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Oramoeba fumarolia gen. nov., sp. nov., a new marine heterolobosean amoeboflagellate growing at 54 °C
European Journal of Protistology 47 (2011) 16–23
Oramoeba fumarolia gen. nov., sp. nov., a new marine heterolobosean amoeboflagellate growing at 54 ◦ C Johan F. De Jonckheerea,b,∗ , Manuela Baumgartnerc , Silvia Eberhardtc , Fred R. Opperdoesa,d , Karl O. Stetterc a
Research Unit for Tropical Diseases, de Duve Institute, B-1200 Brussels, Belgium Scientific Institute of Public Health, B-1050 Brussels, Belgium c Lehrstuhl für Mikrobiologie, Universität Regensburg, D-93053, Germany d Université Catholique de Louvain, B-1200 Brussels, Belgium b
Received 31 March 2010; received in revised form 3 September 2010; accepted 6 September 2010
Abstract An amoeba strain was isolated from marine sediment taken from the beach near a fumarole in Italy. The trophozoites of this new marine species transforms into flagellates with variable numbers of flagella, from 2 to 10. The strain forms round to oval cysts. This thermophilic amoeboflagellate grows at temperatures up to 54 ◦ C. Molecular phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) places the amoeboflagellate in the Heterolobosea. The closest relatives are Stachyamoeba sp. ATCC50324, a strain isolated from an ocean sample, and Vrihiamoeba italica, a recent isolate from a rice field. Like some other heterolobosean species, this new isolate has a group I intron in the SSU rDNA. Because of the unique place in the molecular phylogenetic tree, and because there is no species found in the literature with similar morphological and physiological characteristics, this isolate is considered to be a new genus and a new species, Oramoeba fumarolia gen. nov., sp. nov. © 2010 Elsevier GmbH. All rights reserved. Keywords: Heterolobosea; ITS; New species; SSU rDNA; Thermophilic
Introduction The class Heterolobosea was established for amoebae, which move with eruptive pseudopodia, have intranuclear orthomitosis (promitosis), have mitochondrial cristae which are flattened, often disc-like, and in which stacked Golgi bodies are absent (Page and Blanton 1985). At the time of establishing this class of protists, the Heterolobosea contained amoebae and amoeboflagellates. In the latter there ∗ Corresponding author at: Research Unit for Tropical Diseases, de Duve Institute, B-1200 Brussels, Belgium. Tel.: +32 2 764 74 71; fax: +32 2 7626853. E-mail address: [email protected] (J.F. De Jonckheere).
0932-4739/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2010.09.002
is transformation from an amoeba stage into a temporary flagellate stage, and vice versa. Recently it was found that Percolomonas and Pleurostomum, which were originally placed within the heterotrophic flagellates, and Stephanopogon, originally placed in the Ciliophora, also belong to the Heterolobosea (Nikolaev et al. 2004; Park et al. 2007; Yubuki and Leander 2008; Cavalier-Smith and Nikolaev 2008). These three genera had remained incertae sedis for a long time, but molecular phylogenetic analysis places them within the Heterolobosea, a conclusion which is supported by ultrastructural data. However, in Pleurostomum flabellatum, one of the characteristics of Heterolobosea, mitochondrial cristae, were not observed (Park et al. 2007). In all the Heterolobosea a unique characteristic insertion in the secondary structure (helix 17-1) of the small subunit ribosomal
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DNA (SSU rDNA) is found (Nikolaev et al. 2004), except in ‘Macropharyngomonas halophila’, which has a basal position in the heterolobosean phylogenetic tree. While the majority of heterolobosean species had been found in fresh water habitats and soil (Page 1988), some were isolated from marine (Page 1983) and even hypersaline habitats (Park et al. 2007). We report the isolation of a thermophilic marine amoeboflagellate from a beach near a fumarole. The strain was studied by light microscopy and the SSU rRNA gene sequence analysis shows that it belongs to a new genus in the Heterolobosea.
Materials and Methods The strain under study, SAN2, was isolated from a sediment sample taken in October 2000 in Italy. The sediment, varying in temperature from 55 ◦ C to 70 ◦ C, originated from the beach near a fumarole at San Angelo on the island Ischia (40◦ 43 N, 13◦ 54 E). Ten ml of these sediment samples were incubated at 45, 50 and 55 ◦ C in synthetic seawater BisTris buffered medium (SME-Medium) with 0.1% (v/v) glycerol and 0.05 (w/v) yeast extract. Pseudomonas sp. or Marinobacter sp. (Baumgartner et al. 2002) was added as food source. For growth on agar plates, agar was added to a final concentration of 1.8% to SME-Medium and food bacteria were spread on the plates. The strain was also grown in Page Amoeba Saline (PAS) (Page 1988), supplemented with 3% NaCl, and Escherichia coli as food source. The morphology of moving amoebae attached to glass surfaces was studied at 50 ◦ C using phase contrast optics with a Zeis Axiovert inverse microscope (Baumgartner et al. 2003). Length and breadth dimensions of 100 actively moving trophozoites were determined. The size of 20 cysts was measured after growth on agar plates. The number of flagella was counted on 100 cells. The optimal growth temperature of the isolate was determined in liquid SME-Medium. In addition, agar plates with lawns of Marinobacter sp. were incubated at different temperatures. The diameter of the grazed area was measured at regular intervals (Baumgartner et al. 2003). DNA extraction, amplification of the SSU rRNA gene by PCR, and sequencing were carried out as described previously (Baumgartner et al. 2002). The internal transcribed spacers (ITS), including the 5.8S rDNA, sequences were obtained as described by De Jonckheere and Brown (2005). The SSU rRNA sequence of strain SAN2, omitting 1047 bp of an intron, was placed into an alignment of selected Heterolobosea SSU rRNA sequences obtained from the on-line comprehensive ribosomal RNA database Silva (http://www.arb-silva.de/; Pruesse et al. 2007). The SSU RNA sequence from strain SAN2 was manually aligned to this set. A total of 741 of unambiguously aligned sites common to all sequences was retained for phylogenetic analysis. This alignment is available upon request. Phylogenetic trees were inferred by distance matrix
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neighbor-joining as implemented in ClustalX version 1.8 (Thompson et al. 1997), maximum likelihood (Felsenstein 1981) as implemented by the program PhyML version 2.4.5 (Guindon and Gascuel 2003), and by Bayesian analysis using MrBAYES version 3.1.2 (Huelsenbeck and Ronquist 2001). The general time reversible (GTR) model of evolution (Tavaré 1986) was selected as the best model from 28 using the ModelFind program (http://www.hiv.lanl.gov/ content/sequence/findmodel/findmodel.html). The optimal parameters for the GTR model were estimated using the PhyML program. This model extended with gamma-shaped rate variation (α = 0.89) with four rate categories and a proportion of 0.19 of invariable sites was used for both maximum likelihood (100 bootstrap samplings in PhyML) and Baysian analysis (MrBayes). To estimate Bayesian posterior probabilities, Markov Chain Monte Carlo (MCMC) chains were run for 105,000 generations until convergence and sampled every 100 generations (burn-in: 250 generations). The final dataset for tree construction comprised 29 other Heterlobosea taxa (accession numbers): Tetramitus aberdonicus (AJ224888), T. jugosus (M9805), T. entericus (AJ224889), T. rostratus (M98051), T. lobospinosus (M98052), T. thermacidophilus (AJ621575), Vahlkampfia avara (AJ 22488), V. inornata (AJ22488), Naegleria fowleri (U80059), N. andersoni (U80057), Willaertia magna (X93221, X93223 and AY266315), P. flabellatum (DQ979962), Tulamoeba peronaphora (FJ222603), Acrasis rosea (AF011458), Neovahlkampfia damariscottae (AJ224891), Paravahlkampfia ustiana (AJ224890), Heteramoeba clara (AF011460), ‘Plaesiobystra hypersalinica’ (AF011459), Psalteriomonas lanterna (X94430), Sawyeria marylandensis (AF439351), Monopylocystis visvesvarai (AF011463), Stachyamoeba sp. ATCC 50324 (AF011461), Percolomonas cosmopolitus (AF519443 and AF011464), Stephanopogon minuta (AB365646), ‘M. halophila’ (AF011465), Marinamoeba thermophila (FM244741), Vrihiamoeba italica (AB513360) and the uncultured heterolobosean clone WIM43 (AM114803). The sequences of the Euglenozoa Euglena gracilis (AY029409) and Trypanoplasma borreli (AY028454) were used as outgroups.
Results Most observations were performed on amoebae growing on Marinobacter sp. although the isolate also grows well on Pseudomonas sp. and E. coli. Where indicated, observations were made while organisms were growing on Pseudomonas sp. or E. coli. In liquid medium the isolate grows optimally at a temperature of 50 ◦ C, with an upper limit of 54 ◦ C. The growth rate on agar plates was measured by the distance covered by the migrating ring of amoebae. On agar plates, the isolate grows at temperatures between 25 ◦ C and 52 ◦ C, but optimally at 50 ◦ C. The amoebae move with eruptive pseudopodia and appear in two forms, triangular shaped (Fig. 1a
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Fig. 1. Oramoeba fumarolia. Phase contrast micrographs. (a and b) Throphozoites, triangular form. (c and d) Throphozoites, elongated form. (e and f) Flagellate form. (a, c, e and f) Nomarski; (b and d) phase contrast. Scale bar is 10 m in all figures.
and b), representing up to 84% of the total, and elongated (Fig. 1c and d). In the liquid medium at 50 ◦ C, the average length of the amoebae is 12.6 m (from 7.9 to 17.7 m) with an average width of 7.0 m (from 4.3 to 14.3 m) when in triangular shape. When elongated, the length of the amoebae averages 14.0 m (from 9.9 to 17.3 m) and their width averages 4.4 m (from 3.1 m to 5.7 m). In the single nucleus a centrally located nucleolus can be observed, but it is not as well delineated as it is in the Valkampfiidae. A uroid with trailing filaments is sometimes observed. In liquid medium, flagellated forms were seen (Fig. 1e and f). The food bacteria had an influence on the percentage of flagellates present in the liquid medium. With Pseudomonas sp., flagellates were the predominant stage. However, with Marinobacter sp. the amoeboid form was the predominant stage. With E. coli as food source both stages were present in equal amounts. The
flagellates have a triangular shape and the flagella are at the base of this triangle, in a pocket or groove, but sometimes flagella can be seen at both ends of the organism. The flagellates are 10–15 m long and have a width of 5 m. The number of flagella per cell varies from two up to 10 (Table 1). When two flagella are present, one flagellum has a length of twice the body size and the other is shorter than the cell body. We were unable to observe cell division in amoebae or flagellates. However, bigger cells with four flagella and two nuclei were observed, which may be an indication that flagellates were in the process of division. The flagellate stage is highly motile and swimming occurs in a spiraling way, as observed in flagellates of Naegleria. However, unlike Naegleria, the cell body makes a rocking movement. When amoebae were grown on agar plates, flagellates were not present, but upon their return to liquid medium, flagellates formed again. This
Table 1. Number of flagella in 100 flagellates of the SAN2 strain grown in SME with Marinobacter sp. as food organism. Number of flagella
2
3
4
5
6
7
8
10
Number of organisms
45
12
18
2
7
6
7
3
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Fig. 2. Oramoeba fumarolia. Cysts. (a and b) Young culture. (c and b) Aging culture. (c and d) Nomarski; (b and d) phase contrast. Arrow indicates double wall. Scale bar is 10 m in all figures. There is only one scale bar in Fig. (b).
demonstrates that the flagellates represent another stage of the SAN2 strain, and are not just a contaminating organism. Cysts are formed in liquid medium, but are more abundant when grown on agar plates with E. coli. The cysts are round to oval, and sometimes an outer wall is observed (Fig. 2a and b). As the cysts age, large vacuoles are formed (Fig. 2c and d). The diameter of the cysts ranges from 4.6 m to 7.7 m (mean 6.0 m). No pores are observed in the cyst wall, which is smooth. The total SSU rDNA of strain SAN2 was sequenced (EBI accession No. FN668558). The SSU rRNA gene sequence amplified from SAN2 is 2905 bp long. This large size is due to a group I intron of 1048 bp located between bp 1501 and 2549. The intron contains the remnants of a gene that in a number of Naegleria strains codes for a putative homing endonuclease of 245 amino acids (De Jonckheere 2002). However, this pseudogene has undergone a number of deletions, one of which resulted in the loss of an expected initiation codon while a number of nucleotides coding for amino acid residues essential for catalytic activity have been mutated. The SSU rDNA sequence, without the group I intron sequence, was aligned with a number of selected Heterolobosea SSU rDNA sequences to which the sequences of Euglena gracilis and Trypanoplasma borreli were added as outgroups. Phylo-
genetic analysis using Bayesian inference places this new marine amoeba SAN2 in the same clade as Stachyamoeba sp., V. italica and WIM43 (Fig. 3) estimated from SSU rRNA gene sequence data. Maximum likelihood, maximum parsimony and distance matrix neighbor joining methods all gave essentially identical tree topologies (not shown). We have also sequenced the ITS, including the 5.8S rDNA. Because of the shorter length compared to the SSU rDNA, this sequence allows a more rapid and easy identification of species. The ITS, including the 5.8S rDNA, of strain SAN2 has a total length of 364 bp (EBI accession No. FN668557).
Discussion The new amoeboflagellate shows characteristics of the Heterolobosea as defined by Page and Blanton (1985). The amoebae move by eruptive pseudopodia and the average length to width ratio is <3. The amoeba forms cysts, which fall within the variation of cyst morphology within this class: round to oval with a double wall sometimes visible, without pores. Only the presence of large vacuoles in aged cysts seems to be unusual. The amoeba does transform into flagellates, as several other heterolobosean genera do, and the number of
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Fig. 3. SSU rRNA gene tree (of the Bayesian analysis) showing the phylogenetic position of Oramoeba fumarolia relative to 29 heterolobosean and two outgroup taxa. Support values for the nodes from Bayesian (105.000 generations) and PhyML (100 bootstrap samplings) analyses are indicated. Accession numbers of each taxon are presented in parentheses. The horizontal bar represents 10 substitutions per 100 nucleotides. “A” denotes species which have only an amoebic stage, “AF” denotes amoeboflagellate species, “F” denotes species which have only a flagellate stage. Marine and halophilic species are boxed.
flagella varies from two up to 10. However, flagellate transformation in the Heterolobosea is a variable characteristic even within genera (De Jonckheere 2002), as is the presence of pores in the cysts (De Jonckheere 2008). Phylogenies of the SSU rDNA place the new isolate within the Heterolobosea, together with Stachyamoeba sp., V. italica and an uncultured heterolobosean clone WIM43. This new amoeba is thermophilic as its optimal growth temperature is 50 ◦ C, but it does grow at up to 54 ◦ C. The maximum growth temperature for the related V. italica is 25 ◦ C (Murase et al. 2010). The maximum growth temperature for the related Stachyamoeba sp. is not known, but at the American Type Culture Collection (ATCC) the growth temperature of the strain is stated at 25 ◦ C, which is actually the lower end for our isolate SAN2. The type strain of Stachyamoeba lipophora was isolated from soil (Page 1988), but no molecular analysis of this isolate was performed. The type culture, held by the Culture Collection of Algae and Protozoa (CCAP), was lost precluding any further phylogenetic investigation. Molecular analysis of the SSU rDNA sequence (AF011461) from a poorly described Stachyamoeba sp. ATCC 50423 from seawater showed that this strain belongs to the Heterolo-
bosea, but it is uncertain whether this ATCC 50423 strain is related to the above type strain of Stachyamoeba. Recently, we also detected flagellate transformation in this ATCC Stachyamoeba strain (Murase et al. 2010), an observation which was not reported for the type strain of Stachyamoeba. The SSU rDNA sequence of SAN2 is also different from an environmental sequence from soil, WIM43, which also clusters with the Stachyamoeba sp. sequence (Moon-van der Staay et al. 2006). As the WIM43 sequence was obtained without culturing the amoeba, a morphological comparison is impossible. However, the SSU rDNA sequence of WIM43 and SAN2 shares only 81% identical residues, and therefore these two taxa do belong to different genera. Our new isolate grows in normal, but also in double strength artificial seawater (72‰). P. flabellatum and T. peronaphora are extreme halophiles, with optimal growth at 300‰ and 200‰ salinity, respectively (Park et al. 2007, 2009). Apart from the hypersaline species mentioned, not many Heterolobosea from seawater have been described, and the phylogenetic position of most of them has not been established. Several Vahlkampfia spp. from seawater have been described but the only one whose phylogenetic position was determined turned out to belong to another genus,
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Table 2. Presence of group I introns in the SSU rDNA of Heterolobosea. Species
