The molecular basis of glycogen breakdown and transport in <i>Streptococcus pneumoniae</i>

Abbott, D. Wade; Higgins, M.A.; Hyrnuik, S.; Pluvinage, B.; Bueren, A. Lammerts van; Boraston, A.B.

doi:10.1111/j.1365-2958.2010.07199.x

Cited by 59 publications

(85 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…RafEFG is known to transport raffinose, and MalXCD was shown to efficiently transport maltooligosaccharides with between 3 and 8 glucose repeats (1,30,32). To test if msmK might also play a role in utilization of these carbohydrates, the msmK mutant was tested for growth with either raffinose or maltotetraose as the sole carbon source.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2011

View full text Add to dashboard Cite

Streptococcus pneumoniae is 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 that S. pneumoniae cleaves sialic acid from human glycoconjugates to be used as a carbohydrate source. The satABC genes are required for growth and import of sialic acid. The satABC genes 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 identified msmK as a candidate based on similarity to other known carbohydrate ATPases. Recombinant MsmK hydrolyzed ATP, revealing that MsmK is an ATPase. An msmK mutant 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. pneumoniae contains five other loci that are predicted to encode CUT1 family carbohydrate ABC transporter components; each of these lacks a predicted ATPase. Data indicate that msmK is also required for growth on raffinose and maltotetraose, which are the substrates of two other characterized carbohydrate ABC transporters. Furthermore, an msmK mutant was reduced in airway colonization. Together, these data imply that in vivo, MsmK energizes multiple carbohydrate transporters in S. pneumoniae. This is the first demonstration of a shared ATPase in a pathogenic bacterium.

show abstract

Section: Resultsmentioning

confidence: 99%

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

et al. 2011

View full text Add to dashboard Cite

show abstract

“…The corresponding transporter from the taxonomically related Gram-positive pathogen Streptococcus pneumoniae mediates the uptake of malto-oligosaccharides up to eight units with a preference for maltotetraose (44). The expression of the ABC uptake system may be affected by an inserted transposase (LBA1868) that separates the catabolic genes from the transporter in L. acidophilus NCFM but not in other lactobacilli (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

View full text Add to dashboard Cite

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.

show abstract

“…It is unclear if the same is valid for the group represented by L. acidophilus NCFM, where the GH13_31 resides on a separate locus. It cannot be ruled out, however, that uptake of a shorter IMO, e.g., IG2, occurs via other types of transporters, e.g., phosphoenolpyruvate-dependent phosphotransferase system (PTS), as demonstrated in the case of maltose/maltooligosaccharide utilization in other Gram-positive bacteria, where maltose is internalized via a PTS transporter and where longer maltooligosaccharides are transported via an ABC transporter (1,45). Phosphorylated maltose and maltotriose internalized via a PTS are recognized by a specific GH4 6-phospho-␣-glucosidase (EC 3.2.1.122) (45).…”

Section: Figmentioning

confidence: 99%

Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

et al. 2012

View full text Add to dashboard Cite

b Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode ␣-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and ␣-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of ␣-1,6-and ␣-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-␣-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 Å and revealed additional substrate contacts at the ؉2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents.

show abstract

The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae

Cited by 59 publications

References 60 publications

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

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

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

Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

Contact Info

Product

Resources

About