobacilli match was reported. Additionally, a Smith–Waterman alignment on poorly described substrates provided little relevant information of the query sequence with the highest match was performed using on the capacity of lactobacilli to utilize oligosaccharides as carbon a BLOSUM62 substitution matrix. The score of this alignment was source. The lack of reference compounds also impedes identifica- divided by the score of the alignment of the query sequence to itself tion and quantification of individual compounds in a mixture of resulting in a score ratio. An enzyme was marked as present in a oligosaccharides with chromatographic methods. Only few studies given genome or plasmid if the score ratio is above a threshold of characterized oligosaccharide preparations with regards to com- 0.3–0.4. position,linkage type,and degree of polymerization,or monitored The bioinformatic analysis of genes contributing to oligosac- the metabolism of individual compounds during growth of lac- charide metabolism allows an assessment of the frequency of alter- tobacilli (Gopal et al., 2001; Kaplan and Hutkins, 2003; Saulnier native pathways for oligosaccharide metabolism, identifies genes et al., 2007; Ketabi et al., 2011; Teixeira et al., 2012). Despite the that occur together to form a functional metabolic pathway, and importance of oligosaccharide metabolism for the performance delineates major and convergent or divergent metabolic strategies of lactobacilli in food fermentations and in intestinal habitats, of lactobacilli for niche adaptation by specialized oligosaccha- our understanding of oligosaccharide metabolism in lactobacilli ride metabolism. However, it does not account for silent genes remains thus limited. Particularly the delineation of metabolism or orphan genes that are not expressed or not functional (Obst of disaccharides and higher oligosaccharides is unclear, and a et al., 1992). Moreover, carbohydrate fermentation is highly vari- comprehensive description of metabolic pathways is currently not able within strains of the same species due to the loss of plasmid available. encoded traits (de Vos and Vaughan, 1994) and gene acquisition This review aims to provide an overview on oligosaccharide by lateral gene transfer (Barrangou et al., 2003). For example, gene metabolism by lactobacilli. Hydrolysis of starch, the only poly- cassettes coding for carbohydrate utilization in L. plantarum are saccharide hydrolyzed by extracellular enzymes of lactobacilli, is highly variable and were designated as “lifestyle cassettes” that also discussed. Oligosaccharide fermentation is discussed on the may be added or deleted according to the requirements of specific basis of metabolic studies, the cellular location, and substrate ecological niches (Siezen and van Hylckama Vlieg, 2011). specificity of carbohydrate transport systems, glycosyl hydro- lases and phosphorylases, and the distribution of the genes cod- METABOLISM OF α-GLUCANS (MALTOSE, ISOMALTOSE, ing for metabolic enzymes in selected genomes of lactobacilli. MALTODEXTRINS, ISOMALTO-OLIGOSACCHARIDES, AND Oligosaccharide metabolism is discussed in detail for four major STARCH) groups of compounds (i) starch, maltodextrins, and isomalto- Starch is the major storage polysaccharide in cereal grains, grain oligosaccharides (IMO); (ii) fructo-oligosaccharides (FOS); (iii) legumes, and many roots and tubers (van der Maarel et al., 2002; β-galacto-oligosaccharides (βGOS); (iv) raffinose-family oligosac- Belitz et al., 2004). Starch is composed of amylose and amy- charides (RFO) as well as α-galacto-oligosaccharides (RFO and lopectin. Amylose is a linear α-(1 → 4) glucose chain with a αGOS, respectively). plant-specific degree of polymerization of 200–6000. Amylopectin consists of short linear α-(1 → 4) linked chains with α-(1 → 6) BIOINFORMATIC ANALYSES OF OLIGOSACCHARIDE AND linked side chains (van der Maarel et al., 2002). The degree of STARCH METABOLISM OF LACTOBACILLI branching is specific for the plant origin (van der Maarel et al., To assess the distribution of different oligosaccharide metabolic 2002; Belitz et al., 2004). Amylolytic degradation of amylose by pathways in lactobacilli, this study identified genes related to α- and β-amylase and amyloglucosidase yields α-(1 → 4) linked oligosaccharide metabolism in lactobacilli by bioinformatic analy- maltodextrins, maltose, and glucose, respectively. Hydrolysis of ses. The analysis included 38 genomes of lactobacilli that were amylopectin requires amylopullulanase or pullulanase to cleave assembled to the chromosome level (Table 1). The selection of the α-(1 → 6) linked branching points; amylopectin hydrolysis genomes includes representatives of the six major phylogenetic additionally yields α-d-Glu- α-(1 → 6)-d-Glu (isomaltose) and groups in the genus Lactobacillus, the L. salivarius group, the oligosaccharides with mixed α-(1 → 4) and α-(1 → 6) linkages. l. delbrueckii group, the L. buchneri group, the L. plantarum Linear or branched α-glucans with α-(1 → 2), α-(1 → 3), α- group, the L. casei group, the L. reuteri group, as well as the (1 → 4), and α-(1 → 6) are produced by bacteria and fungi. representatives of L. brevis and L. sakei (Hammes and Hertel, Examples include the predominantly α-(1 → 6) linked dextran 2006). The selection of organisms includes obligately homofer- produced by Leuconostoc mesenteroides, and the α-(1 → 6) and α- mentative species, facultatively heterofermentative species, and (1 → 4) linked polysaccharides reuteran and pullulan, produced obligately heterofermentative species; which were isolates from by L. reuteri and Aureobasidium pullulans,respectively (Belitz et al.,

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Table 1 | Genome and plasmid sequences of Lactobacillus spp. used in this study.

Abbreviation Organism Genome accession no. Plasmid accession no.

Aci1 Lactobacillus acidophilus 30SC CP002559 CP002561; CP002560 Aci2 Lactobacillus acidophilus NCFM CP000033 Amy1 Lactobacillus amylovorus GRL 1112 CP002338 CP002612; CP002613 Amy2 Lactobacillus amylovorus GRL1118 CP002609 CP002610 Bre1 Lactobacillus brevis ATCC 367 NC_008497 CP000417 Buc19 Lactobacillus buchneri NRRL B-30929 CP002652 CP002653; CP002654; CP002655 Cas1 Lactobacillus casei ATCC 334 NC_008526 CP000424 Cas2 Lactobacillus casei BD-II CP002618 CP002619 Cas3 Lactobacillus casei BL23 NC_010999 Cas4 Lactobacillus casei LC2W CP002616 CP002617 Cas5 Lactobacillus casei str. Zhang NC_014334 CP000935 Cri1 Lactobacillus crispatus ST1 FN692037 Del1 Lactobacillus delbrueckii subsp. bulgaricus 2038 CP000156 Del2 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 CR954253 Del3 Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 CP000412 Del4 Lactobacillus delbrueckii subsp. bulgaricus ND02 CP002341 CP002342 Fer1 Lactobacillus fermentum CECT 5716 CP002033 Fer2 Lactobacillus fermentum IFO 3956 NC_010610 Gas1 Lactobacillus gasseri ATCC 33323 CP000413 Hel1 Lactobacillus helveticus DPC 4571 CP000517 Hel2 Lactobacillus helveticus H10 CP002429 CP002430 Joh1 Lactobacillus johnsonii DPC 6026 CP002464 Joh2 Lactobacillus johnsonii FI9785 FN298497 FN357112; AY862141 Joh3 Lactobacillus johnsonii NCC 533 AE017198 Kef1 Lactobacillus kefiranofaciens ZW3 CP002764 CP002765; CP002766 Pla1 Lactobacillus plantarum JDM1 NC_012984 Pla2 Lactobacillus plantarum subsp. plantarum ST-III NC_014554 CP002223 Pla3 Lactobacillus plantarum WCFS1 NC_004567 CR377164; CR377165; CR377166 Reu1 Lactobacillus reuteri 100-23 AAPZ02000001;AAPZ02000002 NC_014553; NC_001757 Reu2 Lactobacillus reuteri DSM 20016 NC_009513 Reu3 Lactobacillus reuteri JCM 1112 NC_010609 Reu4 Lactobacillus reuteri SD2112 NC_015697 CP002848; CP002845; CP002846; CP002847 Rha1 Lactobacillus rhamnosus GG NC_013198 Rha2 Lactobacillus rhamnosus Lc 705 FM179323 FM179324 Sak1 Lactobacillus sakei 23K CR936503 Sal1 Lactobacillus salivarius CECT 5713 CP002034 CP002035; CP002036; CP002037 Sal2 Lactobacillus salivarius UCC118 CP000233 CP000234; AF488831; AF488832 San1 Lactobacillus sanfranciscensis TMW 1.1304 CP002461 CP002462; CP002463

