A total of 82 axenic strains that phenotypically resembled members of the Phylum Planctomycetes (order Planctomycetales, family Planctomycetaceae) of the domain Bacteria and that were confirmed as planctomycetes by 16S rRNA gene sequencing, and 7 mixed cultures that contained at least one member resembling the planctomycetes on the basis of electron microscopic evidence, were isolated from 11 different habitats. These habitats included postlarvae of the giant tiger prawns, brackish water, freshwater, soil, marine sediment and a laboratory antibiotic solution. Some of the mixed cultures were resolved into their planctomycete component and this component culture included with the axenic collection. All planctomycete-like new isolates from this study displayed crateriform structures on their cell wall and this as a diagnostic phenotype feature placed these isolates putatively within the family Planctomycetaceae. Antibiotics including antifungal agents in enrichment and selective media and prolonged incubation were important for isolation of planctomycetes. Besides their budding nature for cell reproduction, planctomycetes are a group of bacteria phenotypically very diverse as well with respect to aspects of cell shapes, cell surface structures and colony pigment. The fact that planctomycete-like organisms were successfully isolated from a soil habitat in which the existence of planctomycetes had only previously been demonstrated via clones of amplified partial 16S rRNA genes confirmed that ecologically, planctomycetes are not confined to aqueous habitats only. It also suggests that some planctomycetes which have not been cultured but known only from direct cloning or microscopy may be able to be cultured in the future.
Partial 16S rDNA sequences were obtained from 78 out of the 82 axenic new planctomycete-like isolates by a routine procedure using the planctomycete specific primer 945f. A total of 77 isolates were confirmed to be members of the family Planctomycetaceae. One was identified as an Acinetobacter sp. Analysis of 220 homologous positions of the partial 16S rDNA sequences classified the 77 planctomycete isolates into 13 phylotypes; 4 phylotypes clustered within the Pirellula group, 4 within the Gemmata group and 5 within the Isosphaera group.
Partial 16S rDNA analysis also clarified the identities of 2 stock cultures of the type strain Pirellula staleyi (ATCC 27377T) at The Australian Collection of Microorganisms (ACM). Both ACM 2488 and ACM 3744 are claimed to be ATCC 27377T on ACM documentation. However, analysis of 16S rDNA sequences identified ACM 2488 as Geodermatophilus sp. and confirmed that only ACM 3744 is identical to strain ATCC 27377T. Complete 16S rDNA sequence was also obtained from strain ATCC 35122 and confirmed it as a strain of Pirellula staleyi. The original identification of strain ATCC 35122 was based only on phenotype and its close relationship to the type strain of Pirellula staleyi ATCC 27377T is now confirmed on molecular grounds by 16S rRNA sequence analysis.
PCR amplification using primers corresponding to repetitive extragenic palindromic (REP) elements was carried out as a strain-typing tool to study both reference planctomycetes and the new isolates. It separated 22 reference planctomycete strains into 17 REP groups and 70 new isolates into 19 REP groups. None of the new isolates were classified into the same REP groups as the reference isolates. This is the first study that demonstrated that planctomycetes possess REP elements in their genome. The hypothesis can also be made from this study that planctomycetes may also possess the other 2 families of repetitive DNA sequences dispersed throughout the genome of diverse species, the ERIC (enterobacterial repetitive intergenic consensus) sequence and the BOX elements. REP-PCR proved to be a very efficient typing technique to eliminate replicates among new isolates.
Near-complete 16S rRNA gene sequences were obtained from 13 new isolates. Six of the 13 new isolates clustered within the Pirellula group. Five of the new isolates clustered within the Gemmata group defined by G. obscuriglobus. Two new isolates clustered within the Isosphaera group defined by /. pallida. Of the 5 new isolates that clustered within the Gemmata group, 2 represent the first planctomycetes successfully cultivated from soil. One of these 2 soil isolates, JW3-8sO, is from the same sampling site as that from which the planctomycete-clustering MC soil clones, MC25, MC55, MCI 113, MC11, MC98, MC1and MC100 (Liesack and Stackebrandt, 1992). It clustered, however, closer to cultured str. 633 - a strain from northern Germany, rather than any of the MC soil clones. The general topology of a previously published phylogenetic tree of 16S rRNA analysis of planctomycetes was not changed after the 16S rRNA sequences of 13 new representative isolates were added. The fact that sequences of these soil isolates differ from those of all MC clones may indicate that diversity of planctomycetes in soil is wider than molecular cloning methods could detect and emphasizes the need for culturing as well as direct PCR retrieval for studying microbial diversity; however, this may also reflect differences in community composition at the same site at different times.
A total of 16 planctomycete strains out of 18 strains studied, 8 reference strains and 10 new isolates covering all four planctomycete genera, possessed capsules on their cell wall indicating that a capsule is a common structure for the family of Planctomycetaceae.
A study of cell compartmentalization was carried out with 18 new planctomycete isolates representing the genera Pirellula, Gemmata and Isosphaera and the results were compared with previous studies that were based on studies of the type species of each genus only. All new isolates displayed cell compartmentalization profiles that supported those of the type species within the same genus. All new Gemmata-like isolates, especially, displayed membrane-bounded nuclear bodies. The uniqueness of this group of bacteria in their cell organization is consistent with their phylogenetic distinctiveness.
A culture-independent molecular cloning survey applying a nested-PCR technique using planctomycete-targeting primers 58f and 926r established a clone library of 125 clones from the freshwater body referred to here as the 'big lake' of The University of Queensland, Australia (also called University Lake) and were designated as LUQ clones. This lake is a habitat known to harbour planctomycete-like bacteria. A total of 79 clones of 64 representative clone profiles chosen by RFLP-screening were sequenced and 78 of the readable sequences were identified as planctomycetes, of which 21 clones were identified as possible chimeras by Check_Chimera, 4 pairs of clones appeared to be identical and 53 clones were included in the final phylogenetic analysis. Thirty (30) LUQ clones clustered within the Pirellula group (i.e. including named reference Pirellula species), 1 within the Planctomyces group, 15 within the Gemmata and 6 within the Isosphaera group. One clone clustered together with the Mt. Coot-tha clone MCI8 in a group separated from all other groups. The topology of the phylogenetic tree for the family Planctomycetaceae was not changed compared to previously published 168 rRNA trees, after 53 LUQ clone sequences were introduced. The 4 major groups, Pirellula, Planctomyces, Gemmata and Isosphaera are still prominent.
The protocols developed in this project proved to be applicable to future studies of molecular diversity of planctomycetes in other habitats. The primer set, 58f and 926r, used for nested- PCR was confirmed to be planctomycete specific for ecological studies. The annealing temperature of 60°C proved to be crucial to prevent partial 16S rDNAs of non-planctomycetes being amplified. The diversity of planctomycetes in the chosen habitat as estimated by the direct molecular approach is much more diverse than the conventional culturing route presented. The limitation of the latter might be within the restrictive nature of the culture media employed.