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The phylogenetic status of Sarcobium lyticum, an obligate intracellular bacterial parasite of small amoebae
FEMS MicrobiologyLetters 96 119921199-2112 © 1992 Federation oi European MicrobiologicalSocieties0378-1097/92/$115.(1~1 Published by Elsevier
199
FEMSLE 05043
The phylogenetic status of Sarcobium lyticum, an obligate intracellular bacterial parasite of small amoebae N i n a S p r i n g e r a, W o l f g a n g Ludwig a, W i n c e n t y Dro~afiski b, R u d o l f A m a n n a a n d Karl H e i n z S c h l e i f e r a a Lehrstuhl J~r Mikrobiologie. Technische Unil'ersitiit Miinchen, Miinchen, FRG. and h Department of General Microbiology. Maria-Ct~rie-Sklodowska Unil'ersity, Lublin, Poland
Received 23 June 1992 Accepted 24 June 1992
Key words: lntracellular bacterial oarasite; Sarcobium lyticum; Legionella; 16S rRNA; Phylogeny; Identification, in situ
1. S U M M A R Y
2. I N T R O D U C T I O N
A 16S r R N A gene of the obligate intraceUular bacterial parasite Sarcobium lyticum was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified D N A was cloned, sequenced and compared with other bacterial 16S r R N A sequences. The analysis revealed that S. lyticum belongs to the gamma subclass of the Proteobacteria and shows the closest relationship to an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia. S. lyticum could be detected in situ with a fluorescent oligonucleotide probe by whole cell hybridization.
An obligate intracellular bacterial parasite was found in various small free-living amoebae isolated from soil and water reservoirs [2-4]. The parasites enter the host by phagocytosis and survive in phagosomes since they are resistant to the bacteriolytic enzymes of the host [5,6]. Living bacteria can escape from phagosomes into the cytoplasm where they can grow and finally cause lysis of the host. The bacterial parasite cannot be cultivated on any artificial medium. Living amoeba cells are necessary for the growth of the bacterium. However, an axenic propagation of the parasite on Acanthamoeba castellani is possible [7]. Therefore, the chemical composition of the cell wall and the G + C content of the D N A could be determined [8,9]. Based on these and other, mostly morphological, data the parasite was described as Sarcobiurn lyticum [9]. Since nothing is known about the phylogenetic alloca-
Correspondence to: K.H. Schleifer, Lehrstuhl fiir Mikrobiologie, Technische Universit~it Miinchen, Arcisstr. 21, D-8000 Miinchen 2, FRG.
200 tion of S. lyticum, we decided to determine the 16S r R N A sequence and identify individual cells in situ by using a specific, fluorescent oligonucleotide probe.
3. M A T E R I A L S A N D M E T H O D S The type strain of S. lyticum, which has been deposited in a two-member culture with the Polish Culture Collection of Microorganisms as culture PCM 2298, was propagated in A. castellani as previously described [9]. D N A was isolated from freeze-dried cells according to the method of Wisotzkey et al. [10]. r D N A homologous to Escherichia coil 16S r R N A positions 7 to 1542 were amplified in vitro using the polymerase chain reaction as previously described [11,12]. The amplified 16S r D N A was cloned in the vector pBluescript (Stratagene, La Jolla, USA) and E. coil J M 83. The cloned D N A was sequenced applying the chain termination method [12] in combination with site specific primers. The sequence data have been submitted to E M B L and assigned the accession n u m b e r X 66835. The sequence was aligned with about 800 bacterial 16S r R N A sequences. Synthesis of tetramethylrhodamine labelled oligonucleotide and in situ detection of whole cells were carried out as previously described [11].
Phylogenetic distances [13] were calculated using the progam S E Q D I S (Struckman and Ludwig, unpublished). Distance matrix trees were reconstructed using the neighbour-joining method of Saitou and Nei [14] as implementor in the program N E I G H B O R of the D H Y C I P program [15]. Parsimony and bootstrapped parsimony analyses were performed applying the program DNAPARS and D N A B O O T of the gene program package.