Criteria for selection of genomes are described in the text.

2004; van Hijum et al., 2006; Cheng et al., 2011). Isomaltose, oligosaccharides in cereal fermentations as well as the upper isomaltotriose, isomaltotetraose as well as panose [α-d-Glu-α- intestinal tract of grain-eating animals (Vogel et al., 1999; Tan- (1 → 6)-α-d-Glu-α-(1 → 4)-d-Glu] and glucosyl-panose occur nock et al., 2012). Virtually all lactobacilli metabolize α-glucans in honey, as degradation products of dextran, or as products and many strains harbor alternative pathways (Figure 1; Table 2). of glucansucrases activity (Belitz et al., 2004; van Hijum et al., Moreover, amylopullulanase is the only extracellular polysaccha- 2006). IMO are commercially applied as prebiotic food ingredi- ride hydrolyzing enzyme in lactobacilli. In lactobacilli harboring ents (Seibel and Buchholz, 2010). Commercial IMO preparations maltose phosphorylase but not hexokinase, which particularly consist predominantly of di-,tri-,and tetrasaccharides and contain includes strains of L. sanfranciscensis, maltose is the only carbo- panose, 60 glucosylpanose, and 6060 diglucosylpanose in addition hydrate that is fermented. At least two alternative systems for to IMO (Ketabi et al., 2011). maltose uptake exist: (i) the ABC-transporter MalEFG/MsmK Owing to starch hydrolysis by amylases derived from cereal (Figure 1; Table 2); and (ii) a maltose-H+ symport system grains or saliva, maltose, and maltodextrins are the most abundant (Neubauer et al., 1994). A maltose phosphotransferase system

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Pullulan Glu Glu Glu n Glu Glu Glu Glu Glu n also: Amylopectin, Glycogen Maltodextrin H2O AmyX Glu Glu Glu n-1 Glu Glu Glu Glu Glu n MalE Glu Glu Glu Glu Glu Glu Glu k n-k

MalG MalF

ATP ATP Maltose-6-Phosphate MsmK MsmK P Glu Glu Glu Glu ADP ADP Pi Glu Glu H2O MalP MalH n

Glucose-1-P Glu Glu Glu Glucose-6-P Glu P Glu Glu Glu P n n Glu H O H2O 2 MalL/MalN DexB Glu P

PgmB Glu Glu Glu Glu Glu Glu n-1 n-1

P Glu

FIGURE 1 | Enzymes of lactobacilli involved in metabolism of trehalose-6-phosphate into d-glucose, and d-glucose-6-phosphate maltose, isomaltose, maltodextrins, isomalto-oligosaccharides, and (Thompson et al., 1998). SimA in L. casei hydrolyzes the phosphorylated starch and their cellular location. The distribution of genes in the sucrose isomers trehalulose, turanose, maltulose, leucrose, and palatinose genomes analyzed is shown inTable 2. MalEFG and MsmK (L. (Thompson et al., 2008).AmyX (L. acidophilus), MalN (L. acidophilus) and acidophilus), four-component ATP-binding cassette (ABC) transport MalL (L. acidophilus), amylopullulanases, hydrolyzes α-(1 → 6)-glucosidic system, imports maltodextrins into the cytosol (Nakai et al., 2009). MalP linkages in pullulan and amylopectin, also hydrolyzes α-(1 → 4)-glucosidic (L. acidophilus), named MapA in L. sanfranciscensis maltose linkages in polysaccharides. AmyX a high level of similarity to phosphorylase, phosphorylyses maltose into d-glucose, and β-d-glucose Bifidobacteria amylopullulanase. AmyX is an extracellular enzyme, MalL 1-phosphate (Ehrmann and Vogel, 1998; Nakai et al., 2009). PgmB (L. and MalN are intracellular (Ryan et al., 2006; Nakai et al., 2009). MalL in B. acidophilus), named PgmA in L. sanfranciscensis β-phosphoglucomutase, subtilis also hydrolyzes sucrose (Schönert et al., 1999). DexB (L. converts β-d-glucose 1-phosphate to β-d-glucose-6-phosphate (Ehrmann acidophilus), α-glucosidase with activity on dextran, hydrolyzes and Vogel, 1998; Nakai et al., 2009). MalH (L. acidophilus), named SimA in α-(1 → 6)-glucosidic linkages, isomalto-oligosaccharides, and panose but L. casei, 6-phospho-α-glucosidase, hydrolyzes maltose-6-phosphate, not maltose (Møller et al., 2012). has not been identified in lactobacilli. Intracellular conversion of Comparable to amylolytic enzymes in bifidobacteria, extracellular α-glucosides occurs alternatively by the amylopullulanases MalL amylases of lactobacilli are endoamylases hydrolyzing α-(1 → 6) and MalN, the dextranase DexB, or maltose phosphorylase MalP as well as α-(1 → 4) glucosidic bonds in amylose, amylopectin, or (Figure 1; Table 2). It is noteworthy that the extracellular and pullulan. The activity increases with increasing degree of polymer- intracellular amylopullulanases AmyX and MalL are homologous ization of the substrate (Talamond et al., 2006; Kim et al., 2008). but differ in their cellular location (Kim et al., 2008; Møller et al., Amylases activity on raw starch was dependent on the sequence of 2012). starch binding domains exhibiting significant sequence diversity Extracellular amylase activity was characterized in several lacto- (Rodriguez-Sanoja et al., 2005). Oligosaccharides with a degree of bacilli, including L. fermentum, L. plantarum, L. mannihotivorans, polymerization of 3 and 4 are the major products of catalysis.Amy- L. amylovorus, and L. gasseri (Giraud and Cuny, 1997; Rodriguez- lase activity in lactobacilli is strain-specific. This study identified Sanoja et al., 2005; Talamond et al., 2006; Kim et al., 2008). an extracellular amylase only in L. acidophilus and L. amylovorus

