Planctomycete diversity and cell biology: perspectives from the molecular, cellular and organism levels

The Planctomycetes are a deep branching phylum of the domain Bacteria that incorporate a diverse group of organisms possessing a number of unusual and distinct characteristics. These features include budding reproduction, the planctomycetecharacteristic crateriform structures on their cell surface, a cell wall that lacks peptidoglycan, internal compartmentalisation and unique molecular features of their rRNA genes. This study chose to investigate a number of aspects of planctomycete cell biology and diversity to further our knowledge of this unique group. In a study of the diversity of ribonuclease P (RNase P) RNA, one molecule of relevance to cell biology and compartmentalisation in planctomycetes, RNase P RNA genes were sequenced for species from all genera of planctomycetes for which a pure culture exists. Secondary structures for RNase P RNA of these strains were deduced, taking to 26 the number of planctomycete RNase P RNA structures. Nucleotide positions were identified in which some planctomycetes possess a less common form, including one thought to be otherwise conserved within all Bacteria and Archaea. Phylogenetic analysis of RNase P RNA genes was relatively consistent with that of 16S rRNA genes with the exception that clustering of Gemmata and anammox sequences occurred, possibly due to either long-branch attraction or lateral gene transfer. Analysis of RNase P RNA secondary structures revealed unusual features of planctomycetes relative to all other bacteria, including an additional helix within the P13 helix of Candidatus Brocadia anammoxidans, Candidatus Kuenenia stuttgartiensis and all Gemmata sequences. The longest P12 helix of any bacteria type A RNase P RNA was found in a Gemmatalike isolate. The short tandem repeats in P12 helices of two Gemmata-like isolates are possibly analogous to short tandem repetitive repeat sequences of some cyanobacteria RNase P RNA. In experiments using Gemmata obscuriglobus as a model for planctomycete cell biology and compartmentalisation functions, electron microscope-level in situ hybridisation (EMISH), and subsequent statistical analysis, was developed to localise 16S rRNA, 23S rRNA and RNase P RNA to particular regions within Gemmata obscuriglobus, the first instance of EMISH being applied in this way to bacteria. Statistical analysis localised 16S rRNA to both nuclear body and to riboplasm outside this region but it was absent from paryphoplasm. While co-localisation of both 16S rRNA and 23S rRNA molecules, which might indicate assembled ribosomes, was rarely observed, 23S rRNA, like 16S rRNA, was distributed in both riboplasm-containing areas of the cell. While statistical analysis revealed minor DNA within riboplasm outside the nuclear body, the majority was localised to that body. These results suggest at least some uncoupling of translation from transcription involving ribosomes in the riboplasm. RNase P RNA was localised both to the nuclear body and to the riboplasm outside this region, suggesting that pre-tRNA processing occurs both within nuclear body, where RNA transcripts are presumably generated, and outside nuclear body, separated from the origin of these transcripts. This is also consistent with the hypothesis that processed tRNA is required in the riboplasm outside the nuclear body, due to occurrence of some uncoupled translation. In research on planctomycetes not yet examined with respect to cell plan or structure, 16S rRNA gene sequencing of isolate ATCC 35122 confirmed its very close relationship to the type strain of Pirellula staleyi and its membership of the phylum Planctomycetes. Morphological characteristics, including polar crateriform structures and the occurrence of a unique internal, single membrane-bounded compartment enclosing nucleoid and ribosome-like particles, the pirellulosome, and a polar cap region, are also consistent with its membership of the planctomycetes and of genus Pirellula. Cells often displayed pointed, hump-like protrusions opposite each other on the cell, constituting prosthecae. Also re-examined using a number of methods were uncultured species Planctomyces bekefii and Pl. guttaeformis. Samples could be enriched for Pl. bekefii via either addition of ferric citrate or ampicillin. An application of a novel approach, laser microdissection and pressure catapulting, was also used physically to enrich P. bekefii rosettes. Fluorescent in situ hybridisation provided the first molecular evidence of Pl. bekefii and Pl. guttaeformis as Planctomycetes. Also confirming Planctomycetes membership of Pl. bekefii was the presence of a cytoplasm divided into two regions by an intracytoplasmic membrane, consistent with membership to the genus Planctomyces. Two new planctomycete-like organisms, MBLW1 and MBLW2, were isolated in this study and possessed a Gemmata-like cell plan. 16S rRNA gene sequencing confirmed these isolates belonged to the Gemmata clade within phylum Planctomycetes, though they may comprise a separate but closely related genus. Via EMISH, both ATCC 35122 and MBLW1 were hybridised with a planctomycete-specific probe, consistent with membership to the planctomycetes. Statistical analysis showed that 16S rRNA was present in both regions of the riboplasm of MBLW1, identical to the distribution observed G. obscuriglobus. This is another example of possible uncoupled translation within a member of the planctomycetes and within organisms in the Gemmata clade of planctomycetes