4. R E S U L T S A N D D I S C U S S I O N D N A was extracted from lyophilized cells of S. tyticum grown in A, castellani. Using a primer
pair specific for bacterial 16S r R N A genes, an almost complete gene was amplified in vitro by the PCR technique. The amplified material was cloned and sequenced. The sequence was compared with 800 16S r R N A sequences of bacteria. Calculated similarity values revealed that the newly obtained 16S r R N A sequence is highly similar (99%) to that of the recently described intracellular Legionella species LLAP-3 recovered by amoeb~,l enrichment from the sputum of a patient with pneumonia [1]. Overall similarity values of 16S r R N A sequences from S. lyticum and members of the Legioneilaceae are shown in Table 1. To prove that the amplified and cloned
Table 1 Overall 16S rRNA sequence similarities (%) of Sarcobiun: lytieum (S.), legionella (L.) anti Eseherichia coil (E.). t subsp, fraseri Los Angeles 1:2 subsp, fraseri Dallas E2; 3 subsp, pneumophila Knoxville: 4 subsp, pneumophila Philadelphia. S. lyacum
LLAP3 L. Iongbeachae L. bozemanii L. pneumophila K L. pneumophila 2 L, pneumophila ~ L. pneumophila 4 L. micdadei
L. hackeliae L. ~piritensis L. epythra E. coil
LLAP 99.0
L.lo 94.4 94.9
L.bo 95.4 95,8 97,2
L.pl 94.9 95.2 96.5 97.0
L.p2 94.7 94.9 96.4 96.5 99.6
L.p3 95.5 95.7 96.9 96.9 99.4 99.1
L.p4 95.5 95.7 96.9 97. I 99.3 99. I 99.9
L.mi 91.6 92.2 92.5 93.1 93.2 93.1 93.7 93.9
L.ha 93.5 93.9 94.9 95.3 95.1 95.0 95.3 95.4 93.6
L.sp 93.3 93.8 95.1 94.8 95.4 95.2 95.8 95.8 93.3 96.3
L.er 93.8 94,0 94.8 95.3 95.2 95.1 95.2 95.4 93.8 95.5 96.3
E.co 82.8 83.0 83.7 84.2 83.5 83.0 83.9 83.9 81.4 83.9 83.8 83.8
sequence originated from S. lyticum and was not an artefact, we synthesized the oligonucleotide Siy23aX ' 5 - X A C T A C C C T C T C C C A T A C T - 3 ' that is complementary to a characteristic region of the corresponding 16S rRNA. T h e oligonucleotide was labelled with tetramethylrhodamine and used for in situ hybridizations [16,17]. Epifluorescence microscopy with a tetramethyirho~amine-specific filter set showed a specific reactk*n with S. lyticum (Fig. 1). A d d e d cells of E. ccJi did not react with the specific probe but could be detected with a bacterial probe (Fig. 1). A distance matrix tree reflecting the affiliation of S. lyticum with members of the genus Legionella is depicted in Fig. 2. T h e close relationship of S. lyticum to legionellae is an interesting finding since it is known that free-living amoebae can support the intracellular replication of Legiorella pneumophila [18,19] and can even be used to isolate legionellae from clinical and environmental material [20,21]. The very close relationship of S. lyticum to strain LLAP-3, that could be enriched in A. polyphaga from the sputum of a patient with persistent p n e u m o n i a but cannot be cultured on standard media for legionellae, is of special interest. Based on the comparative 16S r R N A analysis S. lyticum is closely related to the genus Legionella. The almost identical sequence of 16S r R N A genes of S. lyric,ira and strain LLAP-3 indicates that they may even belong to the same species within the genus Legionella. S. lyticum infects axenic cultures of amoebae at temperatures ranging from 20 to 35°C. It does not grow in mouse flbroblasts but preliminary data indicate that it may replicate at low levels in the h u m a n monocyte line U 937 (B.S. Fields, Center for Infectious Diseases, Atlanta, USA, unpublished observations). In contrast to all o t h e r bac-
Fig. 1. In situ detection of Sarcobhlm lyticum in a mixture of infected Acantamoeba castellani and Escherichiacoli. Bar, 20 /~m. Samples were simultaneously hybridized with the bacterial probe [221labelled with fluorescein and the probe specific for S. lyticum labelled with tetramethylrhodamine. Identical microscopic fields are shown. Phase contrast (A) and epifluoresence micrographs using fluoresceia- (B) and rhodaminespecific (C) filter sets (Zeiss, Oberkochen, FRG).