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Table 2 | Distribution of genes coding for metabolism of maltose, isomaltose, maltodextrins, isomalto-oligosaccharides, and starch in 38 genomes of lactobacilli. Aci1 Aci2 Amy2 Bre1 Cas1 Cas2 Cas3 Cas5 Cri1 Del1 Del2 Del3 Del4 Fer2 Gas1 Hel1 Hel2 Joh1 Joh2 Joh3 Kef1 Pla1 Pla3 Reu2 Reu4 Rha1 Rha2 Sak1 Sal1 San1 Amy1 Buc1 Cas4 Fer1 Pla2 Reu3 Sal2 Reu1 Reu1

malEFG msmK * malL * pgmB * malP * malH * amyX * malN * dexB * glgP *

See Figure 1 for metabolic pathways; genome accession numbers are provided inTable 1. Gray Background: Presence of Gene, white Background: Absence of Gene, * query sequence.

(Table 2). The infrequent occurrence of amylase genes corre- are also present, indicating a functional maltodextrin operon com- sponds to the observation that the majority of lactobacilli are not parable to L. acidophilus and L. casei (Table 2). DexB is the most amylolytic. However, amylolytic lactobacilli are more frequently widely distributed gene coding for conversion of α-glucosides in isolated from cereal fermentations in tropical climates, possibly lactobacilli (Table 2) but is not part of the maltodextrin operon reflecting the lower β-amylase activity in C4 plants when com- (Møller et al., 2012). DexB hydrolyzes isomalto-oligosaccharides, pared to wheat or rye (Gänzle and Schwab, 2009; Turpin et al., panose, and dextran, but not maltose or sucrose (Møller et al., 2011). 2012). Maltose transport by the ABC-transporter MalEFG-MsmK was Maltose phosphorylase (MalP, MapA in L. sanfranciscen- characterized in L. casei and l. acidophilus (Monedero et al., 2008; sis) catalyzes phosphorolysis of maltose to glucose and β-d- Nakai et al., 2009). This ABC-transporter is homologous to mal- glucose-1-phosphate (Stolz et al., 1996; Ehrmann and Vogel, todextrin transport proteins of Bacillus subtilis and has a low 1998). Maltose phosphorylase is invariably associated with affinity for maltose transport. Moreover, several intracellular glu- β-phosphoglucomutase converting β-d-glucose-1-phosphate to canases are co-transcribed with MalEFG-MsmK, indicating the glucose-6-phosphate (Table 2). Phosphorolysis of maltose does transport system functions as oligosaccharide transporter (Mon- not expend ATP for generation of glucose-6-phosphate and is edero et al., 2008; Nakai et al., 2009). The MalEFG/MsmK trans- energetically more favorable than hydrolysis (Stolz et al., 1996). port system is widespread in lactobacilli but noticeably absent During maltose metabolism of L. sanfranciscensis, L. reuteri, and in many lactobacilli that grow rapidly with maltose as the sole L. fermentum, glucose is transiently accumulated in the medium, source of carbon (Table 2). Maltose transport in L. sanfranciscen- indicating that glucose-6-phosphate is preferentially metabo- sis was attributed to a maltose-H+ symport system that was not lized. Maltose phosphorylase is highly specific for maltose and characterized on the genetic level (Neubauer et al., 1994). Mal- does not convert isomaltose, kojibiose, nigerose, or maltodextrins tose phosphotransferase systems were characterized in other lactic (Ehrmann and Vogel, 1998; Nakai et al., 2009). In most obligate acid bacteria (Le Breton et al., 2005) but are absent in lactobacilli heterofermentative lactobacilli (Table 2), i.e., L. brevis, L. buchneri, (Table 2). L. fermentum, and L. reuteri, maltose phosphorylase is the only Three intracellular α-glucosidases hydrolyze maltodextrins or enzyme active on maltose. In other lactobacilli, maltose phos- isomalto-oligosaccharides, MalL, MalN, and DexB (Figure 1; phorylase is part of the MalEFG/MsmK maltodextrin operon, Schönert et al., 1999; Nakai et al., 2009; Møller et al., 2012). together with the α-glucosidases MalL and MalN. The narrow sub- All three are GH13 enzymes. MalL in B. subtilis was described strate specificity of MalP implies that MalL and MalN hydrolyze as sucrose-maltase-isomaltase with broad substrate spectrum maltodextrins and isomalto-oligosaccharides while MalP converts (Schönert et al., 1999). Sequence homologies of MalL and MalN maltose, one of the products of MalN and MalL activities. Sev- to AmyX as well as the amylopullulanase of bifidobacteria suggest eral lactobacilli harbor glycogen phosphorylase (GlgP) in addition that both enzymes also hydrolyze oligosaccharides with α-(1 → 4) to maltose phosphorylase (Table 2). In contrast to MalP, GlgP and α-(1 → 6)glucosidic bonds (Kim et al., 2008; Nakai et al., shows activity with maltotriose, higher maltodextrins, and glyco- 2009; Table 2). In L. acidophilus and L. casei, MalN, MalL, MalP, gen but not with maltose as substrate (Alonso-Casajus et al., 2006). PgmB, and the ABC-transporter MalEFG-MsmK form a single However, the substrate specificity of bacterial glycogen phospho- maltodextrin operon regulated by MalR. Genes in the operon are rylases is poorly characterized and a contribution to carbohydrate induced by maltose and repressed by glucose (Monedero et al., metabolism in lactobacilli remains unclear. 2008; Nakai et al., 2009). In most other lactobacilli harboring the The contribution of MalH, a phospho-α-glucosidase, to mal- ABC-transporter MalEFG-MsmK, MalN, MalL, MalP, and PgmB tose metabolism in lactobacilli is unclear as a corresponding