Between sites
O. fumarolia N. andersoni ‘P. longifilum’ Pl. flabellatum ‘M. halophila’
1501–2549 648–1958 1256–1748 1486–1873 1563–1941 2157–2516 695–1034 1680–1966 2032–2400 687–1996
A. rosea
Strain BA
Length (in bp) 1048 1309 493 388 379 360 340 287 369 759
Neovahlkampfia (Brown and De Jonckheere 1999). Other marine Vahlkampfia spp., which have not been investigated phylogenetically, such as V. dumnonica (Page 1983), V. caledonica (Anderson et al. 1997) and V. anaerobica (Smirnov and Fenchel 1996), are unlikely candidates for our marine amoeba, because none of these species produces cysts, while the latter is also anaerobic. The marine species Pernina chaumonti has no morphological features in common with our amoeba (El Kadiri et al. 1992). The spherical cysts of P. chaumonti have two distinct envelopes and have pores. Although it is not stated by El Kadiri et al. (1992) at what temperature P. chaumonti grows, it is probably not thermophilic as it was grown in co-culture with an alga, Porphyridium, which is most commonly kept at 18 ◦ C (ATCC 50161). Although another marine strain, Heteramoeba clara (Droop 1962), also transforms into flagellates, it is phylogenetically unrelated to our amoeba (Fig. 3). Only amoebae were seen in M. thermophila (De Jonckheere et al. 2009) and this genus is also phylogenetically unrelated to our new isolate, but related to the hypersaline genera Tulamoeba (Park et al. 2009) and Pleurostomum. Also in the marine M. visvesvarai only amoebae were detected while it does not grow under normal aerobic conditions (O’Kelly et al. 2003). The latter genus is phylogenetically unrelated to our new isolate, but rather related to the freshwater amoeboflagellates P. lanterna and S. marylandensis. Our new amoeba is thermophilic as it does not grow below 25 ◦ C, while its optimal temperature of growth is 50 ◦ C, with an upper limit of 54 ◦ C. P. flabellatum grows at 45 ◦ C while a growth temperature was not reported for T. peronaphora. The maximum temperature of growth for the related genera Willaertia and Naegleria is also 45 ◦ C, but in the genus Naegleria there are also several species which do not grow above 30 ◦ C. In both genera Willaertia and Naegleria, marine species are unknown. While P. flabellatum is a flagellate which does not transform into amoebae, T. peronaphora is an amoeba which does not form flagellates, but our new amoeba has both a flagellate and an amoeboid form. When all of these facts are taken into account we conclude that our isolate belongs to a new genus. The SSU rDNA of SAN2 contains a group I intron. A single group I intron, which functions as a ribozyme, is
ORF (No. AA)
EMBL accession
186 245 – – – – – – – 253
FN668558 Z16417 AF011462 DQ979962 AF011465 AF011465 AF011458 AF011458 AF011458 DQ388519
also found in the SSU rDNA of several Naegleria spp., the SSU rDNAs of Pleurostomum flagellatum, and of ‘Pseudomastigamoeba longifilum’. Acrasis rosea has three group I introns and ‘Macropharyngomonass halophila’ has two group I introns (Table 2). In the Heterolobosea, only the group I intron of the Naegleria spp. and an unnamed environmental isolate (BA) contain a putative endonuclease (De Jonckheere 2002). In strain SAN2, the endonuclease has been reduced to a pseudogene. Alignment of this pseudogene to the endonuclease of N. andersoni shows that only 46 amino acids (25%) can be aligned. The endonuclease of the unnamed environmental heterolobosean isolate BA (DQ388519), related to Allovahlkampfia spelaea (Walochnik and Mulec 2009), has 51 amino acids (28%) in common with the SAN2 pseudogene. The secondary structure of the SAN2 SSU rDNA displays the helix 17-1 insertion, which is typical for the Heterolobosea excluding M. halophila. For rapid identification of future isolates of the species we have sequenced the ITS, including the 5.8S rDNA. It has a total length of 364 bp (EBI accession No. FN668557). Variations in the SSU rDNA sequences have been observed in strains (Brown and De Jonckheere 1999), which have an identical ITS sequence (De Jonckheere and Brown 2005; Baumgartner et al. 2009), but strains with identical ITS sequences have been shown to belong all to the same species (De Jonckheere 2002). Because this new amoeboflagellate was isolated from the sea coast (Latin: ora) near a fumarole and grows at very high temperatures we propose the name Oramoeba fumarolia gen. nov., sp. nov.
Description of a New Genus and New Species: Oramoeba fumarolia gen. nov. sp. nov. Diagnosis of the Genus Oramoeba Marine heterolobosean amoebae moving with eruptive pseudopodia. They have a single nucleus, with a single nucleolus, which is not well delineated. Sometimes an uroid with
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trailing filaments is observed. Flagellate stages are observed with a number of flagella, varying from two up to 10. There is an indication that the flagellates do divide. The cysts are round to oval.
Type Species fumarolia With characters of the genus. The trophozoites average 12.5–13.6 m in length and the width averages 7.0–4.2 m depending on the morphological form, triangular or monopodial. The cyst averages 6.0 m. Sometimes an outer wall is visible, but no pores are present. Aging cysts contain big vacuoles. New isolates can easily be identified by determining the ITS, including the 5.8S rDNA sequences (EBI accession No. FN668557). Type Locality Sediments of the beach near a fumarole at Ischia Island (Italy), west of Naples. Type Material Isolate SAN2 is available at the American Type Culture Collection under accession No. PRA-15.
Acknowledgements We acknowledge the help of Patrick Van Der Smissen (de Duve Institute) and Andrey Tsyganov (University of Antwerp) for obtaining micrographs. We thank Mark Rider (de Duve Institute) for editing the English.
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Walochnik, J., Mulec, J., 2009. Free-living amoebae in carbonate precipitating microhabitats of karst caves and a new vahlkampfiid amoeba. Allovahlkampfia spelaea gen. nov. sp. nov. Acta Protozool. 48, 25–33. Yubuki, N., Leander, B.S., 2008. Ultrastructure and molecular phylogeny of Stephanopogon minuta: an enigmatic microeukaryote from marine interstitial environments. Eur. J. Protistol. 44, 241–253.
Oramoeba fumarolia gen. nov., sp. nov., a new marine heterolobosean amoeboflagellate growing at 54 ◦ C Johan F. De Jonckheerea,b,∗ , Manuela Baumgartnerc , Silvia Eberhardtc , Fred R. Opperdoesa,d , Karl O. Stetterc a
Research Unit for Tropical Diseases, de Duve Institute, B-1200 Brussels, Belgium Scientific Institute of Public Health, B-1050 Brussels, Belgium c Lehrstuhl für Mikrobiologie, Universität Regensburg, D-93053, Germany d Université Catholique de Louvain, B-1200 Brussels, Belgium b
Received 31 March 2010; received in revised form 3 September 2010; accepted 6 September 2010
Abstract An amoeba strain was isolated from marine sediment taken from the beach near a fumarole in Italy. The trophozoites of this new marine species transforms into flagellates with variable numbers of flagella, from 2 to 10. The strain forms round to oval cysts. This thermophilic amoeboflagellate grows at temperatures up to 54 ◦ C. Molecular phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) places the amoeboflagellate in the Heterolobosea. The closest relatives are Stachyamoeba sp. ATCC50324, a strain isolated from an ocean sample, and Vrihiamoeba italica, a recent isolate from a rice field. Like some other heterolobosean species, this new isolate has a group I intron in the SSU rDNA. Because of the unique place in the molecular phylogenetic tree, and because there is no species found in the literature with similar morphological and physiological characteristics, this isolate is considered to be a new genus and a new species, Oramoeba fumarolia gen. nov., sp. nov. © 2010 Elsevier GmbH. All rights reserved. Keywords: Heterolobosea; ITS; New species; SSU rDNA; Thermophilic
Introduction The class Heterolobosea was established for amoebae, which move with eruptive pseudopodia, have intranuclear orthomitosis (promitosis), have mitochondrial cristae which are flattened, often disc-like, and in which stacked Golgi bodies are absent (Page and Blanton 1985). At the time of establishing this class of protists, the Heterolobosea contained amoebae and amoeboflagellates. In the latter there ∗ Corresponding author at: Research Unit for Tropical Diseases, de Duve Institute, B-1200 Brussels, Belgium. Tel.: +32 2 764 74 71; fax: +32 2 7626853. E-mail address: [email protected] (J.F. De Jonckheere).
0932-4739/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2010.09.002
is transformation from an amoeba stage into a temporary flagellate stage, and vice versa. Recently it was found that Percolomonas and Pleurostomum, which were originally placed within the heterotrophic flagellates, and Stephanopogon, originally placed in the Ciliophora, also belong to the Heterolobosea (Nikolaev et al. 2004; Park et al. 2007; Yubuki and Leander 2008; Cavalier-Smith and Nikolaev 2008). These three genera had remained incertae sedis for a long time, but molecular phylogenetic analysis places them within the Heterolobosea, a conclusion which is supported by ultrastructural data. However, in Pleurostomum flabellatum, one of the characteristics of Heterolobosea, mitochondrial cristae, were not observed (Park et al. 2007). In all the Heterolobosea a unique characteristic insertion in the secondary structure (helix 17-1) of the small subunit ribosomal
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DNA (SSU rDNA) is found (Nikolaev et al. 2004), except in ‘Macropharyngomonas halophila’, which has a basal position in the heterolobosean phylogenetic tree. While the majority of heterolobosean species had been found in fresh water habitats and soil (Page 1988), some were isolated from marine (Page 1983) and even hypersaline habitats (Park et al. 2007). We report the isolation of a thermophilic marine amoeboflagellate from a beach near a fumarole. The strain was studied by light microscopy and the SSU rRNA gene sequence analysis shows that it belongs to a new genus in the Heterolobosea.
