Heinz Schlesner

The Development of Media Suitable for the Microorganisms Morphologically Resembling Planctomyces spp., Pirellula spp., and other Planctomycetales from Various Aquatic Habitats Using Dilute Media

Summary A total of 257 strains of budding bacteria morphologically resembling members of the order Planctomycetales were isolated from various aquatic habitats by the use of different enrichment procedures and dilute media for isolation and cultivation. The habitats differed in many respects. Salinity varied between freshwater and saline water (62.8%o); pH ranged from 4.2 to 11.6. Isolates were recovered from oligotrophic, eutrophic, very eutrophic and frankly polluted habitats. This indicates the wide distribution of budding, peptidoglycan-less bacteria. Media with N-acetylglucosamine as carbon and nitrogen source and containing the antibiotics ampicillin and cycloheximide proved to be optimal for isolation. The isolates were identified morphologically as Planctomyces spp., Pirellula spp., and as yet undescribed bacteria with spherical or prosthecate cells.

Filobacillus milensis gen. nov., sp. nov., a new halophilic spore-forming bacterium with Orn-D-Glu-type peptidoglycan

A spore-forming, halophilic bacterium was isolated from surface sediment located on the beach of Palaeochori Bay near to a shallow water hydrothermal vent area, Milos, Greece. The bacterium, designated SH 714T, consisted of motile, strictly aerobic rods which contained an Orn-D-Glu type murein and a G+C content of 35 mol%. Thin sections showed a cell wall typical for Gram-positive bacteria; the peptidoglycan layer, however, was very thin. The Gram-reaction of the organism was negative. Comparative 16S rRNA gene sequencing demonstrated that the isolate represents a new line of descent within the spore-forming rods branching at the periphery of the rRNA group 1 Bacillus (Bacillus sensu stricto). The nearest phylogenetic neighbours of the unknown bacterium were Bacillus haloalkaliphilus, Marinococcus albus and Halobacillus species. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Filobacillus milensis gen. nov., sp. nov. The type strain is SH 714T (= DSM 13259T = ATCC 700960T).

The order Planctomycetales, including the genera Planctomyces, Pirellula, Gemmata and Isosphaera and the Candidatus genera Brocadia, Kuenenia and Scalindua

The order Planctomycetales comprises a remarkable group of budding bacteria. They and their nearest relatives, the chlamydiae (Weisburg et al., 1986; see The Genus Chlamydia–Medical in this Volume), are the only known cell-wall containing bacteria that lack peptidoglycan. Furthermore, the planctomycetes are morphologically distinctive because of their budding division, their spherical to ovoid cells with crateriform pits (Figs. 1 and 2), and the nonprosthecate appendages (stalks) produced by some members of the group (Fig. 3). Multicellular aggregates or rosettes are formed by some species that produce polar holdfasts (Fig. 3). One genus, Isosphaera, is a multicellular filamentous bacterium that moves by gliding. Other motile members of the group produce flagella. A relatively recent addition to the morphological oddity of the planctomycetes is the discovery of cellular compartmentalization, posing a challenge to the traditionally held view of the prokaryote:eukaryote dichotomy (Fuerst and Webb, 1991; Lindsay et al., 1997; Lindsay et al., 2001). Knowledge of the order is limited owing to the relatively few species that have been obtained in pure culture and characterized. However, through the application of molecular microbial ecology techniques over the last 10 years, it has become apparent that planctomycetes are ubiquitous in a wide range of terrestrial and aquatic environments; the physiological diversity underlying this geographic ubiquity has not yet been fully explored. The availability of genome sequence data should provide a valuable resource for the future investigation of planctomycete biology and promises to reveal previously unknown aspects of these unique organisms.

Assignment of the genera Planctomyces and Pirella to a new family Planctomycetaceae fam. nov. and description of the order Planctomycetales ord. nov.

Summary The genera Planctomyces and Pirella are assigned to a new family Planctomycetaceae . Members of this family are characterized by a unique morphology, the formation of buds, and a proteinaceous cell wall. Comparative 16S rRNA oligonucleotide cataloguing has shown this family to be a coherent taxon. The branching point of the Planctomyces — Pirella line of descent within the phylogenetic tree of eubacteria is so deep than an order Planctomycetales is described to harbor Planctomycetaceae .