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phosphotransferase system is lacking (Figure 1). GlvA, the cor- in sourdough are generally associated with sucrose positive lac- responding phospho-α-glucosidases in B. subtilis, hydrolyzes tobacilli and thus take advantage of extracellular levansucrase maltose-6-phosphate as well as trehalose-6-phosphate (Thomp- activity of other strains (Tieking et al., 2003). son et al., 1998). The GlvA/MalH homolog SimA in L. casei Glucansucrases and fructansucrases are the only extracellu- contributes to metabolism of sucrose isomers rather than maltose lar enzymes capable of sucrose hydrolysis (Figure 2) but are the (see below and Thompson et al., 2008). enzymes least frequently found in lactobacilli (Table 3). Glucansu- Several strains of L. reuteri harbor GtfB, a GH70 4,-6-α- crases and fructansucrases alternatively catalyze sucrose hydrolysis glucanotransferase active with maltodextrins as substrates. GtfB and oligo- or polysaccharide formation (van Hijum et al., 2006) cleaves α-(1 → 4) linkages in maltotetraose to synthesize α- and clearly serve ecological functions other than carbohydrate (1 → 6) linked oligo- and polysaccharides (Kralj et al., 2011). metabolism. Exopolysaccharide formation by glucansucrases and Glucose and maltose are among the reaction products from mal- fructansucrases contributes to biofilm formation in intestinal totetraose but the in vivo role of the enzyme may relate to poly- ecosystems as well as the resistance of lactobacilli to chemical and saccharide synthesis and modification rather than oligosaccharide physical stressors (Schwab and Gänzle, 2006; Walter et al., 2008). metabolism (Kralj et al., 2011). Correspondingly, their expression is induced by sucrose in some strains of L. reuteri but their expression in L. reuteri and L. san- METABOLISM OF SUCROSE AND franciscensis was also reported to be constitutive or dependent on FRUCTO-OLIGOSACCHARIDES environmental stress (Tieking et al., 2005; Schwab and Gänzle, FOS consist of β-(2 → 1) or β-(2 → 6)-linked d-fructose units 2006; Teixeira et al., 2012). However, glucansucrases and levansu- linked to a terminal d-glucose or d-fructose. Sucrose is wide- crases also contribute to sucrose metabolism. In L. sanfranciscensis, spread in plants and is the most abundant sugar in many levansucrase is the only enzyme capable of sucrose conversion fruits, grain legumes, and ungerminated cereal grains. The (Tieking et al., 2005; Table 3). Disruption of glucansucrases and inulin-type β-(2 → 1) linked FOS 1-kestose, nystose, and 1- levansucrase genes in L. reuteri impaired sucrose metabolism of L. fructofuranosylnystose are also widespread in nature, although reuteri TMW1.106 but not of L. reuteri LTH5448 (Schwab et al., they typically occur at lower concentrations than sucrose. High 2007). concentrations are found in Jerusalem artichoke, onions, and In silico and transcriptome analyses demonstrate that the func- chicory root. Wheat, rye, and barley contain 0.15–0.4% of the tions of sucrose phosphorylase (ScrP) and (phospho-)fructo- FOS with a degree of polymerization of 3–5 (Campbell et al., furanonosidases (BfrA and SacA) in lactobacilli match those of 1997). 1-Kestose is more abundant than nystose and fructofu- sucrose phosphorylase in L. mesenteroides, BfrA in Bifidobacterium ranosylnystose in cereal grains whereas the tri-, tetra-, and pen- lactis, or SacA of B. subtilis (Kawasaki et al., 1996; Ehrmann et al., tasaccharides are approximately equally abundant in onions and 2003; Reid and Abratt, 2005; Barrangou et al., 2006; Saulnier et al., chicory roots (Campbell et al., 1997). Levan-type β-(2 → 6) linked 2007). BfrA is a fructo-furanosidase hydrolyzing sucrose, inulin- FOS, 6-kestose, and higher oligosaccharides, are less abundant in type FOS, or inulin (Ehrmann et al., 2003) that was identified in nature but are formed by degradation of levan-type fructans, or 3 of the 38 genomes analyzed (Table 3). The sucrose-(phosphate) by bacterial levansucrases (Praznik et al., 1992; van Hijum et al., hydrolase SacA was the most frequent fructosidase (Table 3). SacA 2006). Inulin-type FOS are commercially applied as prebiotic is frequently associated with the sucrose phosphotransferase sys- food ingredients (Roberfroid et al., 1998). Commercial produc- tem (Table 3). The two genes are located on the same operon in tion of FOS relies on inulin hydrolysis or synthesis from sucrose L. plantarum and L. acidophilus and both genes are regulated by by fructansucrases (Yun, 1996; Seibel and Buchholz, 2010). ScrR (Reid and Abratt, 2005; Barrangou et al., 2006; Saulnier et al., Three pathways for sucrose metabolism exist in lactobacilli; 2007). However, SacA of B. subtilis shows activity with sucrose (i) extracellular hydrolysis by glucansucrases or fructansucrases; or sucrose-1-phosphate as substrate (Reid and Abratt, 2005) and (ii) transport and concomitant phosphorylation of sucrose by SacA is found without the associated phosphotransferase system the Pts1BCA phosphotransferase system, and hydrolysis by the in strains of L. acidophilus, L. casei and L. rhamnosus (Table 3). A (phospho-)fructo-furanosidase SacA/ScrB; and (iii) transport,fol- SacA homolog in Bacillus stearothermophilus hydrolyzed sucrose lowed by phosphorolysis by sucrose phosphorylase or hydrol- but not sucrose-1-phosphate (Li and Ferenci, 1996), and SacA in L. ysis by the (phospho)-fructo-furanosidases BfrA or SacA/ScrB plantarum hydrolyzed 1-kestose and nystose (Saulnier et al., 2007). (Figure 2; for review,see Reid andAbratt,2005). The (phospho)-α- Thus, SacA homologs in lactobacilli likely cleave FOS-phosphates glucosidase MalL of B. subtilis also recognizes sucrose as substrate as well as FOS. (Schönert et al., 1999) and may contribute to sucrose hydrolysis Sucrose phosphorylase exhibits high specificity for sucrose as in lactobacilli. Levansucrases synthesize FOS from sucrose but do substrate (Goedl et al., 2008). Sucrose phosphorolysis is energet- not contribute to FOS metabolism (Tieking et al., 2005; van Hijum ically more favorable than sucrose hydrolysis because glucose is et al., 2006). With the exception of L. brevis, all genomes of Lac- phosphorylated with inorganic phosphate and not at the expense tobacillus spp. analyzed harbored at least one functional sucrose of ATP (Figure 2). However, sucrose phosphorylase was less fre- metabolic pathway (Table 3). The presence of two or more alterna- quently identified in genomes of lactobacilli than sucrose hydro- tive pathways for metabolism of sucrose and higher FOS in most lases (Table 3). Sucrose phosphorylase is the only intracellular lactobacilli indicates that sucrose and higher FOS are highly pre- sucrose converting enzyme in L. reuteri and significantly con- ferred substrates. Many strains of L. sanfranciscensis do not metab- tributes to sucrose metabolism in this species (Schwab et al., olize sucrose; however, sucrose-negative strains L. sanfranciscensis 2007).

