Crystallization and X-ray Crystallographic Analysis of the Adhesive SpaC Pilin Subunit in the SpaCBA Pilus of Gut-adapted Lactobacillus rhamnosus GG

ABSTRACT Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.

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New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit

Scientific Reports ◽

10.1038/srep28664 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 9

Author(s):

Priyanka Chaurasia ◽

Shivendra Pratap ◽

Ingemar von Ossowski ◽

Airi Palva ◽

Vengadesan Krishnan

Keyword(s):

Crystal Structure ◽

Lactobacillus Rhamnosus ◽

Lactobacillus Rhamnosus Gg ◽

Pilin Subunit

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Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adaptedLactobacillus rhamnosusGG

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17006963 ◽

2017 ◽

Vol 73 (6) ◽

pp. 321-327 ◽

Cited By ~ 7

Author(s):

Arjun K. Mishra ◽

Abhin Kumar Megta ◽

Airi Palva ◽

Ingemar von Ossowski ◽

Vengadesan Krishnan

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Molecular Mechanisms ◽

Recombinant Expression ◽

Lactobacillus Rhamnosus ◽

X Ray Diffraction ◽

X Ray ◽

Pilin Subunit ◽

Sad Phasing

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensalLactobacillus rhamnosusGG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria,L. rhamnosusGG SpaE for crystallization was produced by recombinant expression inEscherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.

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Growth of Lactic Acid Bacteria on Gold—Influence of Surface Roughness and Chemical Composition

Nanomaterials ◽

10.3390/nano10122499 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2499

Author(s):

Joanna Grudzień ◽

Magdalena Jarosz ◽

Kamil Kamiński ◽

Mirosława Kobasa ◽

Karol Wolski ◽

...

Keyword(s):

Surface Roughness ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Lactobacillus Rhamnosus ◽

Well Being ◽

Metallic Surface ◽

Lactobacillus Rhamnosus Gg ◽

Surface Colonization ◽

X Ray ◽

Gold Substrates

The main focus of this work was to establish a correlation between surface topography and chemistry and surface colonization by lactic acid bacteria. For this reason, we chose gold substrates with different surface architectures (i.e., smooth and nanorough) that were characterized by atomic force microscopy (AFM), electron scanning microscopy (SEM), and X-ray diffractometry (XRD). Moreover, to enhance biocompatibility, we modified gold substrates with polymeric monolayers, namely cationic dextran derivatives with different molar masses. The presence of those layers was confirmed by AFM, infrared spectroscopy (IR), and X-ray photoelectron spectroscopy (XPS). In order to determine the adhesion abilities of non-modified and modified gold surfaces, we tested three lactic acid bacteria (LAB) strains (i.e., Lactobacillus rhamnosus GG, Lactobacillus acidophilus, and Lactobacillus plantarum 299v). We have shown that surface roughness influences the surface colonization of bacteria, and the most significant impact on the growth was observed for the Lactobacillus rhamnosus GG strain. What is more, covering the gold surface with a molecular polymeric film by using the layer-by-layer (LbL) method allows additional changes in the bacterial growth, independently on the used strain. The well-being of the bacteria cells on tested surfaces was confirmed by using selective staining and fluorescence microscopy. Finally, we have determined the bacterial metabolic activity by measuring the amount of produced lactic acid regarding the growth conditions. The obtained results proved that the adhesion of bacteria to the metallic surface depends on the chemistry and topography of the surface, as well as the specific bacteria strain.

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Sortases from a Probiotic Lactobacillus rhamnosus GG: Cloning, Expression, Purification, Crystallization and Preliminary X-Ray Diffraction Study

Crystallography Reports ◽

10.1134/s1063774519070162 ◽

2019 ◽

Vol 64 (7) ◽

pp. 1117-1121 ◽

Cited By ~ 2

Author(s):

S. Pratap ◽

A. K. Megta ◽

V. Krishnan

Keyword(s):

Diffraction Study ◽

Lactobacillus Rhamnosus ◽

X Ray Diffraction ◽

Lactobacillus Rhamnosus Gg ◽

X Ray ◽

Probiotic Lactobacillus

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Quaternary structure of Limulus polyphemus hemocyanin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081747 ◽

1978 ◽

Vol 36 (2) ◽

pp. 176-177

Author(s):

T. Wichertjes ◽

E.J. Kwak ◽

E.F.J. Van Bruggen

Keyword(s):

Electron Microscopy ◽

Functional Properties ◽

Horseshoe Crab ◽

Temperature Jump ◽

Quaternary Structure ◽

Crystallographic Analysis ◽

Limulus Polyphemus ◽

Oxygen Binding ◽

X Ray ◽

Intact Molecule

Hemocyanin of the horseshoe crab (Limulus polyphemus) has been studied in nany ways. Recently the structure, dissociation and reassembly was studied using electron microscopy of negatively stained specimens as the method of investigation. Crystallization of the protein proved to be possible and X-ray crystallographic analysis was started. Also fluorescence properties of the hemocyanin after dialysis against Tris-glycine buffer + 0.01 M EDTA pH 8.9 (so called “stripped” hemocyanin) and its fractions II and V were studied, as well as functional properties of the fractions by NMR. Finally the temperature-jump method was used for assaying the oxygen binding of the dissociating molecule and of preparations of isolated subunits. Nevertheless very little is known about the structure of the intact molecule. Schutter et al. suggested that the molecule possibly consists of two halves, combined in a staggered way, the halves themselves consisting of four subunits arranged in a square.

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