Journal: Applied and Environmental Microbiology

Article Title: Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

doi: 10.1128/aem.01853-24

Figure Lengend Snippet: Smlt1473 inhibits the mucoid phenotype of P. aeruginosa , but data suggest most effective enzyme concentration varies by isolate. ( A ) Representative image of phenotypic changes for P. aeruginosa UVA 44618 in the presence and absence of Smlt1473 indicating an inhibitory function. ( B–F ) Each isolate of P. aeruginosa was grown in the presence and absence of Smlt1473, plate contents were collected, and uronic acid concentration, which corresponds to alginate content, was quantified. Uronic acid concentration was determined by measuring the absorbance at 530 nm (A530) of the resulting solution. High A530 corresponds to high alginate content, whereas low A530 corresponds to low alginate content. Y222F is the catalytically inactive form of Smlt1473 used to show that results are due to an active enzyme. The results are means and standard deviations and statistical analysis was performed using a One-way Welch’s ANOVA and Dunnett’s T3 multiple comparison post-hoc test.

Article Snippet: An E. coli codon-optimized nucleotide sequence of Smlt1473 (GenBank accession number CAQ45011 ) was synthesized and cloned into pET-28a(+) using NcoI/XhoI restriction sites (GenScript).

Techniques: Concentration Assay, Comparison

Journal: Applied and Environmental Microbiology

Article Title: Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

doi: 10.1128/aem.01853-24

Figure Lengend Snippet: SEM images of all mucoid P. aeruginosa isolates treated with Smlt1473 and buffer showing enzymatic inhibition of mucoid phenotype. Samples were grown in the presence of enzyme or buffer, transferred to 12 mm glass slides, glutaraldehyde fixed, and imaged using SEM. Each set of images is shown at 50,000× magnification. The left side of the figure panel depicts samples that were grown in the presence of buffer, whereas the right side shows samples that were grown in the presence of Smlt1473. (A and B) UVA 44618, (C and D) UVA 61605, (E and F) UVA 84977, (G and H) UVA 55009, and (I and J) PDO300.

Article Snippet: An E. coli codon-optimized nucleotide sequence of Smlt1473 (GenBank accession number CAQ45011 ) was synthesized and cloned into pET-28a(+) using NcoI/XhoI restriction sites (GenScript).

Techniques: Inhibition

Journal: Applied and Environmental Microbiology

Article Title: Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

doi: 10.1128/aem.01853-24

Figure Lengend Snippet: Smlt1473 degrades the mucoid biofilm of P. aeruginosa after it has been established on a surface. ( A ) UVA 61605 biofilm after 24 h of growth showing a prominent raised, mucoid phenotype. ( B–F ) Each P. aeruginosa isolate was grown on LB agar for 24 h at 37°C with no treatment to develop an established mucoid phenotype, as shown in panel A. Plate contents were collected, and the alginate-containing biofilm was used as the substrate in the TBA assay. Upon addition of enzyme, alginate is depolymerized via β-elimination mechanism where unsaturated products react with thiobarbituric acid to create a pink chromogen with absorbance at 540 nm. High A540 corresponds to greater alginate depolymerization, and low A540 corresponds to minimal alginate depolymerization. The results presented are means and standard deviations, and statistical analysis was performed using a one-way Welch’s ANOVA and Dunnett’s T3 multiple comparison post-hoc tests. ( G ) Representative image displaying the pink chromogen as a result of the addition of Smlt1473 to UVA 55009 biofilm mixture (right) compared with the addition of buffer to the mixture (left).

Article Snippet: An E. coli codon-optimized nucleotide sequence of Smlt1473 (GenBank accession number CAQ45011 ) was synthesized and cloned into pET-28a(+) using NcoI/XhoI restriction sites (GenScript).

Techniques: Comparison

Journal: Applied and Environmental Microbiology

Article Title: Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

doi: 10.1128/aem.01853-24

Figure Lengend Snippet: Stacked 1 H NMR spectra of all five P. aeruginosa isolates showing acetylation and quantitative determination of degree of acetylation. ( A ) Peaks around 2.12ppm are a result of the acetyl group of acetylated sugars, indicating all of the isolates in this study are comprised of acetylated alginate. ( B ) The degree of acetylation was quantitatively determined using a method previously described, further proving that all biofilm samples have some fraction of acetylated alginate. Data in shows that Smlt1473 degrades established mucoid biofilm, and in combination with these NMR and degree of acetylation results, we can conclude that Smlt1473 is effective against acetylated alginate.

Article Snippet: An E. coli codon-optimized nucleotide sequence of Smlt1473 (GenBank accession number CAQ45011 ) was synthesized and cloned into pET-28a(+) using NcoI/XhoI restriction sites (GenScript).

Techniques: