Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli

Johnson, Brant R.; Hymes, Jeffrey P.; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo; Barrangou, Rodolphe; Klaenhammer, Todd R.

doi:10.1128/aem.01968-15

Cited by 48 publications

(84 citation statements)

References 56 publications

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“…This suggests that SLAP domains constitute a general cell attachment scaffold that is fused to a select set of activities destined for cell surface display. The prevalence of this domain in the S-layer exoproteome of distinct lactobacilli is also in agreement with this role (43).…”

Section: Discussionsupporting

confidence: 62%

“…The repression of pullulanase activity in the presence of glucose is suggestive of regulation through global catabolite repression (45). Indeed, this enzyme was not identified in the exoproteome of L. acidophilus NCFM grown on glucose in a recent proteomic analysis of S-layer-associated proteins (43). A similar observation was made in a proteome analysis of L. acidophilus NCFM grown on raffinose, where LaPul13_14 was repressed in the presence of glucose (46) but was clearly detectable in the presence of raffinose, suggesting a level of constitutive expression of the enzyme in the absence of glucose.…”

Section: Discussionmentioning

confidence: 99%

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

Møller

Goh

Rasmussen

et al. 2017

Appl Environ Microbiol

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

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

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

Møller

Goh

Rasmussen

et al. 2017

Appl Environ Microbiol

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“…Most notably, the probiotic activity of L. acidophilus is mediated by cell surface-associated components which interact with the host gastrointestinal mucosa and immune system (9–11). …”

Section: Introductionmentioning

confidence: 99%

AcmB Is an S-Layer-Associated β- N -Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM

Johnson

Klaenhammer

2016

Appl Environ Microbiol

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Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus. Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro. These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species.IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.

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“…This strain consequently degraded xylans more extensively than wild-type C. bescii. The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptor species.Many bacteria (1, 2) and archaea (3) produce a two-dimensional, para-crystalline array of protein that covers the outside of the cell, referred to as a surface layer (S-layer).5 In bacteria, the majority of S-layer proteins are non-covalently associated with the bacterial cell surface via specialized domains at their N or C terminus, typically from one of three distinct but analogous domain categories: surface layer homology (SLH) (4, 5), CWB2 (pfam04122) (6), or NCAD (previously SLAP, pfam03217) (7,8). In archaea, S-layer proteins are most often attached to the cell membrane.…”

mentioning

confidence: 99%

“…5 In bacteria, the majority of S-layer proteins are non-covalently associated with the bacterial cell surface via specialized domains at their N or C terminus, typically from one of three distinct but analogous domain categories: surface layer homology (SLH) (4, 5), CWB2 (pfam04122) (6), or NCAD (previously SLAP, pfam03217) (7,8). In archaea, S-layer proteins are most often attached to the cell membrane.…”

mentioning

confidence: 99%

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

Conway¹,

Pierce

et al. 2016

Journal of Biological Chemistry

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The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis encodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptor species genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique to C. kronotskyensis and contains SLH domains. Calkro_0111 was produced recombinantly in Escherichia coli as two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo-and exoglucanase activity on the ␤-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs in C. kronotskyensis, when produced in E. coli, was active on a variety of xylans and ␤-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptor and among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii. As such, the gene encoding Calkro_0402 was inserted into the C. bescii genome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-type C. bescii. The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptor species.Many bacteria (1, 2) and archaea (3) produce a two-dimensional, para-crystalline array of protein that covers the outside of the cell, referred to as a surface layer (S-layer).5 In bacteria, the majority of S-layer proteins are non-covalently associated with the bacterial cell surface via specialized domains at their N or C terminus, typically from one of three distinct but analogous domain categories: surface layer homology (SLH) (4, 5), CWB2 (pfam04122) (6), or NCAD (previously SLAP, pfam03217) (7,8). In archaea, S-layer proteins are most often attached to the cell membrane. S-layer proteins can be anchored to the membrane via a C-terminal transmembrane helix domain (3, 9) or covalently attached to cell membrane lipid via an archaeosortase, as shown in Haloferax volcanii (10,11). In most microorganisms, the S-layer is composed entirely of one or two proteins (SLPs), which self-assemble. In addition to these SLPs, some Firmicutes produce a family of proteins that also contain SLH, CWB2, or NCAD domains and, as such, are predicted to be associated with the S-layer (12). SLPs and S-layer associated proteins can also contain domains with specialized functions allowing the S-layer to play a role in adherence and biofilm formation (13-16), biogenesis of the cell envelope (17), cell division (18...

show abstract

Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli

Cited by 48 publications

References 56 publications

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

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

AcmB Is an S-Layer-Associated β- N -Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

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