Verrucomicrobium spinosum gen. nov., sp. nov.: a Fimbriated Prosthecate Bacterium

Summary Six new isolates of prosthecate bacteria which morphologically resemble Prosthecomicrobium spp. are described as Verrucomicrobium spinosum . Their most striking morphological characteristic is the possession of bundles of fimbriae which originate from the tips of the prosthecae. Fimbriated strains of prosthecomicrobia or ancalomicrobia have not yet been reported. These heterotrophic Verrucomicrobium strains utilise only a limited spectrum of carbon sources, i. e. mainly hexoses and their derivatives. The G+C content of their DNA is 57.9 – 59.3 mol% and thus more than 5 mol% lower than the range defined for the genus Prosthecomicrobium .

Pirella marina sp. nov., a budding, peptidoglycan-less bacterium from brackish water

Summary Four new isolates of budding peptidoglycan-less eubacteria from brackish water of the Baltic Sea are described as Pirella marina. They are compared to Pirella staleyi, from which they can be differentiated by morphological characteristics such as the shape of buds and the distribution pattern of crateriform structures. They also differ in physiological characteristics for instance in their higher salt tolerance, requirement of NaCl for growth and use of a wider range of organic compounds as a carbon source.

Assignment of hitherto unidentified 16S rDNA species to a main line of descent within the domain Bacteria

Phylogenetic analysis of the almost complete 16S rDNA sequence of the fimbriate prosthecate bacterium Verrucomicrobium spinosum confirms the unique phylogenetic position of this organism, as previously shown by oligonucleotide cataloguing and partial reverse transcriptase sequencing. Comparative 16S rDNA sequence analysis of V. spinosum with a group of environmental clones, considered to represent a novel phylum, reveals their relatedness, and allows assignment of these clones to a known main line of descent. This phylogenetic relationship is supported by the presence in the 16S rDNA sequence of V. spinosum of signature nucleotides previously considered unique for the environmental clone cluster.

The budding bacteria, Pirellula and Planctomyces, with atypical 16S rRNA and absence of peptidoglycan, show eubacterial phospholipids and uniquely high proportions of long chain beta-hydroxy fatty acids in the lipopolysaccharide lipid A

Fatty acids of twelve strains of budding bacteria (Planctomyces and Pirellula spp.), which have atypical 16S rRNA and do not contain peptidoglycan cell walls, were shown to contain typical diacyl polar lipids with no indication of isoprenoid ether lipids suggestive of a relationship with the archaebacteria. The major ester-linked fatty acids of the phospholipids were palmitic, palmitoleic and oleic acids, which are more typical of microeukaryotes than of eubacteria. Lipopolysaccharide lipid A (LPS) was detected; it contained major proportions of long chain normal 3-OH fatty acids (3-OH eicosanoic at 23% and 17% of the total in two strains of Planctomyces, and 3-OH octadecanoic at 18%, and 3-OH palmitic at 11% of the total in one strain of Pirellula). Major portions of long chain 3-OH fatty acids in the LPS are extremely unusual and provide another atypical property of these organisms. Each strain investigated showed a specific total fatty acid composition, reflecting the diversity in 16S rRNA nucleotide catalogues.

Chemotaxonomic Investigation of Various Prosthecate and/or Budding Bacteria

Summary Sixty-three strains of prosthecate and/or budding bacteria were investigated chemotaxonomically using quinones, phospholipids, and fatty acids as chemical markers. Phospholipids and hydroxy fatty acids were most suitable for discrimination of strains at the genus level, whereas quinones were characteristic for the different phyla which the strains of this study belong to. Thus, the quinones of the Proteobacteria Ancalomicrobium, Angulomicrobium, Asticcacaulis, Labrys, Prosthecobacter, Prosthecomicrobium, and Stella were of the ubiquinone type Q-10, the Planctomycetales had menaquinone MK-6, while Verrucomicrobium possessed. MK-9/10/10(H2)/11. The recently reported phylogenetic heterogeneity among the Prosthecomicrobium-like bacteria and among the Planctomycetales was confirmed with these chemotaxonomic methods. Therefore these strains were placed into several subgroups. In some cases unusual features of bacteria were noticed. For example, menaquinones were found in the Gram-negative Planctomycetales and Verrucomicrobium although they were grown under aerobic conditions. Also, a long-chain cyclopropane fatty acid was detected in some Planctomyces strains. Verrucomicrobium contained some unsaturated fatty acids with unusual double bond positions. In addition, relatively high amounts of hydroxy fatty acids were found in Stella strains.

Identification of Planctomycetes with Order-, Genus-, and Strain-Specific 16S rRNA-Targeted Probes

A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp. strain SH1 have been designed and optimized. Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp. strain SH1 in different habitats. With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysinaaerophoba and from the water column of the Kiel Fjord. An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.