Características fenotípicas controladas pelo sistema quorum sensing em Enterobacter cloacae

• Autor(a) principal: Ponce, Adriana dos Reis
• Data de Publicação: 2007
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/5291
• topic: Enterobacter cloacae; Quorum sensing; Lactonase; Enterobacter cloacae; Quorum sensing; Lactonase; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA

Many bacteria present an elaborate system of communication dependent of cell density known as quorum sensing, and the knowledge of the phenotypes controlled by this mechanism may contribute for the elucidation of important processes in food microbiology. The objective of this study was to determine phenotypes regulated by quorum sensing in Enterobacter cloacae 067 isolated from cooled raw milk. Phenotypic characteristics of E. cloacae 067 wild type were compared to the transconjugant strain, that synthesizes the lactonase enzyme, which is responsible for hydrolysis of Nacylhomoserine lactones (AHLs) that are signal molecules of the quorum sensing system. Wild type and transconjugant strains of E. cloacae 067 were grown in temperatures from 4 °C until 43 °C. The optimum temperature was between 36 °C and 43 °C and the maximum population was reached at 30 °C. The optical density of transconjugant strain culture was higher (p < 0,05) than the wild type in both complex and in mineral medium which contained only one organic carbon source. The swarming motility was not verified in E. cloacae strains tested and the twitching motility was not modified in the transconjugant. Biofilms was formed in polystyrene surface and E. cloacae 067 wild type presented higher adhesion than the transconjugant strain when cultivated in Luria Bertani medium (LB), triptona, extract yeast and phosphate medium (TYEP), and in minimal medium (MMS). The addition of calcium to the TYEP medium reduced the biofilm formation by the wild type, but not in the transconjugant. In stainless steel surface, the adhesion of the wild type was approximately one logarithmic cycle higher than the transconjugant until 48 hours of incubation. After this period, differences were not detected in both microscopic observation and in the counting of adhered viable cells. The difference in adhesion of E. cloacae 067 wild type and transconjugant in polystyrene and stainless steel suggests that quorum sensing system regulates biofilm formation by this strain. Amylases, celulases, lipases and pectinases activities were not verified in the wild type and transconjugant strains of E. cloacae 067. However, the transconjugant strain was capable to use gelatin after 12 days of incubation at 30 °C. The transconjugant of E. cloacae 067 presented higher proteolytic activity in casein agar and in LB agar added of 2% (w/v) of skim milk. This strain also presented higher degree of proteolyses than the wild type. The spoilage of reconstituted 12% (w/v) skim milk and of pasteurized milk for E. cloacae 067 transconjugant occurred after 36 hours of incubation, while the milk spoilage for the wild type strain occurred only after 96 hours. These results suggest the existence of a mechanism of partial negative regulation of proteolytic activity in E. cloacae 067 for the quorum sensing system. The proteolytic activity of both strains was not detected in the zimogram azocasein gel. However, when gelatin was used as substrate, it was possible to observe proteolytic activity in protein extract of the wild type and transconjugant strains. Analysis of the free supernatant of cells, concentrated 15 turn, in SDS-PAGE exhibited higher number of extracellular protein bands produced by E. cloacae 067 wild type than the transconjugant. Therefore, this suggests that besides proteases, quorum sensing system may be involved in the regulation of other extracellular proteins.