Seasonal microbial ribotype shifts in the sulfurous karstic lakes Cisó and Vilar, in northeastern Spain / Cambios estacionales en los ribotipos microbianos en los lagos cársticos y sulfurosos Cisó y Vilar, en el nordeste de España

Spatio-temporal changes in two sulfurous lakes from the karstic area of Banyoles (Girona, Spain), holomictic lake Cisó and meromictic lake Vilar, were studied over one year. Samples were collected at different depths from the two lakes on the same days, during each of the four seasons, and several physico-chemical variables (temperature, light, pH, conductivity, sulfide, oxygen concentration, pigment concentrations, etc.) were measured. To fingerprint bacterial populations from each sample, DNA was extracted, bacterial 16S rRNA genes were amplified by PCR, and restriction fragment length polymorphism (RFLP) analyses of the total bacterial 16S rDNAs were performed. Each 16S rDNA pool was independently digested with three restriction endonucleases (AluI, HinfI, and RsaI) and separated electrophoretically. More restriction fragments were obtained from the Lake Vilar samples than from the Lake Cisó samples. Moreover, intrasample and intersample differences were observed in each lake. RFLP patterns were compared by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. In both lakes, results indicated that seasonality was mostly responsible for the observed fluctuations in the RFLP patterns, while the effect of stratification was less pronounced (AU)

Se estudió a lo largo de un año el cambio espacio-temporal que se produjo en dos lagos sulfurosos de la zona cárstica de Banyoles (Girona, España), el lago Cisó, holomíctico, y el Lago Vilar, meromíctico. Se tomaron muestras a diferentes profundidades en los dos lagos los mismos días durante las cuatro estaciones y se midieron algunas variables fisicoquímicas (temperatura, luz, pH, conductividad, sulfuro, concentraciones de oxígeno y de pigmentos, etc.). Para obtener la impronta genética de las poblaciones bacterianas de cada muestra, se extrajo el DNA, se amplificaron los genesdel 16S rRNA mediante PCR y se analizó el polimorfismo en la longitud de los fragmentos de restricción (RFLP) del total de 16S rDNA bacteriano. Los diferentes conjuntos de 16S rDNA bacteriano fueron digeridos de manera independiente con tres endonucleasas de restricción (AluI, HinfI, y RsaI) y separados por electroforesis. Se obtuvieron más fragmentos de restricción de las muestras del lago Vilar que del Cisó. Además, en cada lago se observaron también diferencias dentro de cada muestra y entre las diferentes muestras. Luego se compararon los patrones de RFLP puntuando las similitudes mediante el coeficiente Jaccard y la creación un mapa de escalamiento multidimensional (MDS) a partir de la matriz de similitudes resultante. Los resultados indicaron que la estacionalidad era la principal causa de las fluctuaciones observadas en los patrones de RFLP en ambos lagos, mientras que el efecto de la estratificación era menos pronunciado (AU)

Seasonal microbial ribotype shifts in the sulfurous karstic lakes Cisó and Vilar, in northeastern Spain.

Spatio-temporal changes in two sulfurous lakes from the karstic area of Banyoles (Girona, Spain), holomictic lake Cisó and meromictic lake Vilar, were studied over one year. Samples were collected at different depths from the two lakes on the same days, during each of the four seasons, and several physico-chemical variables (temperature, light, pH, conductivity, sulfide, oxygen concentration, pigment concentrations, etc.) were measured. To fingerprint bacterial populations from each sample, DNA was extracted, bacterial 16S rRNA genes were amplified by PCR, and restriction fragment length polymorphism (RFLP) analyses of the total bacterial 16S rDNAs were performed. Each 16S rDNA pool was independently digested with three restriction endonucleases (AluI, HinfI, and RsaI) and separated electrophoretically. More restriction fragments were obtained from the Lake Vilar samples than from the Lake Cisó samples. Moreover, intrasample and intersample differences were observed in each lake. RFLP patterns were compared by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. In both lakes, results indicated that seasonality was mostly responsible for the observed fluctuations in the RFLP patterns, while the effect of stratification was less pronounced.

Factors affecting interpretation of restriction fragment length polymorphism (RFLP) patterns from PCR-amplified bacterial 16S rRNA genes: operon number and primer mismatching.

PCR methods have been shown to be biased by several factors. In the present study, we have developed a theoretic and practical approximation to elucidate how the presence of mismatches at the primers annealing regions and the different number of rDNA operons per cell can influence PCR and subsequent restriction fragment length polymorphism (RFLP) analyses from bacterial populations. We have performed RFLP analyses of 16S rRNA genes amplified by PCR from mixed bacterial cultures showing different primer identities and number of rDNA operons. Our results clearly corroborate that both factors, number of rDNA operons and primers identity, clearly influence the 16S rDNA-RFLP estimations. It has been demonstrated that a higher number of operons leads to a higher efficiency of detection, but a lower degree of primer complementarity implies a decrease in such efficiency.

Seasonal population changes in the restriction fragment length polymorphism (RFLP) patterns from PCR-amplified 16S rRNA genes of predominant ribotypes in microbial mat samples from the Ebro Delta (Spain).

The stratified benthic microbial communities, which developed as a consequence of the physico-chemical gradients and the physiology of the inhabiting microorganisms, from Ebro Delta microbial mats were analyzed. 16S rRNA eubacterial and archaeal genes were amplified by polymerase chain reaction (PCR). PCR products were separately digested with three different restriction enzymes (AluI, HinfI, and RsaI) and later separated by horizontal electrophoresis to determine whether changes of predominant ribotypes are occurring over a period of a year. Comparison of total restriction patterns was performed by scoring similarities by using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. The three enzymes gave a consistent result, a seasonal distribution instead of a spatial and/or physiological one.