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Sucrose Glu Fru

GtfA FOS

Glu Fru Glu Fru n MsmE Glu Fru n

Glu Fru Glu Fru Ftf MsmF MsmG Pts1BCA PEP ATP ATP MsmK MsmK Pyruvate ADP ADP

P Glu Fru n Glu Fru n

Fru ( P ) Glu Fru Glu Fru n ATP H O ATP SacK1 2 BfrA/SacA ScrP ADP ADP Glucose-1-P Fru P Fru Fru ( P ) Glu Fru Glu P n-1

FIGURE 2 | Enzymes of lactobacilli involved in metabolism of sucrose sucrose phosphotransferase system, transports FOS into the cytosol while and fructo-oligosaccharides and their cellular location. The distribution of transferring a phosphoryl-moiety onto the glucose residue of the FOS. genes in the genomes analyzed is shown inTable 3. MsmEFGK (L. Pts1BCA transports FOS with a degree of polymerization of 3 and 4 (Saulnier acidophilus), four-component ATP-binding cassette (ABC) transport system, et al., 2007). SacK1 (L. plantarum), a fructokinase, transfers a phosphate imports FOS into the cytosol (Barrangou et al., 2003). ABC transport systems group to d-fructose, converting it to a d-fructose-6-phosphate (Saulnier et al., for FOS in lactobacilli transport glucose, fructose, and FOS with a degree of 2007). LevS (L. sanfranciscensis) named FtfA in L. reuteri, cell-wall bound polymerization of 2–4 (Kaplan and Hutkins, 2003). BfrA or ScrB (L. levansucrase, hydrolyzes sucrose to glucose and fructose, also has a acidophilus) and SacA (L. plantarum), fructosidases, hydrolyze terminal transferase activity which catalyzes the transfer of the fructose moiety of β-d-fructofuranosides in (phospho-)β-d-fructofuranoside oligosaccharides sucrose to a fructosyl-acceptor yielding FOS or levan (Tieking et al., 2005; van (Barrangou et al., 2003; Ehrmann et al., 2003; Saulnier et al., 2007). ScrP (L. Hijum et al., 2006). GtfA (L. reuteri), extracellular glucansucrase, hydrolyzes reuteri), named GtfA in L. acidophilus, a sucrose phosphorylase, sucrose to glucose, and fructose, also has a transglucosylation activity which phosphorylyses sucrose to d-fructose, and α-d-glucose-1-phosphate transfers the glucose moiety of sucrose to a glucan chain (van Hijum et al., (Barrangou et al., 2003; Schwab et al., 2007). Pts1BCA (L. plantarum), a 2006).

Transport systems for sucrose in lactobacilli include the et al., 2007). Characterization of an ABC family transporter in oligosaccharide transporter MsmEFGK and the sucrose phos- L. paracasei demonstrated that its affinity strongly decreased photransferase system Pts1BCA (Figure 2; Barrangou et al., in the order kestose > glucose or fructose > sucrose or nys- 2003; Saulnier et al., 2007). MsmEFGK was identified only in tose > fructofuranosylnystose (Kaplan and Hutkins, 2003). Irre- L. acidophilus and L. crispatus (Table 3); its presence in L. spective of the presence of intracellular fructo-furanosidases with acidophilus was attributed to acquisition by lateral gene trans- activity on high molecular weight fructans,transport limits metab- fer (Barrangou et al., 2003). Both transport systems also inter- olism of FOS in lactobacilli to di-, tri-, and tetrasaccharides. nalize FOS with a degree of polymerization of 2 and 3 but However, because many sucrose-metabolizing lactobacilli harbor have a very low affinity for FOS with a degree of polymer- neither MsmEFGK nor Pts1BCA, additional but uncharacterized ization of 4 or higher (Kaplan and Hutkins, 2003; Saulnier sucrose transport systems exist.

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Table 3 | Distribution of genes coding for metabolism of sucrose and fructo-oligosaccharides in 38 genomes of lactobacilli. Aci1 Aci1 Amy2 Cas1 Cas3 Del4 Fer2 Joh2 Pla2 Pla3 Reu3 Rha2 Sal2 Aci2 Amy1 Bre1 Buc1 Cas2 Cas4 Cas5 Cri1 Del1 Del2 Del3 Fer1 Gas1 Hel1 Hel2 Joh1 Joh3 Kef1 Pla1 Reu1 Reu2 Reu4 Rha1 Sak1 Sal1 San1

msmEFGK * bfrA * sacA * scrP * Pts1BCA * sacK1 * levS * gtfA *

See Figure 2 for metabolic pathways; genome accession numbers are provided inTable 1. Gray Background: Presence of Gene, white Background: Absence of Gene, * query sequence.

METABOLISM OF β-GALACTO-OLIGOSACCHARIDES pathway also metabolize higher βGOS (Gopal et al., 2001). (ii) β-Galacto-oligosaccharides (βGOS) consist of β-(1 → 2, 3, 4, or 6) Organisms expressing the LacEF/LacG pathway utilize glucose and linked galactose units with terminal galactose or glucose (Torres galactose simultaneously whereas metabolism by the LacS/LacLM et al., 2010; Gänzle, 2012). The only βGOS occurring widely in or LacZ pathway results in preferential metabolism of glucose nature is lactose [Gal-β-(1 → 4)-Glu], which is present in the milk and excretion of galactose (de Vos and Vaughan, 1994; Francl of mammals at concentrations of 2–10% (Gänzle et al., 2008). Tri- et al., 2012). (iii) Expression of LacEF/LacG is induced by lac- and tetrasaccharides (e.g., β30 galactosyllactose, β40 galactosyllac- tose, and gene expression is transcriptionally linked to galac- tose, or β60 galactosyllactose) are present only in trace amounts tose metabolism. In contrast, LacS and β-galactosidases in lac- in humans and most non-human mammals (Kunz et al., 2000; tobacilli are often constitutively expressed, and are often located Urashima et al.,2001; Kobata,2010). Noticeable exceptions include on the same genetic loci as metabolic enzymes for α-galactosides the milk of some marsupials, e.g., the tammar wallaby, where (Obst et al., 1992; de Vos and Vaughan, 1994; Silvestroni et al., β30 galactosyllactose and corresponding higher oligosaccharides 2002). are a major component. βGOS also occur in nature as degrada- The β-galactosidases LacLM and LacZ, both classified in the tion products of plant galactans or arabinogalactans (Belitz et al., GH2 family, hydrolyze a wide variety of β-(1 → 2, 3, 4, or 6) βGOS, 2004). Moreover, βGOS are commercially produced and applied including oligosaccharides with a degree of polymerization of 3–6 as prebiotic food ingredients (Yun, 1996; MacFarlane et al., 2008; (Gänzle, 2012). LacLM or LacZ enzymes are active as multimeric Seibel and Buchholz, 2010). Commercial βGOS preparations have enzymes (Schwab et al., 2010). The GH42 β-galactosidases LacA a degree of polymerization of 2–6 and contain predominantly β- was cloned and characterized in L. acidophilus and was found to (1 → 3, 4, or 6) linked di- and trisaccharides (Torres et al., 2010; have only low activity on lactose or GOS (Schwab et al., 2010). Gänzle, 2012). In other GH42 family β-galactosidases from bifidobacteria and Initial studies on βGOS metabolism by lactic acid bacteria Carnobacterium piscicola,activity with lactose as substrate is low or aimed to understand lactose metabolism of dairy starter cultures absent. A contribution of LacA to βGOS metabolism in lactobacilli (Premi et al., 1972; Poolman et al., 1992; deVosandVaughan, 1994; thus remains to be demonstrated. Obst et al., 1995). More recent investigations on β-galactosidases βGOS are transported by the lactose permase LacS, which of lactobacilli focused on the production and metabolism of prebi- transports lactose in exchange with galactose, or in symport with otic βGOS (e.g., Nguyen et al., 2007; Schwab et al., 2010; Andersen protons (Poolman et al., 1992). Induction of lacS expression by et al., 2011; for review, see Torres et al., 2010; Gänzle, 2012). Lacto- βGOS with a DP of 2–6 in L. acidophilus was interpreted as indica- bacilli metabolize βGOS by two alternative pathways; (i) transport tion that LacS transports higher βGOS as well as lactose, however, and concomitant phosphorylation by the LacEF phosphotrans- experimental evidence for tri- or tetrasaccharide transport by ferase system and hydrolysis by LacG, a β-phospho-galactosidases, LacS is lacking. Lactose transport by LacS of S. thermophilus is or (ii) transport by the lactose permease LacS and hydrolysis by inhibited by the disaccharide melibiose, indicating that αGOS β-galactosidase (Figure 3). The lactose permease/β-galactosidase are an alternative substrate for the transport enzyme (Poolman pathway is more widespread in lactobacilli (Table 4). βGOS meta- et al., 1992). L. rhamnosus and L. acidophilus were capable of acid bolic genes are frequently encoded on plasmids (Table 4); more- production from galactosyllactose, but preferentially metabolized over, silent β-galactosidases were described (Obst et al., 1992). disaccharides over tri- and tetrasaccharides (Gopal et al., 2001). Metabolism by the LacEF/LacG exhibits several distinct character- Because β-galactosidase exhibits no preference for disaccharides istics from the LacS/β-galactosidase pathway. (i) The β-phospho- over tri- or tetrasaccharides, this preferential metabolism of βGOS galactosidase LacG, classified in the GH1 family, is specific for with a lower degree of polymerization likely reflects transport lactose whereas organisms employing the LacS/β-galactosidase limitations.

