Background: Polysaccharide lyases degrade anionic polysaccharides and are important in bacterial biofilm formation and host-pathogen interactions. Results: Putative alginate lyase (Smlt1473) from Stenotrophomonas maltophillia displays activity toward multiple polysaccharides in a pH-regulated manner. Conclusion: Smlt1473 is a unique polysaccharide lyase with pH-dependent activity toward mammalian, plant, and microbial substrates. Significance: Characterization of Smlt1473 allows for an understanding of possible roles in biofilm formation and pathogenesis.

Background: A polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia degrades multiple anionic polysaccharides in a pH-regulated manner. Results: Mutation of predicted substrate-binding residues significantly increased activity and specificity toward poly-␤-D-mannuronic acid or poly-␤-D-glucuronic acid. Conclusion: Substrate specificity is highly sensitive to conserved, charged, and aromatic residues flanking the active site. Significance: Smlt1473 may serve as a platform for the design of robust, highly specific polysaccharide lyases.

BackgroundMacroalgae represents a promising source of fermentable carbohydrates for use in the production of energy efficient biofuel. The primary carbohydrate in brown algae is the uronic acid-containing alginate, whereas green algae contains a significant amount of glucuronan. A necessary step in the conversion of these polyuronides to bioethanol is saccharification, which can be achieved by enzymatic or chemical degradation.ResultsPolysaccharide lyases are a class of enzymes which cleave uronic acid-containing glycans via a β-elimination mechanism, acting both endo- and exolytically on their substrates. In the present work, we characterize a putative alginate lyase from Stenotrophomonas maltophilia K279a (Smlt2602) and describe a H208F mutant that, in addition to cleaving alginate-based substrates, displays significant, exolytic glucuronan activity.ConclusionsTo our knowledge this is the first polysaccharide lyase to act exolytically on glucuronan and is an attractive candidate for the broad-spectrum digestion of polyuronides into fermentable monomers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0455-8) contains supplementary material, which is available to authorized users.

Stenotrophomonas maltophilia is an emerging, multidrug-resistant pathogen of increasing importance for the immunocompromised, including cystic fibrosis patients. Despite its significance as an emerging pathogen, relatively little is known regarding the specific factors and mechanisms that contribute to its pathogenicity. We identify and characterize a putative ankyrin-repeat protein (Smlt3054) unique to clinical S. maltophilia isolates that binds F-actin in vitro and co-localizes with actin in transfected HEK293a cells. Smlt3054 is endogenously expressed and secreted from clinical S. maltophilia isolates, but not an environmental isolate (R551-3). The in vitro binding of Smlt3054 to F-actin resulted in a thickening of the filaments as observed by TEM. Ectopic expression of Smlt3054-GFP exhibits strong co-localization with F-actin, with distinct, retrograde F-actin waves specifically associated with Smlt3054 in individual cells as well as formation of dense, internal inclusions at the expense of retrograde F-actin waves. Collectively, our results point to an interaction between Smlt3054 and F-actin. Furthermore, as a potentially secreted protein unique to clinical S. maltophilia isolates, Smlt3054 may serve as a starting point for understanding the mechanisms by which S. maltophilia has become an emergent pathogen.

Pseudomonas aeruginosa is a Gram‐negative bacteria and opportunistic pathogen commonly associated with diseases such as corneal keratitis. While antibiotics can be effective at treating primary infections, secondary, host‐induced effects from chronic infection can still lead to major permanent scarring and possibly even blindness. Host‐induced tissue damage is a major complication associated with chronic P. aeruginosa infections, and paradoxically can in some instances enhance the severity of infection and bacterial persistence. This damage has been shown to be a result of the over‐activation of the host's proteases, including cathepsins and MMPs. Genomic screening of P. aeruginosa identified a putative bacterial protease, which may be involved in activating host immune proteases. This protease contains N‐terminal domains of substantial homology to membrane occupancy and recognition nexus (MORN), for which little structural information exists. We have demonstrated in transfected cells that the N‐terminus is required for optimal catalytic activity, which may suggest a role in host‐induced tissue damage. Herein we outline the successful bacterial expression and purification of this MORN domain using an MBP‐T7 fusion vector as well as outline an experimental approach for defining the role of this domain in activity of the putative protease and host induced tissue damage.