Presencia de genes rhlAB, rhlR y rhlC en Pseudomonas aeruginosa nativas sobreproductoras de ramnolípidos

Se realizó la caracterización molecular de los genes asociados a la producción de ramnolípidos (RL), en 61 cepas bacterianas de la colección del Laboratorio de Microbiología y Biotecnología Microbiana (LAMYBIM) de la Universidad Nacional Mayor de San Marcos, Perú. Las cepas provenian de entornos peruanos contaminados con hidrocarburos y fueron catalogadas como sobreproductoras de RL(n= 21), productoras de RL (n=20) y no productoras de RL (n= 20). Las 61 cepas fueron identificadas bioquímicamente con el sistema API 20 NE. La identificación molecular se realizó empleando el gen del RNAr 16S. Se encontró que Pseudomonas aeruginosa fue el microorganismo de mayor prevalencia en los estratos sobreproductores y productores de ramnolípidos. Además, se encontraron: Burkholderia cepacea, Pseudomonas fluorescens, Aeromonas hydrophila y Chryseobacterium indologenes. Los microorganismos no productores de ramnolípidos, también fueron caracterizados bioquímicamente. Mediante amplificación de PCR y electroforesis en gel de agarosa, estandarizados por la UNAM, se evidenció que las cepas seleccionadas poseen los genes: rhlA, rhlB, rhlR y rhlC. Para el secuenciamiento de la región génica rhLABR, se seleccionaron cuatros especies: Pseudomonas aeruginosa T2X-2, Pseudomonas aeruginosa IIIT1P2, Pseudomonas aeruginosa 6K-11 y Pseudomonas aeruginosa ATCC 9027, siguiendo metodología estandarizada por la UNAM y fueron comparados con Pseudomonas aeruginosa PAO1. Nuestros resultados muestran que los genes estudiados en las cepas seleccionadas son sinónimas de sus homólogos en la cepa patrón Pseudomonas aeruginosa PAO1, por lo que las diferencias genotípicas que expliquen la sobreproducción de ramnolípidos deberían hallarse en otros marcadores moleculares no cubiertos en el presente estudio.

Genes associated to rhamnolipids production were molecularly characterized in 61 bacterial strains from LAMYBIM bacterial collection (Laboratorio de Microbiología y Biotecnología Microbiana, Universidad Nacional Mayor de San Marcos, Perú). Strains were isolated from peruvian environments hydrocarbons polluted and were classified as RL overproducers (n= 21), RL producers (n = 20) and non-producers (n = 20) producers. Molecular identification using the 16S rRNA gene was preceded by the biochemical identification of 61 strains selected with the API 20 NE system. Pseudomonas aeruginosa was the most prevalent strain of the RL overproducers and RL producers. Species such as Burkholderia cepacea, Pseudomonas fluorescens, Aeromonas hydrophila and Chryseobacterium indologenes, were found too. In the same way, non-producers microorganisms were also characterized. The PCR amplification and agarose gel electrophoresis techniques, standarized by the UNAM laboratory, showed that the selected strains had the genes: rhlA, rhlB, rhlR and rhlC. For the sequencing of the rhLABR gene region, four strains were selected: Pseudomonas aeruginosa T2K2, Pseudomonas aeruginosa III T1P2, Pseudomonas aeruginosa 6K-11 and Pseudomonas aeruginosa ATCC 9027, applying the methodology standardized by the UNAM and were compared with Pseudomonas aeruginosa PAO1. Our results show that the genes studied in the selected strains are synonymous with their homologues in Pseudomonas aeruginosa PAO1 standard strain. Therefore, genotypical differences that explain the overproduction of rhamnolipid might be found in other molecular markers not covered in this study.

T. thermophilus Rhamnolipids Induce Cytogenetic Damage on Human Lymphocytes and Bind DNA in vitro.

Aims: Bacteria including Pseudomonas and T. thermophilus secretes rhamnose–containing glycolipid biosurfactants called rhamnolipids (RLs), known as bacterial virulence factors. The aim of this investigation was the evaluation of DNA damage induced on human lymphocytes by both RLs itself, secreted in a host organism by pathogens during a bacterial attack or symbiosis and in combination with the camptothecin (CPT), and on calf thymus DNA. Study Design: Human lymphocytes and calf thymus DNA were treated with isolated T. thermophilus RLs for studying DNA damage in vitro. Methodology: RLs DNA damaging action was evaluated by the Sister Chromatid Exchanges (SCEs) methodology, a method for estimating genotoxicity of human exposure to different chemicals or other mutagenic agents and by DNA electrophoretic mobility experiments. Results: RLs at concentrations of 100 and 150 μg/mL reveal significant toxicity. The highest concentration of 200 μg/mL reveals higher genotoxicity. The frequency of SCEs/cell was increased two times over the control level. When CPT, an antineoplastic drug with DNA damaging action, was tested together with RLs the genotoxic activity was reduced significantly (P<0.01) compared to the action caused by CPT itself. Sequential increase in the concentration of RLs results in the proportional reduction of Proliferation Rate Index (PRI) which is a cytostatic index. Also, Mitotic Index (MI), a cytotoxic index, was also significantly decreased at concentration of 200 μg/mL RLs. Addition of RLs in the same concentration together with CPT doesn’t affect the MI so much. Moreover, RLs are obviously capable for strong binding to plasmid or calf thymus DNA in vitro. Conclusion: RLs exert genotoxicity, cytotoxicity and cytostaticity in human lymphocytes and play probably a protective role for cells against CPT due to RLs’ detergent capability to enrobe CPT and DNA, providing a significant property that might support its possible involvement in DNA horizontal transfer phenomena.

Optimización y modificación del método para la detección de ramnolípidos

El uso de biosurfactantes en biorremediación facilita y acelera la degradación microbiana de hidrocarburos. El método del agar CTAB/MB creado por Siegmund y Wagner para el screening de cepas productoras de ramnolípidos (RL), ha sido ampliamente utilizado sin sufrir mejoras significativas en más de 20 años. Con el fin de optimizar la técnica como método cuantitativo, se hicieron placas con agar CTAB/MB y se probaron diferentes variables, tales como tiempo de incubación, refrigeración, concentración de CTAB, presencia de azul de metileno, diámetro de los pozos y volumen del inóculo. Adicionalmente, se desarrolló un nuevo método para detectar el RL de los halos mediante precipitación con HCl, lo cual permite la observación de un nuevo patrón de halos más fáciles de observar y medir. Esta investigación reafirma que este método no es del todo apropiado para un análisis cuantitativo fino, debido a la dificultad de correlacionar de forma precisa la concentración de RL y el área de los halos formados. La difusión del RL no parece tener un comportamiento simple y existen numerosos factores que afectan la velocidad de migración del RL.

Use of biosurfactants in bioremediation, facilitates and accelerates microbial degradation of hydrocarbons. CTAB/MB agar method created by Siegmund & Wagner for screening of rhamnolipids (RL) producing strains, has been widely used but has not improved significantly for more than 20 years. To optimize the technique as a quantitative method, CTAB/MB agar plates were made and different variables were tested, like incubation time, cooling, CTAB concentration, methylene blue presence, wells diameter and inocula volume. Furthermore, a new method for RL detection within halos was developed: precipitation of RL with HCl, allows the formation a new halos pattern, easier to observe and to measure. This research reaffirm that this method is not totally suitable for a fine quantitative analysis, because of the difficulty to accurately correlate RL concentration and the area of the halos. RL diffusion does not seem to have a simple behavior and there are a lot of factors that affect RL migration rate.

In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil

In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019) between TPH concentration (mg/kg) and surface tension (mN/m), When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009) was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil.