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1 Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Am Botanischen Garten 1-9, D-24118 Kiel, Germany
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
3 Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, D-28359 Bremen, Germany
4 Zentrale Mikroskopie, Christian-Albrechts-Universität, Am Botanischen Garten 5, D-24098 Kiel, Germany
Correspondence
Heinz Schlesner
hschlesner{at}t-online.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 20 February 2004 as DOI 10.1099/ijs.0.63113-0.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) comprise the order Planctomycetales, with the single family Planctomycetaceae (Schlesner & Stackebrandt, 1986) and the four genera Planctomyces, Pirellula, Gemmata and Isosphaera (Staley et al., 1992). The planctomycetes were considered as unculturable bacteria for many decades. Following the first report of a pure culture (Staley, 1973), however, a large number of strains have been isolated from a variety of aquatic habitats differing considerably in salinity, pH or the availability of nutrients (Schlesner, 1994). Other sources of strains were tissue cultures (Fuerst et al., 1991) and postlarvae of the giant tiger prawn Penaeus monodon (Fuerst et al., 1997) and the tissue of the Mediterranean sponge Aplysina aerophoba (Gade et al., 2004). Recently, the isolation of Gemmata-like bacteria from soil was reported (Wang et al., 2002). Detailed information on the history and biology of planctomycetes can be found in the excellent review of Fuerst (1995).
Studies on the evolutionary position of these organisms indicated a great inter- and intrageneric heterogeneity in the 16S rRNA gene sequences (Stackebrandt et al., 1986; Ward et al., 1995; Fuerst et al., 1997; Griepenburg et al., 1999). Sequence-similarity values for the 16S rRNA genes of Pirellula staleyi, Pirellula marina (here described as Blastopirellula marina gen. nov., comb. nov.) and Pirellula sp. SH 1T (here described as Rhodopirellula baltica gen. nov., sp. nov.) were below 90 % (see Table 4), supporting the differentiation into three genera. At present, it is generally accepted that individual species of the same genus should have 16S rRNA gene sequence similarity of more than 95 % (Devereux et al., 1990; Fry et al., 1991; Stackebrandt & Goebel, 1994). The 16S rRNA gene sequence heterogeneity was further supported by analysis of the cell-wall components (König et al., 1984; Liesack et al., 1986), phospholipids (Kerger et al., 1988; Sittig & Schlesner, 1993) and polyamines (Griepenburg et al., 1999).
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). These results have been confirmed by the recent publication of the complete genome sequence of R. baltica (Glöckner et al., 2003; http://www.regx.de). With a genome size of 7·145 Mb, it is one of the largest circular bacterial genomes known to date. In addition, ongoing genome sequencing projects with Gemmata obscuriglobus (http://www.tigr.org) and Gemmata sp. Wa 1-1 (http://wit.integratedgenomics.com) support the unusually large genome sizes of planctomycete species. It is generally assumed that early genomes were probably small in size and that larger genomes arose later in evolution as a result of events such as gene duplication or the acquisition of additional genes from other organisms. While it is also known that ‘organisms' such as obligate intracellular symbionts or plastids may also reduce their genome size by gene loss (evolutionary reversal), a prerequisite is that the ancestors must have passed through a stage in which the genome size was larger (Anderson & Anderson, 1999; Blanchard & Lynch, 2000; Gil et al., 2002). While planctomycetes have been variously described as rapidly evolving or ancient, it would appear that the large genome size, at least, is probably not a ‘primitive’ feature.
In the light of these studies on the genomes of various planctomycetes, it is becoming important to re-examine our appreciation of the biology and diversity of this group of organisms. In this study, we investigated the taxonomic positions of 97 pigmented and unpigmented Pirellula-, Planctomyces- and Gemmata-like strains isolated from various aquatic habitats, including the type strains of Pirellula staleyi, B. marina, Planctomyces maris and G. obscuriglobus. R. baltica was selected as representative of a group of isolates that were genetically related at the species level. This group contained 22 pigmented isolates from brackish water and two strains isolated from the tissue of the sponge A. aerophoba (Gade et al., 2004). Here, we describe Pirellula sp. SH 1T as the type strain of R. baltica gen. nov., sp. nov. Because of the diversity of the respective strains at the genetic and phenotypic levels, we propose that Pirellula marina be excluded from the genus Pirellula and be transferred to the new genus Blastopirellula gen. nov. as Blastopirellula marina comb. nov. This also means that the description of the genus Pirellula must be emended to reflect its changed circumscription.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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).
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).
Microscopy.
Morphological studies involving phase-contrast microscopy and electron microscopy were performed as described previously (Schlesner, 1986; Gade et al., 2004). For cryofixation and cryosubstitution, bacteria were fixed using high-pressure freezing (Hohenberg et al., 1994). Samples in 1 % (w/v) osmium tetroxide in acetone were dehydrated in a freeze-substitution unit (AFS Leica) at temperatures of –90, –60 and –30 °C (for 8 h each). Finally, the temperature was increased to 4 °C. The samples were infiltrated with Epon 812 (Epon Kit 45359; Fluka) by incubation with 30 % and 70 % (w/v) resin in acetone for 2 h each and then in pure resin for 12 h at 20 °C. Polymerization occurred at 60 °C for 24 h.
Physiological studies.
Growth experiments with liquid media were carried out essentially as described by Schlesner (1986, 1994). Cultivation was done with 100 ml Erlenmeyer flasks containing 50 ml medium. Cultures were incubated at 25 °C for 3 weeks on a shaker. To test for carbon sources (0·1 %, w/v) that supported growth, medium M40 was used for R. baltica (SH 1T) and B. marina (DSM 3645T), whereas medium M40c was used for Pirellula staleyi (ATCC 27377T). Anaerobic growth was tested under fermentative conditions and in the presence of nitrate as electron acceptor. Techniques for the preparation of media and cultivation under anoxic conditions were described previously (Widdel & Bak, 1992). Ascorbate was added to the media as an additional reductant (Rabus & Widdel, 1995).
Hydrolysis of gelatin was visualized by flooding the plates with hot (70–80 °C) saturated ammonium sulfate instead of mercury chloride. Lipase activity was tested according to Kouker & Jaeger (1987), using plates that contained trioleic acid and rhodamine B. Activities for the hydrolysis of starch, casein, aesculin and DNA were tested according to standard procedures (Smibert & Krieg, 1994). Formation of hydrogen sulfide from thiosulfate was tested as described previously (Schlesner, 1986).
Salinity tolerance was studied with liquid media containing increasing proportions of artificial sea water (ASW; Lyman & Fleming, 1940) to give final concentrations of 0, 6, 12 and 25 %; 25 % steps were then used to reach a maximum of 300 % ASW (100 % ASW=34·5 
salinity).
Phospholipids and quinones.
These cell components were extracted from lyophilized cells and analysed by TLC as described by Sittig & Hirsch (1992). In addition, polar lipids, respiratory lipoquinones and fatty acids were extracted and analysed as described previously (Tindall, 1990a, b; Strömpl et al., 1999).
DNA base ratio and DNA–DNA hybridization.
G+C content determinations and DNA–DNA hybridization experiments were performed as described previously (Rathmann, 1992; Gade et al., 2004).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Initially, we categorized all strains mainly according to pigmentation of colonies, the motility of daughter cells and DNA–DNA hybridization groups (Table 2). The novel isolates were obtained from a variety of aquatic habitats, including freshwater, brackish water and marine water. Most habitats had a slightly alkaline pH (around 8), while water bodies of chalk mines reached maximal pH values of 11·6 (Schlesner, 1994). Hypertrophic water samples were retrieved from Apetlon village pond (Austria) and Schrevenpark Pond (Kiel, Germany). The following section deals with the categorization of the entire set of strains investigated.