202 REFERENCES
L. mh'dad¢i L ha,.k,,ii,,,. I..spiritcnsis
/
S. lyticum
I ] I / I t '~3 L. b,ngheachae
|
\11//L,,:,
....... ,,
/ E. coli Fig. 2. Distance matrix tree showing the intrageneric phylogeneic relationships of Legionella[I.23]. The 16S rRNA of E. coli was used as an out-group sequence. All positions shich were determined unambiguously for all reference sequences we~'e included for the calculation of phylogenetic distances. Bar. 0.05 phylogenetic distance [13].
teria that can thrive in a m o e b a e , S. lyticum d o e s not provide benefit to the host but c a u s e s its death. Currently it is not k n o w n if it is also h a r m f u l to h i g h e r animals.
ACKNOWLEDGEMENT This w o r k was s u p p o r t e d by a g r a n t f r o m the E u r o p e a n E c o n o m i c C o m m u n i t y No. B I O T cr91-0294.
[1] Fry, N.K., Rowbotham, T.J., Saunders, N.A. and Embley, T.M. (1991) FEMS Microbiol. Lett. 83, 165-168. [2] Dro~afiski. W. (1956) Acta Microbiol. Pol. 5. 315-317. [3] Dro2afiski, W. (1963) Acta Microbiol. Pol. 12, 3-8. [4l Dro~afiski, W. (1965) Abstr. Second Int. Conf. Protozoply, pp. 254-255, London. [5] Dro~afiski, W. and Chmielewski, I'. (1979) Aeta Microbiol. Pol. 28, 123-133. In] DroSafiski, W., Madra, L., Chmielessski, T., Schlecht, S. and Golecki, J.R. (1990) Syst. Appl. Microbiol. 13. 220226. [7] Schlecht. S. and Dro2afiski, W. (1987) Syst. Appl. Microbiol. 10, 92-97. [8] Dro~afiski. W., Dro~afiska, D. and Wicifiska (1984) Aeta Microbiol. Pol. 33, 195-196. [9] Dro~afiski, W. (1991) Int. J. Syst. Bacteriol. 41, 82-87. [10] Wisotzkey, J.D.. Jurtshuk, Jr., P. and Fox, G.E. (1990) Curr. Microbiol. 21. 325-327. [I 1] Amann, R.I., Springer, N., Ludwig, W., G6rtz, H.-D. and Schleifer, K.H. (1991) Nature 351. 161-164. [12] Ludwig. W., Kirchhof, G., Weizenegger, M, and Weiss, N. (1992)J. Int. Syst. Bacteriol. 42. 161-165. [13] Jukes, T.H. and Cantor, C.R. (1969) In: Mammalian Protein Metabolism {Munro, H.N., Ed.), pp. 21-131. Academic Press, N.Y. [14] Saitou, N. and Nei, M. (1987) Mol. Biol. Evol. 4, 406-425. [15] Felsenstein, J. (1982) Q. Rev. Biol, 57, 379-404. [16] DeLong, E.F., Wickham. G.S. and Pace, N.R. (1989) Science 243, 1360-1363. [17] Amann, R.I., Krumholz, L. and Stahl, D.A. (1990) J. Bacteriol. 172, 762-770. [18] Kurtz, J.B., Barthlett, C.L.R., Newton, U.H., White, R.A. and Jones, W.L. (1982) J. Hyg. 88. 369-381. [19] Barbaree, J.M., Fields, B.S.. Feeley, J.C.. Gormau, G.W. and Martin, W.T. (1986) Appl. Environ. Microbiol. 51. 422-424. [20] Rowbotham, T.J. (1983)J. Clin. Pathol. 36, 978-986. [21] Fallon, R.J. and Rowbotham, T.J. (1990) J. Clin. Pathol. 43, 479-483. [22] Amann, R.I., Binder, B.J., OIson, R.J., Chisholm, S.W., Devereax, R. and Stahl, D.A. (1990) Appl. Environ. Microbiol. 56, 1919-1926. [23] Fry, N.K., Warwick. S.. Saunders, N.A. and Embley, T.M. (1991) J. Gen. Microbiol. 137, 1215-|222.
199
FEMSLE 05043
The phylogenetic status of Sarcobium lyticum, an obligate intracellular bacterial parasite of small amoebae N i n a S p r i n g e r a, W o l f g a n g Ludwig a, W i n c e n t y Dro~afiski b, R u d o l f A m a n n a a n d Karl H e i n z S c h l e i f e r a a Lehrstuhl J~r Mikrobiologie. Technische Unil'ersitiit Miinchen, Miinchen, FRG. and h Department of General Microbiology. Maria-Ct~rie-Sklodowska Unil'ersity, Lublin, Poland
Received 23 June 1992 Accepted 24 June 1992
Key words: lntracellular bacterial oarasite; Sarcobium lyticum; Legionella; 16S rRNA; Phylogeny; Identification, in situ
1. S U M M A R Y
2. I N T R O D U C T I O N
A 16S r R N A gene of the obligate intraceUular bacterial parasite Sarcobium lyticum was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified D N A was cloned, sequenced and compared with other bacterial 16S r R N A sequences. The analysis revealed that S. lyticum belongs to the gamma subclass of the Proteobacteria and shows the closest relationship to an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia. S. lyticum could be detected in situ with a fluorescent oligonucleotide probe by whole cell hybridization.
An obligate intracellular bacterial parasite was found in various small free-living amoebae isolated from soil and water reservoirs [2-4]. The parasites enter the host by phagocytosis and survive in phagosomes since they are resistant to the bacteriolytic enzymes of the host [5,6]. Living bacteria can escape from phagosomes into the cytoplasm where they can grow and finally cause lysis of the host. The bacterial parasite cannot be cultivated on any artificial medium. Living amoeba cells are necessary for the growth of the bacterium. However, an axenic propagation of the parasite on Acanthamoeba castellani is possible [7]. Therefore, the chemical composition of the cell wall and the G + C content of the D N A could be determined [8,9]. Based on these and other, mostly morphological, data the parasite was described as Sarcobiurn lyticum [9]. Since nothing is known about the phylogenetic alloca-
Correspondence to: K.H. Schleifer, Lehrstuhl fiir Mikrobiologie, Technische Universit~it Miinchen, Arcisstr. 21, D-8000 Miinchen 2, FRG.
200 tion of S. lyticum, we decided to determine the 16S r R N A sequence and identify individual cells in situ by using a specific, fluorescent oligonucleotide probe.
3. M A T E R I A L S A N D M E T H O D S The type strain of S. lyticum, which has been deposited in a two-member culture with the Polish Culture Collection of Microorganisms as culture PCM 2298, was propagated in A. castellani as previously described [9]. D N A was isolated from freeze-dried cells according to the method of Wisotzkey et al. [10]. r D N A homologous to Escherichia coil 16S r R N A positions 7 to 1542 were amplified in vitro using the polymerase chain reaction as previously described [11,12]. The amplified 16S r D N A was cloned in the vector pBluescript (Stratagene, La Jolla, USA) and E. coil J M 83. The cloned D N A was sequenced applying the chain termination method [12] in combination with site specific primers. The sequence data have been submitted to E M B L and assigned the accession n u m b e r X 66835. The sequence was aligned with about 800 bacterial 16S r R N A sequences. Synthesis of tetramethylrhodamine labelled oligonucleotide and in situ detection of whole cells were carried out as previously described [11].
Phylogenetic distances [13] were calculated using the progam S E Q D I S (Struckman and Ludwig, unpublished). Distance matrix trees were reconstructed using the neighbour-joining method of Saitou and Nei [14] as implementor in the program N E I G H B O R of the D H Y C I P program [15]. Parsimony and bootstrapped parsimony analyses were performed applying the program DNAPARS and D N A B O O T of the gene program package.