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METABOLISM OF RAFFINOSE-FAMILY OLIGOSACCHARIDES to sucrose. RFO are widely distributed in plants, for example, (RFO) AND α-GALACTO-OLIGOSACCHARIDES (α-GOS) AND ungerminated wheat and rye grains contain 0.1–0.5% raffinose; α-GALACTOSIDES OF LACTOSE the seeds of grain legumes contain 2–10% RFO. α-Galactosides of The RFO raffinose, stachyose, and verbascose consist of one, lactose, particularly α30 galactosyllactose, are found in the milk of two, and three α-(1 → 6) d-galactose units, respectively, linked several non-human mammals (Urashima et al., 2001). The αGOS melibiose [α-Gal-(1 → 6)- Glu], manninotriose [α-Gal-(1 → 6)- α-Gal-(1 → 6)-Glu], and manninotetraose occur as degradation GOS products of RFO, or as products of transgalactosylation (Mital Lactose et al., 1973; Belitz et al., 2004; Teixeira et al., 2012). Glu Gal Glu Gal n=1-2 Different from FOS and βGOS, which are considered prebi- otic oligosaccharides, RFO are considered anti-nutritive factors, H+ causing dose-dependent flatulence, and gastro-intestinal discom- fort (Oku and Nakamura, 2002). Accordingly, studies on RFO LacS LacEF metabolism by lactobacilli mainly aimed at allowing their fer- PEP mentative removal (Mital et al., 1973; de Giori et al., 2010). H+ However, conversion of RFO to αGOS by lactobacilli (Teixeira Pyruvate et al., 2012) may prevent gastro-intestinal discomfort without eliminating prebiotic properties. The use of galactosidases for Lactose-6-P transgalactosylation, which was a focus of recent research on β- Glu Gal Glu Gal P α n galactosidases, was also explored for -galactosidases (Tzortzis et al., 2004). α-Galactosidase activity of lactobacilli was initially described by Mital et al.(1973). α-Galactosidase of L. plantarum Glu Gal and L. acidophilus is encoded by melA, a glycosyl hydrolase in Glu Gal P n the GH36 family (Figure 4). MelA is widely distributed in lacto- H2O bacilli (Table 5), reflecting the relevance of α-galactosides in plants LacZ/LacLM H2O LacG and intestinal ecosystems. The enzyme is active as homotetramer, and recognizes unbranched oligosaccharides, including melibiose, Glu Gal Gal raffinose, and stachyose, as substrate (Silvestroni et al., 2002; Fred- n-1 Glu Gal P slund et al., 2011). Interestingly, the galactoside metabolism gene cluster in L. plantarum and L. fermentum encodes melA as well FIGURE 3 | Enzymes of lactobacilli involved in metabolism of lactose as a LacLM type β-galactosidase (Silvestroni et al., 2002; Carrera- and β-galacto-oligosaccharides and their cellular location. The Silva et al., 2006). This arrangement may reflect that melibiose distribution of genes in the genomes analyzed is shown inTable 4. LacS (L. acidophilus), named RafP in L. plantarum lactose permease, imports di- and rarely occurs in nature. Naturally occurring oligosaccharides with 0 trisaccharide GOS into the cytosol, functions either as a proton symport α-galactosidic bonds also contain β-galactosidic bonds (e.g., α3 system or as a lactose-galactose antiporter (de Vos and Vaughan, 1994; galactosyllactose) or β-(1 → 2) fructosides (e.g., raffinose). αGOS Silvestroni et al., 2002). LacZ (L. acidophilus) and LacLM (L. plantarum), and βGOS are transported by LacS/RafP,the lactose transport pro- β-galactosidases, hydrolyze terminal β-d-galactose in β-d-galactosides tein that also shows affinity for melibiose (Poolman et al., 1992; (Schwab et al., 2010). LacEF (L. casei), a lactose phosphotransferase system, transports lactose into the cytosol while transferring a phosphoryl Silvestroni et al., 2002). However, melibiose transport and uti- onto the galactose residue of the lactose (de Vos and Vaughan, 1994). LacG lization in mutants of L. plantarum that are deficient in lactose (L. casei), a phospho-β-galactosidase, hydrolyzes terminal units which are transport demonstrates that alternative transport enzymes exist bound to a phospho-β-galactose (de Vos and Vaughan, 1994). (Tamura and Matsushita, 1992), potentially homologs of MelB, a

Table 4 | Distribution of genes coding for metabolism of lactose and β-galacto-oligosaccharides in 38 genomes of lactobacilli. Aci1 Aci2 Amy1 Bre1 Cas1 Cas2 Cas4 Cas5 Del1 Del2 Gas1 Hel1 Joh1 Joh2 Kef1 Pla1 Pla2 Pla3 Reu1 Reu2 Reu3 Reu4 Sal2 Amy2 Amy2 Buc1 Cas3 Cri1 Del3 Del4 Fer1 Fer2 Hel2 J Rha1 Rha2 Sak1 Sal1 San1 oh3 oh3

lacS * LacZ * LacL * 2 LacM * 2 LacEF * 1 1 LacG 1 * 1 1

See Figure 3 for metabolic pathways; genome accession numbers are provided inTable 1. Gray Background: Presence of Gene, white Background: Absence of Gene, * query sequence. 1 Gene is present on genome and on associated plasmid, 2 Gene is present only on associated plasmid, not on genome.