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) indicates the genetic diversity of this group. The G+C value for most strains was in the range reported for the genera Planctomyces (50–58 mol%), Pirellula (54–57 mol%) or Gemmata (64 mol%; Staley et al., 1992). Strains SH 404 and SH 449, however, displayed considerably higher values: 69 and 68 mol%, respectively.
DNA–DNA hybridization experiments with labelled DNA of 20 selected strains (underlined in Table 2) resulted in 19 hybridization groups. Definition of hybridization groups was based on 70 % DNA binding (different strains belonging to the same species; Wayne et al., 1987; Stackebrandt & Goebel, 1994). Six hybridization groups were represented by one strain only. The DNA of 18 strains did not hybridize with labelled DNA of any of the 20 selected strains, with Planctomyces maris (ATCC 29201T) or with G. obscuriglobus (DSM 5831T). These results also point to the genetic heterogeneity among the studied strains. Pigmentation proved to be a useful taxonomic marker, since individual hybridization groups contained exclusively either pigmented or unpigmented strains.
Hybridization group I contained 25 pigmented strains of R. baltica, which displayed the typical Pirellula morphology. All of them possessed motile daughter cells. The majority of strains (19) were isolated from Kiel Fjord (Baltic Sea) over a period of 25 years, suggesting that these bacteria belong to the autochthonous microbial community of this habitat. Interestingly, two novel strains were recently isolated from the Mediterranean sponge A. aerophoba (Gade et al., 2004), indicating the widespread occurrence of this species. In addition, DeLong et al. (1993) obtained molecular clones of 16S rRNA gene sequences with high similarity to R. baltica from marine snow (Pacific Ocean).
Hybridization group II consisted of eight strains of B. marina, including the type strain (DSM 3645T). The main difference with respect to hybridization group I was the lack of pigmentation. While four strains were isolated from Kiel Fjord, others originated from other brackish habitats.
Hybridization group III consisted of six strains of Pirellula staleyi, including the type strain (ATCC 27377T). Like those in hybridization group II, the colonies were unpigmented. While members of hybridization groups I and II originated from brackish to marine habitats, group III strains were isolated from freshwater habitats in northern Germany. Pirellula staleyi originated from Lake Lansing (Michigan, USA; Staley, 1973).
A detailed differentiation of the 19 hybridization groups according to salinity tolerance, hydrolysis of polymers and the presence of phosphatidylcholine is summarized in Table 3. Strains within an individual hybridization group displayed only minor phenotypic differences. Although the presence of MK-6 as the major respiratory quinone did not allow differentiation within the group, it allows this group to be distinguished from all other prokaryotes that produce menaquinones of longer chain length. Similar results were found by Sittig & Schlesner (1993). Detailed studies on the fatty acids and polar lipids of a range of strains indicated that it was possible to differentiate distinct groups, highlighting the evolutionary heterogeneity of this group. Of the 97 strains investigated here, only six had immotile daughter cells. In the following sections, we focus on the taxonomic differentiation of R. baltica, B. marina and Pirellula staleyi.
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]. The morphology of R. baltica resembled that of other members of the Planctomycetales, in particular of the group of Pirellula-like bacteria, as indicated by the mode of budding, the presence of fimbriae and crateriform structures and the holdfast substance excreted directly from the smaller cell pole (Schlesner & Hirsch, 1984; Fig. 1a). In contrast to that of B. marina (Schlesner, 1986), the bud was not bean-shaped but was a smaller mirror image of the mother cell. Thin cryosubstituted sections of cells (Fig. 1b) showed membranous structures surrounding the nuclear material and the majority of the ribosomes. Analysis of cross-sections of strain SH 1T as well as SH 796 (Gade et al., 2004) revealed several small structures in addition to a large central one. This microscopic appearance differs from that of the pirellulosome structure described for Pirellula staleyi and B. marina (Lindsay et al., 2001).
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R. baltica and B. marina can be considered as marine bacteria since they do not grow in freshwater media. Growth of R. baltica occurred in media containing rising concentrations (12–175 %) of ASW (100 % ASW corresponds to a salinity of 34·5 ). Similar values were observed for B. marina. Essential components of ASW were Ca2+, Na+ and Cl–. In contrast, the freshwater bacterium Pirellula staleyi tolerated only up to 50 % ASW. R. baltica required the addition of vitamin B12 to the medium, while B. marina and Pirellula staleyi were able to grow in vitamin-free media (Table 4).
Despite the 16S rRNA gene sequence difference between R. baltica, B. marina and Pirellula staleyi, their physiological properties were very similar. Substrates serving as carbon and energy sources were mainly carbohydrates. N-Acetylglucosamine also served as a nitrogen source. Chondroitin sulfate was an excellent carbon source for R. baltica and B. marina. All three organisms displayed catalase and cytochrome oxidase activities, but no urease activity; they produced H2S from thiosulfate, but did not produce acetoin or indole. Initial tests on the mesophilic cells of R. baltica indicated that they appeared to be strictly aerobic, since they were unable to use nitrate as an electron acceptor or to grow fermentatively with glucose. In agreement with a strictly aerobic metabolism, the genome of R. baltica revealed no evidence for an anaerobic ribonucleotide reductase. Thus, the predicted capacity for lactic acid fermentation (Glöckner et al., 2003) may serve maintenance only.
Chemotaxonomic characteristics
Chemotaxonomic markers proved to be more useful than physiological properties for differentiating between R. baltica, B. marina and Pirellula staleyi. The chemotaxonomic markers that have been analysed are the fatty acid and phospholipid profiles (Kerger et al., 1988; Sittig & Schlesner, 1993), the amino acid composition of cell walls (König et al., 1984; Liesack et al., 1986) and the polyamine patterns (Griepenburg et al., 1999). In contrast, analysis of the quinone profile was not useful within the group, since all planctomycetes investigated so far have possessed MK-6 as the only quinone (Sittig & Schlesner, 1993; B. J. Tindall & H. Schlesner, unpublished). However, the presence of this short-chain lipoquinone is useful for delineating this group and also in distinguishing it from other menaquinone-producing prokaryotes with longer isoprenoid side chains. It should be noted that the chemical composition of the cells (polar lipids, fatty acids and respiratory lipoquinone composition) provides a way of differentiating organisms within this group, but also indicates that the planctomycetes are chemically distinct from any other taxa examined to date. Detailed analyses of the fatty acids examined in this study are given in Table 4. All species produce 16 : 1
9, 16 : 0, 18 : 19 and 18 : 0 fatty acids. While these fatty acids are fairly common in members of the 
-, 
- and 
-subclasses of the Proteobacteria, this combination, together with the presence of MK-6, clearly distinguishes these species from these major evolutionary groups. Among the strains examined, the presence/absence of 14 : 0, 15 : 0, i-16 : 0, 17 : 19, 17 : 0 and 20 : 111 can be used to distinguish between different taxa. While Kerger et al. (1988) reported the presence of hydroxy fatty acids in planctomycetes, they only deduced that they originated from lipopolysaccharides, providing no direct proof. In this study, the methods used would detect the presence of lipopolysaccharide-derived hydroxy fatty acids present in Escherichia coli. Thus we conclude that the absence of measurable amounts of hydroxy fatty acids is indicative of the absence of significant amounts of lipopolysaccharides in the cell wall, despite the fact that these organisms are Gram-negative. Sittig & Schlesner (1993) provided the first indications of the chemical heterogeneity of this group, but the full significance of this is now evident when different (phenotypic and genetic) datasets are integrated.
