Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Fri, 20 May 2016 10:29:04 Microbiology (1 998), 144, 3257-3266 Printed in Great Britain Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16s rRNA-targeted probes Alexander Neef,’t Rudolf Arnann,’r2 Heinz Schlesner3 and Karl-Heinz Schleiferl Author for correspondence: Alexander Neef. Tel : + 49 641 99 37373. Fax : + 49 641 99 37359. e-mail : alexander.neef @agrar.uni-giessen.de 1 Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Arcisstr. 16, D-80290 Munchen, Germany 2 Max-Planck-lnstitutfur Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany 3 lnstitut fur Allgemeine Mikrobiologie, Christian- Albrechts-Universittit Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany 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. Keywords : planctomycetes, rRNA-targeted probes, fluorescence in situ hybridization (FISH), in situ identification INTRODUCTION Planctomycetes are aerobic, Gram-negative, chemo- heterotrophic bacteria with mostly spherical, ovoid or pear-shaped cell morphology. They share some unique characters that distinguish them from other bacteria, most importantly the lack of peptidoglycan in their cell walls and the budding type of reproduction. Some members have typical acellular appendages. Main eco- logical features of the group have been recently reviewed (Fuerst, 1995). Pure cultures have been isolated from diverse aquatic habitats such as freshwater lakes, brackish water and sea water as well as from a hot spring (Schmidt, 1978; Schlesner, 1986, 1994; Gio- vannoni f?t al-7 1987) - SO far, four genera, PlanCtOmyCeS, pirellula, Gemmata and Isosphaera, are validly de- .............................................................................................. , .................................................. t Present address: lnstitut fur Angewandte Mikrobiologie, Justus-Liebig- Universitat Giessen, Senckenbergstr. 3, D-35390 Giessen, Germany. Abbreviations: BOD,, biological oxygen demand within 5 d incubation; DAPI, 4’,6-diamidino-2-phenylindole; FLUOS, 5(6)-carboxy-fluorescein-l\l- hydroxysuccinimide ester; TAMRA, 5(6)-carboxy-tetramethylrhodamine-N- hydroxysuccinimide ester; W P , wastewater treatment plant. scribed (Staley et al., 1992; Holt et al., 1994). A new 0002-2624 0 1998 SGM 3257