Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation

ABSTRACT Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C8-HSL and 3-OH-C14-HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C8-HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C8-HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C8-HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1.(AU)

Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk

Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Identificação e caracterização de micro-organismos potencialmente benéficos para o cultivo intensivo do camarão marinho Litopenaeus vannamei em sistema de bioflocos

O cultivo intensivo de L. vannamei em sistemas de Bioflocos tem se mostrado como uma alternativa biossegura e mais sustentável para a carcinicultura moderna. Tal sistema é regulado principalmente por micro-organismos, em especial pelas bactérias e o processo de formação dos flocos microbianos e a estrutura das comunidades microbianas sobre a qual os sistemas de cultivo intensivo são fortemente dependentes ainda é pouco conhecido. O objetivo deste trabalho foi identificar bactérias cultiváveis potencialmente benéficas durante o processo de formação do floco microbiano para sua utilização durante o cultivo de camarões marinhos. No o primeiro estudo, foi construída uma estufa com 6 tanques de 14 m3 e após dois ciclos de cultivo de 75 dias foram isoladas e identificadas morfologicamente 120 cepas, sendo que destas, 91 foram posteriormente identificadas por taxonomia molecular. Em seguida, as cepas GRAM positivas identificadas foram testadas quanto ao seu potencial benéfico através de provas enzimáticas e de inibição in vitro frente a cepas patogênicas, assim como também foram analisadas para a presença do gene aii que codifica para a produção de lactonase (N-acyl homoserine lactonases). Das 91 cepas testadas, 23 apresentaram resultados positivos para a produção de Protease, 43 para Amilase, 75 para Lipase e 3 para a presença do gene Aii. Ao final do primeiro estudo 6 cepas foram identificadas como potencialmente benéficas por apresentarem o maior número de resultados positivos em todas as provas executadas, sendo elas, Enterococcus thailandicus, Bacillus purgationiresistens, Bacillus horneckiae, Exiguobacterium indicum, Exiguobacterium acetylicum (isolada do intestino do L. vannamei) e Exiguobacterium acetylicum (isolada do Bioflocos). Na primeira etapa do segundo estudo as cepas com o maior potencial benéfico foram submetidas às provas de tolerância a salinidade, pH, amônia, nitrito e nitrato. Os resultados in vitro da primeira etapa indicaram a Exiguobacterium acetylicum isolada do intestino, como a cepa com maior potencial benéfico e capacidade de adaptação às condições de cultivo. A segunda etapa do estudo consistiu em padronizar a metodologia de produção em massa da cepa escolhida e avaliar a sua aplicação em um cultivo iniciado em água clara durante o processo de formação do floco microbiano. Após a avaliação do crescimento da cepa em questão em diferentes meios de cultura foi padronizado o processo de produção do inóculo a ser utilizado no estudo em dois tratamentos em um meio de cultura alternativa composto por 1,5% de melaço de cana de açúcar e 0,5% de Uréia, sendo T1 com aplicação semanal do inóculo (na concentração de 107 UFC/mL) e T2 com aplicação quinzenal e o C como controle sem aplicação do inóculo. Estocados em tanques de fibra de vidro com volume útil de 800 L em triplicata, a uma densidade de 125 indivíduos/m2 os camarões apresentavam peso inicial de 6,4 g +- 0,5 g. Ao final do experimento a sobrevivência média foi T1 = 77%, T2 = 91% e C = 71% e os animais apresentaram um crescimento médio semanal de T1 = 1,1 g, T2 = 1,0 g e C = 1,4 g. Com relação à qualidade de água foi observado uma formação de sólidos suspensos totais (SST) na terceira semana nos tratamentos com inóculo, sendo T1 = 342 mg/L, T2 = 352 mg/L e C = 262 mg/L.

The intensive production of L. vannamei in Bioflocos tecnology have been shown as a biosecure and more sustainable alternative to modern shrimp farming. This system is mainly regulated by micro-organisms, especially by bacteria and the process of formation of the flocs and the structure of it´s microbial communities, on which the intensive farming systems are heavily dependente, is still little known. The objective of this study was to identify potentially beneficial culturable bacteria in the microbial floc formation process to use for the intensive marine shrimp production. In the first study, a greenhouse was built with 6 tanks of 14 m3 and after two cycles of 75 days were isolated and morfologialy identify 184 bacterial strains, 152 these being identified by molecular taxonomy. Furthermore, the Gram positive strains were tested in vitro for their potential by enzymatic and inhibition assays and, were also analyzed for the presence of the gene aii encoding for the production of lactonase (N-acyl homoserine lactonases). Of 95 strains tested, 23 were positive for protease, 43 Amylase, 75 for lipase production and 3 for the presence of the IIA gene. At the end of the first study 6 strains were identified as potentially beneficial for having the highest number of positive results in all the tests performed, which were, Enterococcus thailandicus, Bacillus purgationiresistens, Bacillus horneckiae, Exiguobacterium indicum, Exiguobacterium acetylicum (isolated from L. vannamei gut) and Exiguobacterium acetylicum (isolated from Bioflocos). In the first stage of the second study strains with the greatest potential benefit underwent tests of tolerance to salinity, pH, ammonia, nitrite and nitrate. In vitro results of the first phase indicated Exiguobacterium acetylicum isolated from the gut, as the strain with the greatest potential and adaptability to growing conditions

The proteolytic activity of Pseudomonas fluorescens 07A isolated from milk is not regulated by quorum sensing signals

The proteolytic activity of Pseudomonas fluorescens 07A was investigated, and was optimal on tryptone-calcium medium. N-acyl-homoserine lactones (AHLs) were not detected on supernatants of late-exponential and stationary-phase culture broths. Synthetic AHLs or bacterial cell extracts added to the medium did not influence growth or proteolytic activity suggesting that quorum sensing might not regulate protease production in this strain.

2(5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition

Biofouling of membranes demands costly periodic cleaning and membrane replacement. A sustainable and environmentally friendly solution for maintenance is not available and would be of great interest for many purposes including economical. As complex biofilm formation by environmental strains is the major cause of biofouling and biofilm formation in most cases are controlled by N-Acylhomoserine lactone (AHL)mediated Quorum Sensing (QS). An effort was made to understand the appropriateness of 2(5H)-furanone, to use against biofouling of membranes. QS inhibition activity by 2(5H)-furanone was studied using bioindicator strains and known AHLs of different acyl chain lengths. The biofilm inhibition was studied by growth analysis on polystyrene plate of Aeromonas hyrdrophila, an environmental biofilm strain isolated from a bio-fouled reverse osmosis (RO) membrane. Results showed a QS inhibition activity against a wide range of AHLs and also biofilm formation by 2(5H)-furanone, which is believed to act as a potential quorum inhibition agent in a bacterial biofilm community.