The polar lipid patterns determined by two-dimensional TLC are shown in Fig. 2. The polar lipid compositions of the three type strains are clearly different. Investigations performed on a wider range of strains (Sittig & Schlesner, 1993; B. J. Tindall and H. Schlesner, unpublished) indicate that this chemical diversity also correlates well with the 16S rRNA gene diversity. Thus the presence of phosphatidylcholine in R. baltica is not just a feature of this species, but is also found in other strains that group with this species in 16S rRNA gene studies. Similarly, the polar lipid patterns of Pirellula staleyi and B. marina also indicate features that allow them to be differentiated not only from R. baltica, but also from one another. The use of spray reagents that render all lipid-like material visible shows that a significant percentage of the cellular lipids are novel. To date, there are no indications that these unidentified lipids are present in any other taxa beyond planctomycetes.
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Genome of R. baltica
The complete sequence of the 7·145 Mb genome of R. baltica (SH 1T) was only recently reported (Glöckner et al., 2003). Sequence analysis revealed the complete genetic blueprint for glycolysis, the pentose phosphate cycle and the tricarboxylic acid cycle, which agrees with the specialization in carbohydrate utilization found for this strain in the present study. A surprising finding was the presence of more than 100 genes possibly encoding sulfatases. It could be speculated that growth with chondroitin sulfate, as found here, may require the activity of a specific sulfatase that liberates the carbohydrate moiety. The first insights into the regulation of carbohydrate metabolism were recently obtained by means of a proteomic approach (Rabus et al., 2002).
The presence of known phospholipids (phosphatidylcholine and phosphatidylglycerol), together with the presence of novel compounds (of as yet unknown structures), will provide a stimulus for examining the biosynthesis and gene regulation of these cellular components, and for locating the genes responsible. The presence of phosphatidylcholine in R. baltica as the sole nitrogen-containing phospholipid is interesting, since it is generally accepted that phosphatidylcholine is synthesized via progressive methylation of phosphatidylethanolamine (Pieringer, 1989). Polar lipid patterns containing phosphatidylethanol and phosphatidylcholine are typical of certain actinomycetes and some major evolutionary groups within the -subclass of the Proteobacteria (Ratledge & Wilkinson, 1989). However, there are currently no reliable reports of prokaryotes containing phosphatidylcholine as the sole nitrogen-containing phospholipid, and this may be indicative of an alternative pathway leading to the synthesis of phosphatidylcholine in these organisms.
In re-examining the taxonomy of the Pirellula group within the planctomycetes, we have attempted to integrate as much current knowledge about this group as possible. Although DNA–DNA hybridization studies are generally considered to be problematic from a methodological standpoint, as well as with respect to determining which fragments of DNA are involved in binding, this method still serves as one of the best ways of gaining an indirect insight into overall similarities between genomes. The results presented in this work clearly indicate that the strains currently available in pure culture not only constitute a diverse range of strains (according to 16S rRNA gene studies), but also that they are represented by numerous, different species. Differences in 16S rRNA gene sequences of more than 3 % (Stackebrandt & Goebel, 1994) are generally indicative of different species (when the species groups are tested by DNA–DNA hybridization). Thus, differences in the 16S rRNA gene sequence are indicative of differences at the ‘genetic level’, which will certainly be evident in the genomes of the organisms concerned. Unfortunately, this genetic (and of course evolutionary) diversity does not appear to be reflected in the physiology of the organisms concerned, as was experimentally determined using classical phenotypic tests. While there are clearly marine and freshwater strains, the ability of the organisms to utilize the range of substrates tested would suggest that members of this group are rather uniform. However, this uniformity at the level of substrate utilization does not tell us anything about the potential diversity in the underlying biochemical pathways, or about the structural diversity of the enzymes concerned. Further studies on genomes of different species and different strains within the planctomycetes will help to elucidate this point.