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Melibiose Raffinose Gal Gal Glu Glu Fru Gal Gal Glu Fru Glu

Ftf Fru [1] [2]

Gal Gal Glu Fru Glu

Gal Gal Glu Glu Fru

H2O H2O MelA

Sucrose Glu Gal Gal Glu Fru

Glu Fru Glu Fru

ATP H O ScrP 2 BfrA/SacA ADP Glucose-1-P Fru Fru Glu Glu P

FIGURE 4 | Enzymes of lactobacilli involved in metabolism of phosphorylase, phosphorylyses sucrose resulting from raffinose hydrolysis melibiose, raffinose, and raffinose-family oligosaccharides, and their by MelA (Barrangou et al., 2003; Schwab et al., 2007). [1][2], import of cellular location. The distribution of genes in the genomes analyzed is melibiose and raffinose into cytosol. Transport enzymes of lactobacilli shown inTable 5. LevS (L. sanfranciscensis), named FtFA in L. reuteri, specific for melibiose or raffinose were not characterized biochemically; cell-wall bound levansucrase, hydrolyzes sucrose, raffinose, stachyose, candidates include LacS and MelB. LacS (lactose transport protein, rafP in and verbascose to fructose and glucose, meliobiose, manninotriose, and L. plantarum) was shown in Streptococcus thermophilus to have an manninotetratose, respectively. Levansucrases also have a transferase affinity not only for β-galactosides and galactose but also for melibiose and activity which catalyzes the transfer of the fructose moiety of sucrose to a to a lesser extent for raffinose (Poolman et al., 1992; Silvestroni et al., fructosyl-acceptor yielding FOS or levan (Tieking et al., 2005; van Hijum 2002). L. plantarum transports melibiose independent of LacS, a possible et al., 2006; Teixeira et al., 2012). MelA (L. acidophilus), α-galactosidase, candidate gene is the predicted melibiose permease with homology to hydrolyzes terminal α-d-galactose residues in α-d-galactosides (Fredslund MelB (Na+/Sugar symporter) in Escherichia coli (Tamura and Matsushita, et al., 2011). ScrP (L. reuteri), named GtfA in L. acidophilus, a sucrose 1992; Pourcher et al., 1995). melibiose permease characterized in E. coli (Pourcher et al., 1995). Metabolism of oligosaccharides with mixed α- and β- α-Galactosidase activity was initially characterized in cells grown galactosidic linkages requires combined activity of α-galactosidase with glucose (Mital et al., 1973); melA expression in L. plantarum and β-galactosidase (Figures 3 and 4). Likewise, hydrolysis of is induced by melibiose but not repressed by glucose (Silvestroni RFO by α-Gal releases sucrose and complete degradation of RFO et al., 2002). is dependent on sucrose metabolic enzymes (Figure 4). Of the

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Table 5 | Distribution of genes coding for metabolism of melibiose, raffinose, and raffinose-family oligosaccharides in 38 genomes of lactobacilli. Hel1 Hel1 Kef1 Reu4 Sal1 Aci1 Aci1 Aci2 Bre1 Cas1 Cas2 Cas5 Cri1 Del2 Del4 Pla1 Reu2 Amy1 Amy2 Buc1 Cas3 Cas4 Del1 Del3 Fer1 Fer2 Gas1 Joh1 Joh2 Joh3 Pla2 Pla3 Reu1 Reu3 Rha1 Rha2 Sak1 Sal2 San1 Hel2

levS * melA * scrP * sacA *

See Figure 4 for metabolic pathways; genome accession numbers are provided inTable 1. Gray Background: Presence of Gene, white Background: Absence of Gene, * query sequence. sucrose metabolic enzymes shown in Figure 2, levansucrase and higher oligosaccharides in lactobacilli are lacking. Trehalose, cel- the fructo-furanosidase BfrA also show activity with RFO as sub- lobiose, and α-d-glucosyl-d-fructose isomers are all metabolized strates (Ehrmann et al., 2003; van Hijum et al., 2006; Teixeira et al., by phosphotransferase systems and intracellular phospho-glycosyl 2012). Few strains of lactobacilli harbor levansucrase but not α- hydrolases (Barrangou et al., 2006; Thompson et al., 2008; Francl Gal, e.g., L. sanfranciscensis LTH2590. These strains convert RFO et al., 2010). Corresponding to other phosphotransferase sys- to αGOS by levansucrase activity without further metabolism of tems, the expression of the operons is induced by the respective the galactosides (Table 5; Teixeira et al., 2012). Glucansucrases substrates. The spectrum of disaccharides that is metabolized and sucrose phosphorylase do not cleave RFO (Kim et al., 2003; by individual phosphotransferase systems can be quite diverse. van Hijum et al., 2006). In strains expressing melA, levansucrase, For example, the phospho-α-glucosyl hydrolase in the sucrose and sucrose phosphorylase or fructo-furanosidase, two alternative isomer metabolism (SIM) operon of L. casei recognizes the five α- pathways for RFO degradation exist: (i) extracellular conversion linked sucrose isomers (see above) as well as maltose-6-phosphate, of RFO to the corresponding αGOS and fructose by levansu- isomaltose-6-phosphate, and trehalose-6-phosphate as substrates. crase, followed by αGOS uptake and hydrolysis; and (ii) RFO Lactobacillus plantarum and L. gasseri harbor several systems uptake, followed by hydrolysis to sucrose and galactose through for transport and metabolism of β-glucosides (Andersson et al., MelA activity, and sucrose conversion by sucrose phosphorylase 2005; Francl et al., 2010). Bioinformatics analyses predicted the or fructo-furanosidase (Teixeira et al., 2012). Extracellular conver- presence of two β-glucoside/cellobiose specific phosphotrans- sion by levansucrase is the preferred metabolic route in L. reuteri ferase systems with associated phospho-β-glucosidase in addition (Teixeira et al., 2012), presumably because of facilitated transport. to a β-glucosidase in L. plantarum WCFS1 (Andersson et al., 2005). However, raffinose induces the expression of sucrose phosphory- It remains to be established whether this multitude of transport lase in L. reuteri (Teixeira et al., 2012) and B. lactis (Trindade et al., and enzyme systems reflects adaptation to substrates differing in 2003), demonstrating that both pathways exist in parallel. their linkage type or degree of polymerization. Human milk contains about 1% oligosaccharides. Human METABOLISM OF TREHALOSE, CELLOBIOSE, AND HUMAN milk oligosaccharides consist of d-glucose, d-galactose, N - MILK OLIGOSACCHARIDES acetylglucosamine, L-fucose, and sialic acid and have a degree of Lactobacilli also have the strain- or species-specific ability to polymerization of 3–32. The combination of different monomers, metabolize the disaccharides cellobiose [Glu-β-(1 → 4)-d-Glu], linkage types, and different degrees of branching or polymeriza- gentiobiose [Glu-β-(1 → 6)-d-Glu], trehalose [Glu-α-(1 → 1)-d- tion allows for a vast number of different structures. More than 100 α-Glu],and the α-d-glucosyl-d-fructose isomers trehalulose,tura- different structures were identified and the composition of milk nose, maltulose, leucrose, and palatinose. Trehalose is produced in oligosaccharides is dependent on the mother (Kunz et al., 2000; response to osmotic stress, or to survive dehydration by organisms Kobata, 2010). Human milk oligosaccharides generally consist of in all kingdoms (Crowe et al., 2001). Cellobiose is the degradation lactose at the reducing end and are elongated with galactose, N - product of cellulose or related plant β-glucans. Sucrose isomers, acetylglucosamine, fucose, and sialic acid. Core structures include particularly leucrose, may occur in nature as a product of bacterial galactosyllactose, fucosyllactose, lacto-N -fucopentaose, and sia- glucansucrase activity (van Hijum et al., 2006). lyllactose (Kunz et al., 2000; Kobata, 2010). These oligosaccha- Diverse disaccharide hydrolases or disaccharide-phosphate rides are not degraded by β-galactosidases of lactobacilli (Schwab hydrolases are found in L. plantarum, L. johnsonii, L. casei, and and Gänzle, 2011). Studies with purified human milk oligosac- L. acidophilus while L. brevis, L. reuteri, and L. delbrueckii have charides demonstrate that lactobacilli utilize fucose and N - a much more restricted spectrum of disaccharide-(phosphate) acetylglucosamine but not human milk oligosaccharides as carbon hydrolases (Andersson et al., 2005; Barrangou et al., 2006; Thomp- source (Ward et al., 2006; Schwab and Gänzle, 2011). Weak growth son et al., 2008; Francl et al., 2010). However, it was noted that of L. acidophilus on human milk oligosaccharides was observed for the annotated specificity of sugar transport systems is gener- L. acidophilus (Marcobal et al., 2010), which may reflect metab- ally inadequate and few studies provide functional analyses of olism of α-galactosyllactose or β-galactosyllactose, or the ability disaccharide metabolism (Thompson et al., 2008; Francl et al., to release few of the fucosyl- or N -acetylglucosaminyl-residues 2010). Moreover, studies on the metabolism of corresponding after cell lysis and release of intracellular glycosyl hydrolases. The