4. R E S U L T S A N D D I S C U S S I O N D N A was extracted from lyophilized cells of S. tyticum grown in A, castellani. Using a primer
pair specific for bacterial 16S r R N A genes, an almost complete gene was amplified in vitro by the PCR technique. The amplified material was cloned and sequenced. The sequence was compared with 800 16S r R N A sequences of bacteria. Calculated similarity values revealed that the newly obtained 16S r R N A sequence is highly similar (99%) to that of the recently described intracellular Legionella species LLAP-3 recovered by amoeb~,l enrichment from the sputum of a patient with pneumonia [1]. Overall similarity values of 16S r R N A sequences from S. lyticum and members of the Legioneilaceae are shown in Table 1. To prove that the amplified and cloned
Table 1 Overall 16S rRNA sequence similarities (%) of Sarcobiun: lytieum (S.), legionella (L.) anti Eseherichia coil (E.). t subsp, fraseri Los Angeles 1:2 subsp, fraseri Dallas E2; 3 subsp, pneumophila Knoxville: 4 subsp, pneumophila Philadelphia. S. lyacum
LLAP3 L. Iongbeachae L. bozemanii L. pneumophila K L. pneumophila 2 L, pneumophila ~ L. pneumophila 4 L. micdadei
L. hackeliae L. ~piritensis L. epythra E. coil
LLAP 99.0
L.lo 94.4 94.9
L.bo 95.4 95,8 97,2
L.pl 94.9 95.2 96.5 97.0
L.p2 94.7 94.9 96.4 96.5 99.6
L.p3 95.5 95.7 96.9 96.9 99.4 99.1
L.p4 95.5 95.7 96.9 97. I 99.3 99. I 99.9
L.mi 91.6 92.2 92.5 93.1 93.2 93.1 93.7 93.9
L.ha 93.5 93.9 94.9 95.3 95.1 95.0 95.3 95.4 93.6
L.sp 93.3 93.8 95.1 94.8 95.4 95.2 95.8 95.8 93.3 96.3
L.er 93.8 94,0 94.8 95.3 95.2 95.1 95.2 95.4 93.8 95.5 96.3
E.co 82.8 83.0 83.7 84.2 83.5 83.0 83.9 83.9 81.4 83.9 83.8 83.8
sequence originated from S. lyticum and was not an artefact, we synthesized the oligonucleotide Siy23aX ' 5 - X A C T A C C C T C T C C C A T A C T - 3 ' that is complementary to a characteristic region of the corresponding 16S rRNA. T h e oligonucleotide was labelled with tetramethylrhodamine and used for in situ hybridizations [16,17]. Epifluorescence microscopy with a tetramethyirho~amine-specific filter set showed a specific reactk*n with S. lyticum (Fig. 1). A d d e d cells of E. ccJi did not react with the specific probe but could be detected with a bacterial probe (Fig. 1). A distance matrix tree reflecting the affiliation of S. lyticum with members of the genus Legionella is depicted in Fig. 2. T h e close relationship of S. lyticum to legionellae is an interesting finding since it is known that free-living amoebae can support the intracellular replication of Legiorella pneumophila [18,19] and can even be used to isolate legionellae from clinical and environmental material [20,21]. The very close relationship of S. lyticum to strain LLAP-3, that could be enriched in A. polyphaga from the sputum of a patient with persistent p n e u m o n i a but cannot be cultured on standard media for legionellae, is of special interest. Based on the comparative 16S r R N A analysis S. lyticum is closely related to the genus Legionella. The almost identical sequence of 16S r R N A genes of S. lyric,ira and strain LLAP-3 indicates that they may even belong to the same species within the genus Legionella. S. lyticum infects axenic cultures of amoebae at temperatures ranging from 20 to 35°C. It does not grow in mouse flbroblasts but preliminary data indicate that it may replicate at low levels in the h u m a n monocyte line U 937 (B.S. Fields, Center for Infectious Diseases, Atlanta, USA, unpublished observations). In contrast to all o t h e r bac-
Fig. 1. In situ detection of Sarcobhlm lyticum in a mixture of infected Acantamoeba castellani and Escherichiacoli. Bar, 20 /~m. Samples were simultaneously hybridized with the bacterial probe [221labelled with fluorescein and the probe specific for S. lyticum labelled with tetramethylrhodamine. Identical microscopic fields are shown. Phase contrast (A) and epifluoresence micrographs using fluoresceia- (B) and rhodaminespecific (C) filter sets (Zeiss, Oberkochen, FRG).