In contrast to the apparently ‘limited’ physiological diversity, the diversity in chemical composition indicates that the use of this polyphasic taxonomic approach, as in other prokaryotes, reflects the evolutionary diversity that we also detect via constrained elements, such as the ribosome. Thus, strains that share a high degree of genetic similarity (as reflected by DNA–DNA hybridization and high 16S rRNA gene similarity values) are difficult to distinguish chemically, but, with increasing genetic diversity (as reflected by decreasing DNA–DNA hybridization values and decreasing 16S rRNA gene sequence similarity), the chemical differences become increasingly evident. In such instances, changes in the chemical composition may reflect changes either in regulatory mechanisms or in the biochemical pathways leading to the synthesis of the end-products. Such changes are as significant in the evolution of the cell as the changes in the sequences of genes, such as the 16S rRNA gene. Taking these aspects into consideration, we suggest that it is possible to examine their significance in the evolution of prokaryotes and the taxonomy upon which this is based.
While planctomycetes have been described as rapidly evolving or ancient, it is evident from the genome size that this is probably representative of a later stage in evolution and it is debatable whether this is a feature of a ‘primitive’ group of organisms. Arguments centring on the fact that planctomycetes are rapidly evolving (Liesack et al., 1992) are based on Simpson's work on ‘tempo and mode’ in evolution (Simpson, 1944). Key arguments in favour of rapid evolution are the large 16S rRNA gene differences between species within the genera Planctomyces and Pirellula as well as the low 16S rRNA gene similarity values between genera (Liesack et al., 1992). In addition, idiosyncrasies in the 16S rRNA gene sequence, together with other peculiarities of planctomycetes, are used in support of the hypothesis that these organisms are evolving rapidly. However, when evaluating rates of evolution, both Simpson (1944) and Mayr (1969) have considered the geological time-scale, a feature usually missing from the majority of gene sequence comparisons. The problem is compounded by the fact that calibration rates are ultimately based on a known fossil record (Doolittle, 1997; Doolittle et al., 1996; Feng et al., 1997; Lee, 1999), and even then rates may vary between lineages (Ochman et al., 1999; Soltis et al., 2002). In addition, there are no reliable reports of ‘molecular fossils' that are more than 50–100 million years old (Poinar et al., 1996; Willerslev et al., 2003). Such problems have been highlighted by Sneath (1974) and are beginning to be discussed again.
In recent years, detailed three-dimensional structures of ribosomes have been published: these have indicated the significance not only of secondary structure (in both proteins and RNA molecules), but also of the diverse close RNA–RNA and RNA–protein interactions (Ban et al., 2000; Brimacombe, 2000; Schluenzen et al., 2000; Wimberly et al., 2000). Thus, the idiosyncrasies in the 16S rRNA gene sequence should be considered in the light of these interactions, rather than the gene sequence alone. Similarly, the fact that planctomycetes are characterized by a set of other peculiarities (to which we can also add the polar lipid composition) certainly serves to underline the uniqueness of this group of organisms, but are not necessarily indicative of rapid rates of evolution. Morse et al. (1996) have also called into question the suggestion that Oenococcus oeni is evolving rapidly.
On the basis of the data presented here, we consider that planctomycetes are diverse across a range of properties, and that this diversity is a reflection of extensive evolution. While we cannot easily say whether the evolution of this group has taken place over short or long periods of geological time, it is evident that the current taxonomy does not reflect the diversity that we find in this group. We propose, therefore, a number of taxonomic changes, based on the closer study of a limited number of strains, which may also further serve as a basis for evaluating the systematics of this group of organisms.
