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Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronic infection of the lungs of cystic fibrosis patients. Chaperone-usher systems in P. aeruginosa are known to translocate and assemble adhesive pili on the bacterial surface and contribute to biofilm formation within the host. Here, we report the crystal structure of the tip adhesion subunit CupB6 from the cupB1-6 gene cluster. The tip domain is connected to the pilus via the N-terminal donor strand from the main pilus subunit CupB1. Although the CupB6 adhesion domain bears structural features similar to other CU adhesins it displays an unusual polyproline helix adjacent to a prominent surface pocket, which are likely the site for receptor recognition., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain., (© 2015 The Protein Society.)

Understanding gene function is far easier when tools are available to engineer a bacterial strain lacking a specific gene and phenotypically compare its behavior with the corresponding parental strain. Such mutants could be selected randomly, either by natural selection under particular stress conditions or by random mutagenesis using transposon delivery as described elsewhere in this book. However, with the advent of the genomic era there are now hundreds of bacterial genomes whose sequence is available, and thus, genes can be identified, chosen, and strategies designed to specifically inactivate them. This can be done by using suicide plasmids and is most convenient when the bacterium of interest is easily amenable to genetic manipulation. The method presented here will describe the use of a suicide vector, pKNG101, which allows the selection of a double-recombination event. The first event results in the integration of the pKNG101 derivative carrying the "mutator" fragment onto the chromosome, and could be selected on plates containing appropriate antibiotics. The pKNG101 carries the sacB gene, which induces death when cells are grown on sucrose. Growth on sucrose plates will thus select the second homologous recombination event, which results in removing the plasmid backbone and leaving behind the mutated target gene. This method has been widely used over the last 20 years to inactivate genes in a wide range of gram-negative bacteria and in particular in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/ cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain. [ABSTRACT FROM AUTHOR]