Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

Abstract

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.

Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit

“…B. cepacia complex was the most frequently isolated microorganism and recA-RFLP pointed to a polyclonal nature of the outbreak, which was confirmed by other molecular typing data. The results are consistent with bacterial diversity found in environmental samples (Salles et al, 2002).…”

Mycophagous growth of Collimonas bacteria in natural soils, impact on fungal biomass turnover and interactions with mycophagous Trichoderma fungi

“…The Pseudomonas-specific primer sets used were PSf (5 0 -GGTCTGAGAGGATGATCAGT-3 0 ) and PSr (5 0 -TTAGCTCCACCTCGCGGC-3 0 ) (Widmer et al, 1998). The Burkholderia-specific primer sets used were Burk3 (5 0 -CTGCGAAAGCCGGAT-3 0 ) and BurkR (5 0 -TGCCATACTCTAGCYYGC-3 0 ) (Salles et al, 2002). For quantification of Trichoderma spp., we used a modified protocol of Hagn et al (2007).…”

“…The nested PCR reactions were performed for Burkholderia spp. with primers Burk3 and R1378 (Salles et al, 2002). The products from the first PCR were diluted at 1:1000 and used as the template in the second PCR with primers Burk3-GC and BurkR (Salles et al, 2002).…”

“…All PCR reactions and DGGE were carried out as described elsewhere (Salles et al, 2002;Yergeau et al, 2007). The primers 968f-GC and 1378r (Heuer et al, 1997), andFR1-GC andFF390r (Vainio andHantula, 2000) were used to analyze bacterial and fungal communities, respectively.…”

Soil microbial community analysis in the environmental risk assessment of transgenic plants

“…Furthermore, in the case of the rhizosphere, several microbial groups, such as Burkhoderia and Pseudomonas, have been shown to be important community members and specific primer sets have thus been developed for these species (Salles et al 2002;Gyamfi et al 2002). Holben et al (2004) reported the use of DGGE in combination with GϩC fractionation to assess microbial community diversity and to detect minority populations of bacteria in the digestive tracts of chickens.…”