Adaptive Evolution of Thermotoga maritima Reveals Plasticity of the ABC Transporter Network

ABSTRACTThermotoga maritimais a hyperthermophilic anaerobe that utilizes a vast network of ABC transporters to efficiently metabolize a variety of carbon sources to produce hydrogen. For unknown reasons, this organism does not metabolize glucose as readily as it does glucose di- and polysaccharides. The leading hypothesis implicates the thermolability of glucose at the physiological temperatures at whichT. maritimalives. After a 25-day laboratory evolution, phenotypes were observed with growth rates up to 1.4 times higher than and glucose utilization rates exceeding 50% those of the wild type. Genome resequencing revealed mutations in evolved cultures related to glucose-responsive ABC transporters. The native glucose ABC transporter, GluEFK, has more abundant transcripts either as a result of gene duplication-amplification or through mutations to the operator sequence regulating this operon. Conversely, BglEFGKL, a transporter of beta-glucosides, is substantially downregulated due to a nonsense mutation to the solute binding protein or due to a deletion of the upstream promoter. Analysis of the ABC2 uptake porter families for carbohydrate and peptide transport revealed that the solute binding protein, often among the transcripts detected at the highest levels, is predominantly downregulated in the evolved cultures, while the membrane-spanning domain and nucleotide binding components are less varied. Similar trends were observed in evolved strains grown on glycerol, a substrate that is not dependent on ABC transporters. Therefore, improved growth on glucose is achieved through mutations favoring GluEFK expression over BglEFGKL, and in lieu of carbon catabolite repression, the ABC transporter network is modulated to achieve improved growth fitness.

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Novel Mechanisms of Efflux-Mediated Levofloxacin Resistance and Reduced Amikacin Susceptibility in Stenotrophomonas maltophilia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01284-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01284-20

Author(s):

Punyawee Dulyayangkul ◽

Karina Calvopiña ◽

Kate J. Heesom ◽

Matthew B. Avison

Keyword(s):

Abc Transporter ◽

Abc Transporters ◽

Stenotrophomonas Maltophilia ◽

Efflux Pump ◽

Regulatory System ◽

Fluoroquinolone Resistance ◽

Content Type ◽

Antimicrobial Drug Resistance ◽

Regulatory Systems ◽

Selection Of

ABSTRACTFluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr. Overproduction of the other ABC transporter, Smlt2642/3 (renamed SmaAB), and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVr. Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

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ABC Transporters Required for Hexose Uptake byClostridium phytofermentans

Journal of Bacteriology ◽

10.1128/jb.00241-19 ◽

2019 ◽

Vol 201 (15) ◽

Cited By ~ 2

Author(s):

Tristan Cerisy ◽

Alba Iglesias ◽

William Rostain ◽

Magali Boutard ◽

Christine Pelle ◽

...

Keyword(s):

Abc Transporters ◽

Extracellular Enzymes ◽

Plant Biomass ◽

Carbon Sources ◽

Anaerobic Bacteria ◽

Model Organism ◽

Industrial Transformation ◽

Content Type ◽

Plant Matter

ABSTRACTThe mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal howClostridium(Lachnoclostridium)phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based onin vitroactivities. These mechanisms used byClostridiato uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCEPlant-fermentingClostridiaare anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine.Clostridiadegrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organismClostridium phytofermentans. We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

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Molecular Basis of Unexpected Specificity of ABC Transporter-Associated Substrate-Binding Protein DppA from Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.00400-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 1

Author(s):

Mohammad M. Rahman ◽

Mayra A. Machuca ◽

Mohammad F. Khan ◽

Christopher K. Barlow ◽

Ralf B. Schittenhelm ◽

...

Keyword(s):