Materials and Methods The strain under study, SAN2, was isolated from a sediment sample taken in October 2000 in Italy. The sediment, varying in temperature from 55 ◦ C to 70 ◦ C, originated from the beach near a fumarole at San Angelo on the island Ischia (40◦ 43 N, 13◦ 54 E). Ten ml of these sediment samples were incubated at 45, 50 and 55 ◦ C in synthetic seawater BisTris buffered medium (SME-Medium) with 0.1% (v/v) glycerol and 0.05 (w/v) yeast extract. Pseudomonas sp. or Marinobacter sp. (Baumgartner et al. 2002) was added as food source. For growth on agar plates, agar was added to a final concentration of 1.8% to SME-Medium and food bacteria were spread on the plates. The strain was also grown in Page Amoeba Saline (PAS) (Page 1988), supplemented with 3% NaCl, and Escherichia coli as food source. The morphology of moving amoebae attached to glass surfaces was studied at 50 ◦ C using phase contrast optics with a Zeis Axiovert inverse microscope (Baumgartner et al. 2003). Length and breadth dimensions of 100 actively moving trophozoites were determined. The size of 20 cysts was measured after growth on agar plates. The number of flagella was counted on 100 cells. The optimal growth temperature of the isolate was determined in liquid SME-Medium. In addition, agar plates with lawns of Marinobacter sp. were incubated at different temperatures. The diameter of the grazed area was measured at regular intervals (Baumgartner et al. 2003). DNA extraction, amplification of the SSU rRNA gene by PCR, and sequencing were carried out as described previously (Baumgartner et al. 2002). The internal transcribed spacers (ITS), including the 5.8S rDNA, sequences were obtained as described by De Jonckheere and Brown (2005). The SSU rRNA sequence of strain SAN2, omitting 1047 bp of an intron, was placed into an alignment of selected Heterolobosea SSU rRNA sequences obtained from the on-line comprehensive ribosomal RNA database Silva (http://www.arb-silva.de/; Pruesse et al. 2007). The SSU RNA sequence from strain SAN2 was manually aligned to this set. A total of 741 of unambiguously aligned sites common to all sequences was retained for phylogenetic analysis. This alignment is available upon request. Phylogenetic trees were inferred by distance matrix
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neighbor-joining as implemented in ClustalX version 1.8 (Thompson et al. 1997), maximum likelihood (Felsenstein 1981) as implemented by the program PhyML version 2.4.5 (Guindon and Gascuel 2003), and by Bayesian analysis using MrBAYES version 3.1.2 (Huelsenbeck and Ronquist 2001). The general time reversible (GTR) model of evolution (Tavaré 1986) was selected as the best model from 28 using the ModelFind program (http://www.hiv.lanl.gov/ content/sequence/findmodel/findmodel.html). The optimal parameters for the GTR model were estimated using the PhyML program. This model extended with gamma-shaped rate variation (α = 0.89) with four rate categories and a proportion of 0.19 of invariable sites was used for both maximum likelihood (100 bootstrap samplings in PhyML) and Baysian analysis (MrBayes). To estimate Bayesian posterior probabilities, Markov Chain Monte Carlo (MCMC) chains were run for 105,000 generations until convergence and sampled every 100 generations (burn-in: 250 generations). The final dataset for tree construction comprised 29 other Heterlobosea taxa (accession numbers): Tetramitus aberdonicus (AJ224888), T. jugosus (M9805), T. entericus (AJ224889), T. rostratus (M98051), T. lobospinosus (M98052), T. thermacidophilus (AJ621575), Vahlkampfia avara (AJ 22488), V. inornata (AJ22488), Naegleria fowleri (U80059), N. andersoni (U80057), Willaertia magna (X93221, X93223 and AY266315), P. flabellatum (DQ979962), Tulamoeba peronaphora (FJ222603), Acrasis rosea (AF011458), Neovahlkampfia damariscottae (AJ224891), Paravahlkampfia ustiana (AJ224890), Heteramoeba clara (AF011460), ‘Plaesiobystra hypersalinica’ (AF011459), Psalteriomonas lanterna (X94430), Sawyeria marylandensis (AF439351), Monopylocystis visvesvarai (AF011463), Stachyamoeba sp. ATCC 50324 (AF011461), Percolomonas cosmopolitus (AF519443 and AF011464), Stephanopogon minuta (AB365646), ‘M. halophila’ (AF011465), Marinamoeba thermophila (FM244741), Vrihiamoeba italica (AB513360) and the uncultured heterolobosean clone WIM43 (AM114803). The sequences of the Euglenozoa Euglena gracilis (AY029409) and Trypanoplasma borreli (AY028454) were used as outgroups.
Results Most observations were performed on amoebae growing on Marinobacter sp. although the isolate also grows well on Pseudomonas sp. and E. coli. Where indicated, observations were made while organisms were growing on Pseudomonas sp. or E. coli. In liquid medium the isolate grows optimally at a temperature of 50 ◦ C, with an upper limit of 54 ◦ C. The growth rate on agar plates was measured by the distance covered by the migrating ring of amoebae. On agar plates, the isolate grows at temperatures between 25 ◦ C and 52 ◦ C, but optimally at 50 ◦ C. The amoebae move with eruptive pseudopodia and appear in two forms, triangular shaped (Fig. 1a
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Fig. 1. Oramoeba fumarolia. Phase contrast micrographs. (a and b) Throphozoites, triangular form. (c and d) Throphozoites, elongated form. (e and f) Flagellate form. (a, c, e and f) Nomarski; (b and d) phase contrast. Scale bar is 10 m in all figures.
and b), representing up to 84% of the total, and elongated (Fig. 1c and d). In the liquid medium at 50 ◦ C, the average length of the amoebae is 12.6 m (from 7.9 to 17.7 m) with an average width of 7.0 m (from 4.3 to 14.3 m) when in triangular shape. When elongated, the length of the amoebae averages 14.0 m (from 9.9 to 17.3 m) and their width averages 4.4 m (from 3.1 m to 5.7 m). In the single nucleus a centrally located nucleolus can be observed, but it is not as well delineated as it is in the Valkampfiidae. A uroid with trailing filaments is sometimes observed. In liquid medium, flagellated forms were seen (Fig. 1e and f). The food bacteria had an influence on the percentage of flagellates present in the liquid medium. With Pseudomonas sp., flagellates were the predominant stage. However, with Marinobacter sp. the amoeboid form was the predominant stage. With E. coli as food source both stages were present in equal amounts. The
flagellates have a triangular shape and the flagella are at the base of this triangle, in a pocket or groove, but sometimes flagella can be seen at both ends of the organism. The flagellates are 10–15 m long and have a width of 5 m. The number of flagella per cell varies from two up to 10 (Table 1). When two flagella are present, one flagellum has a length of twice the body size and the other is shorter than the cell body. We were unable to observe cell division in amoebae or flagellates. However, bigger cells with four flagella and two nuclei were observed, which may be an indication that flagellates were in the process of division. The flagellate stage is highly motile and swimming occurs in a spiraling way, as observed in flagellates of Naegleria. However, unlike Naegleria, the cell body makes a rocking movement. When amoebae were grown on agar plates, flagellates were not present, but upon their return to liquid medium, flagellates formed again. This
Table 1. Number of flagella in 100 flagellates of the SAN2 strain grown in SME with Marinobacter sp. as food organism. Number of flagella
2
3
4
5
6
7
8
10
Number of organisms
45
12
18
2
7
6
7
3
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Fig. 2. Oramoeba fumarolia. Cysts. (a and b) Young culture. (c and b) Aging culture. (c and d) Nomarski; (b and d) phase contrast. Arrow indicates double wall. Scale bar is 10 m in all figures. There is only one scale bar in Fig. (b).
