Surface Hardness Impairment of Quorum Sensing and Swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns—formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the “standard” swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent

Rhamnolipids: diversity of structures, microbial origins and roles

Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C10-C10), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C10), as well as their mono-rhamnolipid congeners Rha-C10-C10 and Rha-C10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development

Cloning and expression of meta-cleavage enzyme (CarB) of carbazole degradation pathway from Pseudomonas stutzeri

In this work, the 1082bp PCR product corresponding to carBaBb genes that encode the heterotetrameric enzyme 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarB), involved in the Pseudomonas stutzeri ATCC 31258 carbazole degradation pathway, was cloned using the site-specific recombination system. Recombinant clones were confirmed by PCR, restriction enzyme digestion and sequencing. CarB dioxygenase was expressed in high levels and in active form in Escherichia coli BL21-SI using the His-tagged expression vector pDEST TM17 and salt induction for 4h.<br>Carbazol e seus derivados são compostos nitrogenados aromáticos, presentes comumente em petróleo e potencialmente poluentes. A rota de biodegradação de carbazol a ácido antranílico em Pseudomonas sp. é composta por três enzimas responsáveis, respectivamente, pelas reações de dioxigenação angular, meta-clivagem e hidrólise. A segunda enzima da rota, 2'-aminobifenil-2,3-diol 1,2-dioxigenase (CarB), codificada por dois genes (carBa e carBb), é um heterotetrâmero com atividade catalítica na quebra do anel catecol do susbtrato na posição meta. Neste trabalho, foi clonado o produto de PCR de 1082pb correspondente aos genes carBaBb da bactéria degradadora de carbazol Pseudomonas stutzeri ATCC 31258. A estratégia de clonagem empregada foi a de recombinação sítio-específica e a construção dos plasmídeos foi confirmada por PCR, digestão com enzima de restrição e seqüenciamento. A enzima ativa foi expressa em altas concentrações em vetor pDEST TM17 com cauda de histidina e promotor T7 em Escherichia coli BL21-SI com indução por NaCl durante 4h