Properties that differentiate the genera Rhodopirellula, Blastopirellula and Pirellula
Properties of the type strains of R. baltica, B. marina and Pirellula staleyi that allow differentiation of the three genera are summarized in Table 4. Pigmented cells are observed only for R. baltica. Growth of R. baltica and B. marina requires high concentrations of sodium chloride and calcium, whereas Pirellula staleyi has only limited tolerance to ASW. Even though all type strains grow with carbohydrates, some difference can be noted. R. baltica and Pirellula staleyi are not able to utilize fucose and chondroitin sulfate, respectively. Only Pirellula staleyi can utilize glutamic acid and hydrolyse casein, while lipase activity can be observed only in B. marina. Polyamine patterns differ between the type strains. While all contain sym-homospermidine, putrescine and cadaverine are found only in R. baltica, and spermidine is found only in Pirellula staleyi. Only R. baltica possesses phosphatidylcholine. Differences are also observed with respect to the molar ratios of the cell-wall amino acids. The fatty acid patterns of the three organisms differ as follows: R. baltica and Pirellula staleyi lack i16 : 0, 19 : 111 and 19 : 0, which are found in B. marina. In contrast, 17 : 19 and 17 : 0 are present in the former two but absent in the latter. The intracellular compartmentalization is formed by different structures. The pirellulosomes described for B. marina and Pirellula staleyi (Lindsay et al., 2001) show a single large structure in the electron microscopic image, whereas multiple smaller structures are visible in addition to a large one in the case of R. baltica. These considerable differences between the type strains are also reflected at the genetic level, as indicated by DNA–DNA hybridization (Table 2) and the low similarity of the 16S rRNA gene sequences (<90 %; Table 4).
Emended description of the genus Pirellula Schlesner and Hirsch 1987
The description of the genus Pirellula is largely based on physiological, biochemical and morphological properties, and it would be appropriate to emend the description to take into account both additional data and our changing appreciation of the taxonomy of this group. The biochemical, physiological and morphological characteristics are described by Schlesner & Hirsch (1984). The major polyamine is sym-homospermidine. The major respiratory lipoquinone present is MK-6. The major phospholipid present is phosphatidylglycerol. A number of other lipids are present that have characteristic Rf values, but whose structures are not currently known. However, the lipid pattern is characteristic of this genus. The major fatty acids present are 14 : 0, 16 : 19, 16 : 0, 18 : 19, 18 : 111, 18 : 0 and 20 : 19. It is also evident that 16S rRNA gene sequence similarity values are of significance in delineating this genus. Nevertheless, the extent cannot be defined at present, since strains with less than 95 % sequence similarity to members of this genus should probably be placed in separate genera. The type species of the genus is Pirellula staleyi.
Description of Rhodopirellula gen. nov.
Rhodopirellula (Rho.do.pi.rel'lu.la. Gr. neut. n. rhodon a rose; N.L. fem. n. Pirellula name of a bacterial genus; N.L. fem. n. Rhodopirellula a red Pirellula).
Cells are ovoid, ellipsoidal or pear-shaped, occurring singly or in rosettes by attachment at the smaller cell pole. Buds are formed at the broader cell pole. Buds may have a single flagellum inserted subpolarly at the proximal pole. Adult cells are immobile. Crateriform structures and fimbriae are found in the upper cell region. Colonies are pink to red in colour. Non-sporulating. Strictly aerobic. Catalase- and cytochrome oxidase-positive. The proteinaceous cell wall lacks peptidoglycan. The major polyamines are putrescine, cadaverine and sym-homospermidine. The major menaquinone is MK-6. The major fatty acids are 16 : 19, 16 : 0, 17 : 19, 17 : 0, 18 : 19, 18 : 111 and 18 : 0. The major phospholipids are phosphatidylcholine and phosphatidylglycerol. Additional, unidentified polar lipids are also present. This genus is a member of the phylum Planctomycetes, order Planctomycetales, family Planctomycetaceae, as currently defined primarily on the basis of 16S rRNA gene sequence analysis. The type species is Rhodopirellula baltica.
Description of Rhodopirellula baltica sp. nov.
Rhodopirellula baltica (bal'ti.ca. L. fem. adj. baltica pertaining to the Baltic Sea, the place of isolation).