Helicobacter Pylori ◽

Abc Transporter ◽

Binding Protein ◽

Substrate Binding ◽

Binding Pocket ◽

Amino Acid Sequences ◽

Content Type ◽

H Pylori ◽

Substrate Binding Protein ◽

Broad Specificity

ABSTRACT The gastric pathogen Helicobacter pylori has limited ability to use carbohydrates as a carbon source, relying instead on exogenous amino acids and peptides. Uptake of certain peptides by H. pylori requires an ATP binding cassette (ABC) transporter annotated dipeptide permease (Dpp). The transporter specificity is determined by its cognate substrate-binding protein DppA, which captures ligands in the periplasm and delivers them to the permease. Here, we show that, unlike previously characterized DppA proteins, H. pylori DppA binds, with micromolar affinity, peptides of diverse amino acid sequences ranging between two and eight residues in length. We present analysis of the 1.45-Å-resolution crystal structure of its complex with the tetrapeptide STSA, which provides a structural rationale for the observed broad specificity. Analysis of the molecular surface revealed a ligand-binding pocket that is large enough to accommodate peptides of up to nine residues in length. The structure suggests that H. pylori DppA is able to recognize a wide range of peptide sequences by forming interactions primarily with the peptide main chain atoms. The loop that terminates the peptide-binding pocket in DppAs from other bacteria is significantly shorter in the H. pylori protein, providing an explanation for its ability to bind longer peptides. The subsites accommodating the two N-terminal residues of the peptide ligand make the greatest contribution to the protein-ligand binding energy, in agreement with the observation that dipeptides bind with affinity close to that of longer peptides. IMPORTANCE The World Health Organization listed Helicobacter pylori as a high-priority pathogen for antibiotic development. The potential of using peptide transporters in drug design is well recognized. We discovered that the substrate-binding protein of the ABC transporter for peptides, termed dipeptide permease, is an unusual member of its family in that it directly binds peptides of diverse amino acid sequences, ranging between two and eight residues in length. We also provided a structural rationale for the observed broad specificity. Since the ability to import peptides as a source of carbon is critical for H. pylori, our findings will inform drug design strategies based on inhibition or fusion of membrane-impermeant antimicrobials with peptides.

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A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02547-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 3934-3941 ◽

Cited By ~ 14

Author(s):

Hansjürg Engel ◽

Moana Mika ◽

Dalia Denapaite ◽

Regine Hakenbeck ◽

Kathrin Mühlemann ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Abc Transporter ◽

Binding Protein ◽

Population Analysis ◽

Resistance Testing ◽

Penicillin Binding Protein ◽

Secondary Resistance ◽

Content Type ◽

Abc Transporter Genes

ABSTRACTHeteroresistance to penicillin inStreptococcus pneumoniaeis the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found thatpbp2x, but notpbp2borpbp1aalone, conferred heteroresistance to R6. However, a change ofpbp2xexpression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP onciaRdisruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinitypbp2xundergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded bypstS,phoU,pstB, andpstCin these highly resistant subpopulations. In conclusion, the presence of low-affinitypbp2xenables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

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Cryo-electron Microscopy Structure and Transport Mechanism of a Wall Teichoic Acid ABC Transporter

mBio ◽

10.1128/mbio.02749-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 8

Author(s):

Li Chen ◽

Wen-Tao Hou ◽

Tao Fan ◽

Banghui Liu ◽

Ting Pan ◽

...

Keyword(s):

Abc Transporter ◽

Conformational Changes ◽

Abc Transporters ◽

Rational Design ◽

Teichoic Acid ◽

Inhibitory Mechanism ◽

Wall Teichoic Acid ◽

Content Type ◽

Design And Optimization ◽

Cryo Electron Microscopy

ABSTRACT The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a “crankshaft conrod” mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA. IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a “crankshaft conrod” mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.

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Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.00792-15 ◽

2015 ◽

Vol 81 (12) ◽

pp. 4173-4183 ◽

Cited By ~ 10

Author(s):

Akira Watanabe ◽

Kazumi Hiraga ◽

Masako Suda ◽

Hideaki Yukawa ◽

Masayuki Inui

Keyword(s):

Heterologous Expression ◽

Corynebacterium Glutamicum ◽

Abc Transporter ◽

Binding Protein ◽

Functional Characterization ◽

Product Inhibition ◽

Gene Encoding ◽

Content Type ◽

Industrial Strains ◽

Efficient Fermentation

ABSTRACTTheCorynebacterium alkanolyticumxylEFGDgene cluster comprises thexylDgene that encodes an intracellular β-xylosidase next to thexylEFGoperon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover forp-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover forp-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Kifor xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutivetacpromoter in theCorynebacterium glutamicumxylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended oncgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguousxylDgene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability ofC. glutamicumto take up xylooligosaccharides, an ability that is enhanced by in the presence of a functionalxylEFG-encoded xyloside ABC transporter. The finding thatxylEFGimparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.