202 REFERENCES
L. mh'dad¢i L ha,.k,,ii,,,. I..spiritcnsis
/
S. lyticum
I ] I / I t '~3 L. b,ngheachae
|
\11//L,,:,
....... ,,
/ E. coli Fig. 2. Distance matrix tree showing the intrageneric phylogeneic relationships of Legionella[I.23]. The 16S rRNA of E. coli was used as an out-group sequence. All positions shich were determined unambiguously for all reference sequences we~'e included for the calculation of phylogenetic distances. Bar. 0.05 phylogenetic distance [13].
teria that can thrive in a m o e b a e , S. lyticum d o e s not provide benefit to the host but c a u s e s its death. Currently it is not k n o w n if it is also h a r m f u l to h i g h e r animals.
ACKNOWLEDGEMENT This w o r k was s u p p o r t e d by a g r a n t f r o m the E u r o p e a n E c o n o m i c C o m m u n i t y No. B I O T cr91-0294.
[1] Fry, N.K., Rowbotham, T.J., Saunders, N.A. and Embley, T.M. (1991) FEMS Microbiol. Lett. 83, 165-168. [2] Dro~afiski. W. (1956) Acta Microbiol. Pol. 5. 315-317. [3] Dro2afiski, W. (1963) Acta Microbiol. Pol. 12, 3-8. [4l Dro~afiski, W. (1965) Abstr. Second Int. Conf. Protozoply, pp. 254-255, London. [5] Dro~afiski, W. and Chmielewski, I'. (1979) Aeta Microbiol. Pol. 28, 123-133. In] DroSafiski, W., Madra, L., Chmielessski, T., Schlecht, S. and Golecki, J.R. (1990) Syst. Appl. Microbiol. 13. 220226. [7] Schlecht. S. and Dro2afiski, W. (1987) Syst. Appl. Microbiol. 10, 92-97. [8] Dro~afiski. W., Dro~afiska, D. and Wicifiska (1984) Aeta Microbiol. Pol. 33, 195-196. [9] Dro~afiski, W. (1991) Int. J. Syst. Bacteriol. 41, 82-87. [10] Wisotzkey, J.D.. Jurtshuk, Jr., P. and Fox, G.E. (1990) Curr. Microbiol. 21. 325-327. [I 1] Amann, R.I., Springer, N., Ludwig, W., G6rtz, H.-D. and Schleifer, K.H. (1991) Nature 351. 161-164. [12] Ludwig. W., Kirchhof, G., Weizenegger, M, and Weiss, N. (1992)J. Int. Syst. Bacteriol. 42. 161-165. [13] Jukes, T.H. and Cantor, C.R. (1969) In: Mammalian Protein Metabolism {Munro, H.N., Ed.), pp. 21-131. Academic Press, N.Y. [14] Saitou, N. and Nei, M. (1987) Mol. Biol. Evol. 4, 406-425. [15] Felsenstein, J. (1982) Q. Rev. Biol, 57, 379-404. [16] DeLong, E.F., Wickham. G.S. and Pace, N.R. (1989) Science 243, 1360-1363. [17] Amann, R.I., Krumholz, L. and Stahl, D.A. (1990) J. Bacteriol. 172, 762-770. [18] Kurtz, J.B., Barthlett, C.L.R., Newton, U.H., White, R.A. and Jones, W.L. (1982) J. Hyg. 88. 369-381. [19] Barbaree, J.M., Fields, B.S.. Feeley, J.C.. Gormau, G.W. and Martin, W.T. (1986) Appl. Environ. Microbiol. 51. 422-424. [20] Rowbotham, T.J. (1983)J. Clin. Pathol. 36, 978-986. [21] Fallon, R.J. and Rowbotham, T.J. (1990) J. Clin. Pathol. 43, 479-483. [22] Amann, R.I., Binder, B.J., OIson, R.J., Chisholm, S.W., Devereax, R. and Stahl, D.A. (1990) Appl. Environ. Microbiol. 56, 1919-1926. [23] Fry, N.K., Warwick. S.. Saunders, N.A. and Embley, T.M. (1991) J. Gen. Microbiol. 137, 1215-|222.