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inability of lactobacilli to grow on human milk oligosaccharides and L. plantarum. Oligosaccharides are preferentially metabolized contrasts the metabolic toolset of bifidobacteria, which are highly by phosphotransferase/phospho-glycosyl hydrolase systems and adapted to growth on human milk oligosaccharides as carbon oligosaccharide metabolism is repressed by glucose (Andersson source (González et al., 2008; Sela et al., 2008). et al., 2005; Barrangou et al., 2006; Monedero et al., 2008; Francl et al., 2010). Other species in this continuum of metabolic diver- CONCLUSION sity, however, exhibit more restricted carbohydrate fermentation Lactobacilli are well-equipped to metabolize oligosaccharides that patterns. An extreme is the “nothing but maltose or sucrose” diet occur in their habitats, including plants, milk, and the (upper) of several strains of L. sanfranciscensis, which is partially reflected intestinal tract of humans and animals. This metabolic diver- in the genome-sequenced strain L. sanfranciscensis TMW1.304 sity is remarkable for a group of organisms that have evolved (Table 1). In this group of strains, oligosaccharides are pref- by reduction of genome size (Makarova et al., 2006). Metabolic erentially metabolized by permease/phosphorylase systems and pathways for disaccharide metabolism often also enable the metab- oligosaccharide metabolic enzymes are not repressed by glucose olism of tri- and tetrasaccharides. However, with the exception (Tieking et al., 2005; Schwab et al., 2007; Teixeira et al., 2012). of amylase and levansucrase, metabolic enzymes for oligosaccha- Remarkably, both groups – broad versus narrow spectrum of ride conversion are intracellular and oligosaccharide metabolism oligosaccharide fermentation – are represented in intestinal habi- is limited by transport. Starch and related α-glucans are the only tats (e.g., L. acidophilus and L. reuteri) as well as food fermenta- group of compounds for which a metabolic pathway dedicated to tions (e.g., L. plantarum and L. sanfranciscensis). Further insight oligo- and polysaccharide metabolism was retained. This general into oligosaccharide metabolism in lactobacilli is dependent on the restriction to intracellular glycosyl hydrolases clearly differentiates biochemical characterization of metabolic enzymes and their sub- lactobacilli from other bacteria that adapted to intestinal habitats, strate specificity – particularly transport enzymes – and the sound particularly Bifidobacterium spp., which maintain a more exten- quantification of oligosaccharide consumption during metabo- sive toolset for extracellular hydrolysis and transport of complex lism of lactobacilli in model substrates, and in food or intestinal carbohydrates (Sela et al., 2008; van den Broek et al., 2008). The ecosystems. divergent approach of bifidobacteria and lactobacilli to carbohy- Glycosyl hydrolases and glycosyl phosphorylases of lactic acid drate fermentation may reflect their respective dominance in the bacteria have evolved as an important tool in the (chemo-)- human colon, characterized by a limited availability of mono- enzymatic synthesis of functional oligosaccharides or sugar deriv- and disaccharides, and the upper intestinal tract of animals, which atives (e.g., van Hijum et al., 2006; Goedl et al., 2008; Black et al., offers a rich supply of oligosaccharides (Sela et al., 2008; Walter, 2012). Further insight into the diversity and catalytic properties of 2008). carbohydrate-active enzymes of lactobacilli will further improve The capacity of individual strains and species of lactobacilli for this toolset for food-related and other applications. oligosaccharide metabolism differs substantially. This metabolic diversity conforms to the phylogenetic diversity in the genus Lac- ACKNOWLEDGMENTS tobacillus. Several species metabolize a large diversity of different We wish to thank Ali Ketabi for helpful discussions during carbon sources, including all major categories of oligosaccharides. manuscript preparation. Michael Gänzle acknowledges financial Well-characterized representatives include L. acidophilus, L. casei, support from the Canada Research Chairs program.

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Walter, J., Schwab, C., Loach, D. of breast milk oligosaccharides by conducted in the absence of any com- This article was submitted to Frontiers in M., Gänzle, M. G., and Tan- Bifidobacterium infantis and Lacto- mercial or financial relationships that Food Microbiology, a specialty of Fron- nock, G. W. (2008). Glucosyl- bacillus gasseri. Appl. Environ. Micro- could be construed as a potential con- tiers in Microbiology. transferase A (GtfA) and inulosu- biol. 72, 4497–4499. flict of interest. Copyright © 2012 Gänzle and Fol- crase (Inu) of Lactobacillus reuteri Yun, J. W. (1996). Fructooligosac- lador. This is an open-access arti- TMW1.106 contribute to cell aggre- charides – occurrence, Received: 23 July 2012; accepted: 04 Sep- cle distributed under the terms of the gation, in vitro biofilm formation, preparation, and application. tember 2012; published online: 26 Sep- Creative Commons Attribution License, and colonization of the mouse gas- Enzyme Microb. Technol. 19, tember 2012. which permits use, distribution and trointestinal tract. Microbiology 154, 107–117. Citation: Gänzle MG and Follador reproduction in other forums, pro- 72–80. R (2012) Metabolism of oligosac- vided the original authors and source Ward, R. E., Ninonuevo, M., Mills, D. charides and starch in lactobacilli: are credited and subject to any copy- A., Lebrilla, C. B., and German, Conflict of Interest Statement: The a review. Front. Microbio. 3:340. doi: right notices concerning any third-party J. B. (2006). In vitro fermentation authors declare that the research was 10.3389/fmicb.2012.00340 graphics etc.

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