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Ligands of Thermophilic ABC Transporters Encoded in a Newly Sequenced Genomic Region of Thermotoga maritima MSB8 Screened by Differential Scanning Fluorimetry

Applied and Environmental Microbiology ◽

10.1128/aem.05418-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6395-6399 ◽

Cited By ~ 16

Author(s):

Nathalie Boucher ◽

Kenneth M. Noll

Keyword(s):

Abc Transporter ◽

Thermotoga Maritima ◽

Binding Constants ◽

Genomic Region ◽

Genomic Research ◽

Gene Encoding ◽

Content Type ◽

Differential Scanning Fluorimetry ◽

Beta Glucosidase ◽

Abc Transporter Genes

ABSTRACTThe chromosome ofThermotoga maritimastrain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 μM, 0.300 μM, and 56.78 μM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound withKd(dissociation constant) values of 0.042 μM, 0.059 μM, and 1.436 μM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars:T. maritimaMSB8 genomovar TIGR andT. maritimaMSB8 genomovar DSM 3109, respectively.

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Identification of an ATPase, MsmK, Which Energizes Multiple Carbohydrate ABC Transporters in Streptococcus pneumoniae

Infection and Immunity ◽

10.1128/iai.05290-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4193-4200 ◽

Cited By ~ 40

Author(s):

Carolyn Marion ◽

Andrew E. Aten ◽

Shireen A. Woodiga ◽

Samantha J. King

Keyword(s):

Streptococcus Pneumoniae ◽

Sialic Acid ◽

Abc Transporter ◽

Abc Transporters ◽

Community Acquired Pneumonia ◽

Bacterial Survival ◽

Carbohydrate Source ◽

Content Type ◽

Airway Colonization

ABSTRACTStreptococcus pneumoniaeis the leading cause of community-acquired pneumonia and results in over 1 million deaths each year worldwide. Asymptomatic colonization of the airway precedes disease, and acquisition of carbohydrates from the host environment is necessary for bacterial survival. We previously demonstrated thatS. pneumoniaecleaves sialic acid from human glycoconjugates to be used as a carbohydrate source. ThesatABCgenes are required for growth and import of sialic acid. ThesatABCgenes are predicted to encode components of an ABC transporter but not the ATPases essential to energize transport. As this subunit is essential, an ATPase must be encoded elsewhere in the genome. We identifiedmsmKas a candidate based on similarity to other known carbohydrate ATPases. Recombinant MsmK hydrolyzed ATP, revealing that MsmK is an ATPase. AnmsmKmutant was reduced in growth on and transport of sialic acid, demonstrating that MsmK is the ATPase energizing the sialic acid transporter. In addition to satABC,S. pneumoniaecontains five other loci that are predicted to encode CUT1 family carbohydrate ABC transporter components; each of these lacks a predicted ATPase. Data indicate thatmsmKis also required for growth on raffinose and maltotetraose, which are the substrates of two other characterized carbohydrate ABC transporters. Furthermore, anmsmKmutant was reduced in airway colonization. Together, these data imply thatin vivo, MsmK energizes multiple carbohydrate transporters inS. pneumoniae. This is the first demonstration of a shared ATPase in a pathogenic bacterium.

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Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

Eukaryotic Cell ◽

10.1128/ec.00171-13 ◽

2013 ◽

Vol 12 (12) ◽

pp. 1619-1628 ◽

Cited By ~ 43

Author(s):

Sanjoy Paul ◽

Daniel Diekema ◽

W. Scott Moye-Rowley

Keyword(s):

Drug Resistance ◽

Aspergillus Fumigatus ◽

Abc Transporter ◽

Abc Transporters ◽

Sequence Similarity ◽

Atp Binding ◽

Content Type ◽

Transporter Proteins ◽

Encoding Genes ◽

Atp Binding Cassette

ABSTRACTIn yeast cells such as those ofSaccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of twoAspergillus fumigatusproteins that share high sequence similarity withS. cerevisiaePdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The twoA. fumigatusgenes encoding the ABC transporters sharing the highest sequence similarity toS. cerevisiaePdr5 are calledabcAandabcBhere. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains ofA. fumigatus. Loss ofabcBinvariably elicited increased azole susceptibility, whileabcAdisruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from thehspApromoter inA. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane inA. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen.

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Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

Applied and Environmental Microbiology ◽

10.1128/aem.03442-16 ◽

2017 ◽

Vol 83 (7) ◽

Cited By ~ 1

Author(s):

Hoi Yee Keung ◽

Tsz Kai Li ◽

Lok To Sham ◽

Man Kit Cheung ◽

Peter Chi Keung Cheung ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Abc Transporter ◽

Transport System ◽

Binding Protein ◽

Atp Binding ◽

Carbohydrate Utilization ◽

Content Type ◽

Bifidobacterium Breve ◽

Abc Transport ◽

Atp Binding Protein

ABSTRACT Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.

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