Pseudomonas aeruginosais an opportunistic pathogen which causes a variety of diseases, including respiratory tract infections in patients suffering from cystic fibrosis. Therapeutic treatment ofP. aeruginosainfections is still very difficult because the bacteria exhibit high intrinsic resistance against a variety of different antibiotics and, in addition, form stable biofilms, e.g. in the human lung. Several virulence factors are produced byP. aeruginosa, among them the two lectins LecA and LecB, which exert different cytotoxic effects on respiratory epithelial cells and presumably facilitate bacterial adhesion to the airway mucosa. Here, the physiology has been studied of the lectin LecB, which binds specifically tol-fucose. A LecB-deficientP. aeruginosamutant was shown to be impaired in biofilm formation when compared with the wild-type strain, suggesting an important role for LecB in this process. This result prompted an investigation of the subcellular localization of LecB by cell fractionation and subsequent immunoblotting. The results show that LecB is abundantly present in the bacterial outer-membrane fraction. It is further demonstrated that LecB could be released specifically by treatment of the outer-membrane fraction withp-nitrophenylα-l-fucose, whereas treatment withd-galactose had no effect. In contrast, a LecB protein carrying the mutation D104A, which results in a defective sugar-binding site, was no longer detectable in the membrane fraction, suggesting that LecB binds to specific carbohydrate ligands located at the bacterial cell surface. Staining of biofilm cells using fluorescently labelled LecB confirmed the presence of these ligands.

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14^T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 Å resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical α/β-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30°C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 Å resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.

Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens . One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis , and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis , which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.