Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes

Abstract

The group of planctomycetes represents a separate line of descent within the domain Bacteria. Two phylum-specif ic 16s rRNA-targeted oligonucleotide probes for planctomycetes have been designed, optimized for in situ hybridization and used in different habitats to detect members of the group in situ. The probes, named PLA46 and PLA886, are targeting all or nearly all members of the planctomycete line of descent. Planctomycetes could be detected in almost all samples examined, e.g. a brackish water lagoon, activated sludge, and other wastewater habitats. In situ probing revealed quite uniform morphology and spatial arrangement of the detected cells but profound differences in abundance ranging from less than 01 YO to several percentage of the total cells. Single coccoid cells with diameters between I and 2.5 pm were dominating in most samples with the exception of the lagoon, in which rosettes of pear-shaped cells were abundant. The planctomycetes showed generally no hybridization signals with the bacterial probe EUB338, which is in accordance with base changes in their 165 rRNA sequences. A discrete ultrastructure of planctomycete cells was suggested by double staining with rRNA-targeted probes and the DNA-binding dye 4',6-diamidino-2-phenylindole (DAPI). The probe-conferred fluorescence was distributed in a ring-shaped manner around a central DAPI spot. The two probes developed extend the existing set of group-specif ic rRNA-targeted probes and help to elucidate the basic composition of bacterial communities in a first step of differential analysis. In situ hybridization of environmental samples indicated widespread presence of planctomycetes in different ecosystems.

Anaerobic ammonia oxidation in a fertilized paddy soil

“…The initial amplification was carried out using the PLA46f-630r primer combination with a thermal profile of 96 1C for 10 min, followed by 35 cycles of 60 s at 96 1C, 1 min at 56 1C, 1 min at 72 1C (Juretschko et al, 1998;Neef et al, 1998). After the first step, a 500-times diluted (1 ml) PCR product was used as template for the second amplification with Amx368f-Amx820r primers using a thermal profile of 96 1C for 10 min, followed by 25 cycles of 30 s at 96 1C, 1 min at 58 1C, 1 min at 72 1C .…”

Performance and microbial community of simultaneous anammox and denitrification (SAD) process in a sequencing batch reactor

“…Besides, a nested PCR was performed to amplify the anammox 16S rRNA gene. The initial amplification was carried out using the Pla46f-630r primer set (Neef et al, 1998) with a thermal profile of 96°C for 10 min followed by 35 cycles of 1 min at 96°C, 1 min at 56°C, and 1 min at 72°C. Afterwards, a 500-fold diluted PCR product was used as template for the second amplification with the Amx368f-Amx820r primer set (Schmid et al, 2005) using a thermal profile of 96°C for 10 min followed by 25 cycles of 30 s at 96°C, 1 min at 58°C, and 1 min at 72°C.…”

Distinct flavobacterial communities in contrasting water masses of the North Atlantic Ocean

“…Samples were taken at the German North Sea island Helgoland (54111.3 0 N; 07154.0 0 E) and were filtered on polycarbonate filters (0.2 mM pore size) in increasing amounts from 10, 25, 50, 100, 250 to 400 ml in triplicates for each volume. Hybridizations were performed as described above using probes ROS537, for members of the Roseobacter clade (Eilers et al, 2001) at 35% formamide; NOR5-730, for members of the gammaproteobacterial NOR5/OM60 clade (Eilers et al, 2001) at 50% formamide and PLA46, for Plantomycetes (Neef et al, 1998) at 30%. The NON338 probe was used as negative control at 35% formamide.…”