demonstrates that the flagellates represent another stage of the SAN2 strain, and are not just a contaminating organism. Cysts are formed in liquid medium, but are more abundant when grown on agar plates with E. coli. The cysts are round to oval, and sometimes an outer wall is observed (Fig. 2a and b). As the cysts age, large vacuoles are formed (Fig. 2c and d). The diameter of the cysts ranges from 4.6 m to 7.7 m (mean 6.0 m). No pores are observed in the cyst wall, which is smooth. The total SSU rDNA of strain SAN2 was sequenced (EBI accession No. FN668558). The SSU rRNA gene sequence amplified from SAN2 is 2905 bp long. This large size is due to a group I intron of 1048 bp located between bp 1501 and 2549. The intron contains the remnants of a gene that in a number of Naegleria strains codes for a putative homing endonuclease of 245 amino acids (De Jonckheere 2002). However, this pseudogene has undergone a number of deletions, one of which resulted in the loss of an expected initiation codon while a number of nucleotides coding for amino acid residues essential for catalytic activity have been mutated. The SSU rDNA sequence, without the group I intron sequence, was aligned with a number of selected Heterolobosea SSU rDNA sequences to which the sequences of Euglena gracilis and Trypanoplasma borreli were added as outgroups. Phylo-
genetic analysis using Bayesian inference places this new marine amoeba SAN2 in the same clade as Stachyamoeba sp., V. italica and WIM43 (Fig. 3) estimated from SSU rRNA gene sequence data. Maximum likelihood, maximum parsimony and distance matrix neighbor joining methods all gave essentially identical tree topologies (not shown). We have also sequenced the ITS, including the 5.8S rDNA. Because of the shorter length compared to the SSU rDNA, this sequence allows a more rapid and easy identification of species. The ITS, including the 5.8S rDNA, of strain SAN2 has a total length of 364 bp (EBI accession No. FN668557).
Discussion The new amoeboflagellate shows characteristics of the Heterolobosea as defined by Page and Blanton (1985). The amoebae move by eruptive pseudopodia and the average length to width ratio is <3. The amoeba forms cysts, which fall within the variation of cyst morphology within this class: round to oval with a double wall sometimes visible, without pores. Only the presence of large vacuoles in aged cysts seems to be unusual. The amoeba does transform into flagellates, as several other heterolobosean genera do, and the number of
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Fig. 3. SSU rRNA gene tree (of the Bayesian analysis) showing the phylogenetic position of Oramoeba fumarolia relative to 29 heterolobosean and two outgroup taxa. Support values for the nodes from Bayesian (105.000 generations) and PhyML (100 bootstrap samplings) analyses are indicated. Accession numbers of each taxon are presented in parentheses. The horizontal bar represents 10 substitutions per 100 nucleotides. “A” denotes species which have only an amoebic stage, “AF” denotes amoeboflagellate species, “F” denotes species which have only a flagellate stage. Marine and halophilic species are boxed.
flagella varies from two up to 10. However, flagellate transformation in the Heterolobosea is a variable characteristic even within genera (De Jonckheere 2002), as is the presence of pores in the cysts (De Jonckheere 2008). Phylogenies of the SSU rDNA place the new isolate within the Heterolobosea, together with Stachyamoeba sp., V. italica and an uncultured heterolobosean clone WIM43. This new amoeba is thermophilic as its optimal growth temperature is 50 ◦ C, but it does grow at up to 54 ◦ C. The maximum growth temperature for the related V. italica is 25 ◦ C (Murase et al. 2010). The maximum growth temperature for the related Stachyamoeba sp. is not known, but at the American Type Culture Collection (ATCC) the growth temperature of the strain is stated at 25 ◦ C, which is actually the lower end for our isolate SAN2. The type strain of Stachyamoeba lipophora was isolated from soil (Page 1988), but no molecular analysis of this isolate was performed. The type culture, held by the Culture Collection of Algae and Protozoa (CCAP), was lost precluding any further phylogenetic investigation. Molecular analysis of the SSU rDNA sequence (AF011461) from a poorly described Stachyamoeba sp. ATCC 50423 from seawater showed that this strain belongs to the Heterolo-
bosea, but it is uncertain whether this ATCC 50423 strain is related to the above type strain of Stachyamoeba. Recently, we also detected flagellate transformation in this ATCC Stachyamoeba strain (Murase et al. 2010), an observation which was not reported for the type strain of Stachyamoeba. The SSU rDNA sequence of SAN2 is also different from an environmental sequence from soil, WIM43, which also clusters with the Stachyamoeba sp. sequence (Moon-van der Staay et al. 2006). As the WIM43 sequence was obtained without culturing the amoeba, a morphological comparison is impossible. However, the SSU rDNA sequence of WIM43 and SAN2 shares only 81% identical residues, and therefore these two taxa do belong to different genera. Our new isolate grows in normal, but also in double strength artificial seawater (72‰). P. flabellatum and T. peronaphora are extreme halophiles, with optimal growth at 300‰ and 200‰ salinity, respectively (Park et al. 2007, 2009). Apart from the hypersaline species mentioned, not many Heterolobosea from seawater have been described, and the phylogenetic position of most of them has not been established. Several Vahlkampfia spp. from seawater have been described but the only one whose phylogenetic position was determined turned out to belong to another genus,
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Table 2. Presence of group I introns in the SSU rDNA of Heterolobosea. Species