Cells are 1·0–2·5x1·2–2·3 µm in size. A single flagellum is subpolarly inserted at the proximal pole. Colonies are round, smooth and pink to red in colour. Growth is optimal between 28 and 30 °C. Growth is not observed above 32 °C. Vitamin B12 and sea water are required for growth. The bacterium is strictly aerobic. Glucose is not fermented. Nitrate cannot serve as an electron acceptor. Carbon sources utilized are as follows: cellobiose, fructose, galactose, glucose, lactose, lyxose, maltose, mannose, melibiose, melezitose, raffinose, rhamnose, ribose, sucrose, trehalose, xylose, dextrin, N-acetylglucosamine, glycerol, aesculin, amygdalin, gluconate, glucuronate, salicin and chondroitin sulfate. Carbon sources not utilized are as follows: fucose, sorbose, methylamine, methylsulfonate, methanol, ethanol, erythritol, adonitol, arabitol, dulcitol, inositol, mannitol, sorbitol, acetate, adipate, benzoate, caproate, citrate, formate, fumarate, glutarate, lactate, malate, 2-oxoglutarate, phthalate, propionate, pyruvate, succinate, tartrate, alanine, arginine, asparagine, aspartate, cysteine, cystine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, urea, indole, inulin and pectin. Peptone, Casamino acids, yeast extract, gelatin, ammonium, nitrate and N-acetylglucosamine are each utilized as a nitrogen source, but neither nicotinate nor urea is utilized. Aesculin, gelatin and starch are hydrolysed, but alginate, casein, cellulose, chitin and Tween 80 are not hydrolysed. No haemolytic activity is found with horse, calf or sheep blood. Catalase and cytochrome oxidase activities are observed, but urease activity is not observed. H2S, but not acetoin or indole, is produced. Cells are sensitive to tetracycline, but resistant to streptomycin, ampicillin and penicillin. The G+C content of the DNA is 53–57 mol% (55 mol% for the type strain). The chemical composition is identical to that in the genus description. The main habitat is brackish water of Kiel Fjord (Baltic Sea).
The type strain is SH 1T (=IFAM 1310T=DSM 10527T=NCIMB 13988T).
Description of Blastopirellula gen. nov.
Blastopirellula (Blas.to.pi.rel'lu.la. Gr. masc. n. blastos bud, shoot; N.L. fem. n. Pirellula name of a bacterial genus; N.L. fem. n. Blastopirellula a budding Pirellula).
Cells are ovoid, ellipsoidal or pear-shaped, occurring singly or in rosettes by attachment at the smaller cell pole. Buds are formed at the broader, proximal cell pole. Adult cells are immobile. Crateriform structures and fimbriae are found in the upper cell region. Colonies are greyish to brownish white. Non-sporulating. Strictly aerobic. Catalase- and cytochrome oxidase-positive. The proteinaceous cell wall lacks peptidoglycan. The major polyamine is sym-homospermidine. The major menaquinone is MK-6. The major phospholipid present is phosphatidylglycerol. Additional unidentified polar lipids are also present, at least one of which appears to be identical (in Rf value) to one of the major components present in members of the genus Pirellula. The major fatty acids present are 15 : 0, i-16 : 0, 16 : 19, 16 : 0, 17 : 19, 17 : 0, 18 : 19, 18 : 111, 18 : 0, 19 : 111 and 20 : 19. This genus is a member of the phylum Planctomycetes, order Planctomycetales, family Planctomycetaceae, as currently defined primarily on the basis of 16S rRNA gene sequence analysis. The type species is Blastopirellula marina.
Description of Blastopirellula marina comb. nov.
Blastopirellula marina (ma.ri'na. L. fem. adj. marina of, or belonging to, the sea, marine).
Basonym: Pirellula marina (Schlesner 1987) Schlesner and Hirsch 1987
The description is the same as that published for Pirellula marina (Schlesner, 1986), with the following additions. The species is apparently strictly aerobic, as glucose is not metabolized anaerobically either by fermentation or with nitrate as the electron acceptor. The chemical composition is identical to that in the genus description.
The type strain is SH 106T (=IFAM 1313T=DSM 3645T=ATCC 49069T).
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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