Between sites
O. fumarolia N. andersoni ‘P. longifilum’ Pl. flabellatum ‘M. halophila’
1501–2549 648–1958 1256–1748 1486–1873 1563–1941 2157–2516 695–1034 1680–1966 2032–2400 687–1996
A. rosea
Strain BA
Length (in bp) 1048 1309 493 388 379 360 340 287 369 759
Neovahlkampfia (Brown and De Jonckheere 1999). Other marine Vahlkampfia spp., which have not been investigated phylogenetically, such as V. dumnonica (Page 1983), V. caledonica (Anderson et al. 1997) and V. anaerobica (Smirnov and Fenchel 1996), are unlikely candidates for our marine amoeba, because none of these species produces cysts, while the latter is also anaerobic. The marine species Pernina chaumonti has no morphological features in common with our amoeba (El Kadiri et al. 1992). The spherical cysts of P. chaumonti have two distinct envelopes and have pores. Although it is not stated by El Kadiri et al. (1992) at what temperature P. chaumonti grows, it is probably not thermophilic as it was grown in co-culture with an alga, Porphyridium, which is most commonly kept at 18 ◦ C (ATCC 50161). Although another marine strain, Heteramoeba clara (Droop 1962), also transforms into flagellates, it is phylogenetically unrelated to our amoeba (Fig. 3). Only amoebae were seen in M. thermophila (De Jonckheere et al. 2009) and this genus is also phylogenetically unrelated to our new isolate, but related to the hypersaline genera Tulamoeba (Park et al. 2009) and Pleurostomum. Also in the marine M. visvesvarai only amoebae were detected while it does not grow under normal aerobic conditions (O’Kelly et al. 2003). The latter genus is phylogenetically unrelated to our new isolate, but rather related to the freshwater amoeboflagellates P. lanterna and S. marylandensis. Our new amoeba is thermophilic as it does not grow below 25 ◦ C, while its optimal temperature of growth is 50 ◦ C, with an upper limit of 54 ◦ C. P. flabellatum grows at 45 ◦ C while a growth temperature was not reported for T. peronaphora. The maximum temperature of growth for the related genera Willaertia and Naegleria is also 45 ◦ C, but in the genus Naegleria there are also several species which do not grow above 30 ◦ C. In both genera Willaertia and Naegleria, marine species are unknown. While P. flabellatum is a flagellate which does not transform into amoebae, T. peronaphora is an amoeba which does not form flagellates, but our new amoeba has both a flagellate and an amoeboid form. When all of these facts are taken into account we conclude that our isolate belongs to a new genus. The SSU rDNA of SAN2 contains a group I intron. A single group I intron, which functions as a ribozyme, is
ORF (No. AA)
EMBL accession
186 245 – – – – – – – 253
FN668558 Z16417 AF011462 DQ979962 AF011465 AF011465 AF011458 AF011458 AF011458 DQ388519
also found in the SSU rDNA of several Naegleria spp., the SSU rDNAs of Pleurostomum flagellatum, and of ‘Pseudomastigamoeba longifilum’. Acrasis rosea has three group I introns and ‘Macropharyngomonass halophila’ has two group I introns (Table 2). In the Heterolobosea, only the group I intron of the Naegleria spp. and an unnamed environmental isolate (BA) contain a putative endonuclease (De Jonckheere 2002). In strain SAN2, the endonuclease has been reduced to a pseudogene. Alignment of this pseudogene to the endonuclease of N. andersoni shows that only 46 amino acids (25%) can be aligned. The endonuclease of the unnamed environmental heterolobosean isolate BA (DQ388519), related to Allovahlkampfia spelaea (Walochnik and Mulec 2009), has 51 amino acids (28%) in common with the SAN2 pseudogene. The secondary structure of the SAN2 SSU rDNA displays the helix 17-1 insertion, which is typical for the Heterolobosea excluding M. halophila. For rapid identification of future isolates of the species we have sequenced the ITS, including the 5.8S rDNA. It has a total length of 364 bp (EBI accession No. FN668557). Variations in the SSU rDNA sequences have been observed in strains (Brown and De Jonckheere 1999), which have an identical ITS sequence (De Jonckheere and Brown 2005; Baumgartner et al. 2009), but strains with identical ITS sequences have been shown to belong all to the same species (De Jonckheere 2002). Because this new amoeboflagellate was isolated from the sea coast (Latin: ora) near a fumarole and grows at very high temperatures we propose the name Oramoeba fumarolia gen. nov., sp. nov.
Description of a New Genus and New Species: Oramoeba fumarolia gen. nov. sp. nov. Diagnosis of the Genus Oramoeba Marine heterolobosean amoebae moving with eruptive pseudopodia. They have a single nucleus, with a single nucleolus, which is not well delineated. Sometimes an uroid with
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trailing filaments is observed. Flagellate stages are observed with a number of flagella, varying from two up to 10. There is an indication that the flagellates do divide. The cysts are round to oval.
Type Species fumarolia With characters of the genus. The trophozoites average 12.5–13.6 m in length and the width averages 7.0–4.2 m depending on the morphological form, triangular or monopodial. The cyst averages 6.0 m. Sometimes an outer wall is visible, but no pores are present. Aging cysts contain big vacuoles. New isolates can easily be identified by determining the ITS, including the 5.8S rDNA sequences (EBI accession No. FN668557). Type Locality Sediments of the beach near a fumarole at Ischia Island (Italy), west of Naples. Type Material Isolate SAN2 is available at the American Type Culture Collection under accession No. PRA-15.
Acknowledgements We acknowledge the help of Patrick Van Der Smissen (de Duve Institute) and Andrey Tsyganov (University of Antwerp) for obtaining micrographs. We thank Mark Rider (de Duve Institute) for editing the English.
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