- Email: [email protected]
Complete Primary Structure of the 23S rRNA Coding Gene from Thiobacillus cuprinus and its Similarity with that of Burkholderia cepacia
System. Appl. Microbiol. 17,481-483 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Complete Primary Structure of the 23S rRNA Coding Gene from Thiobacillus cuprinus and its Similarity with that of Burkholderia cepacia DAVID MOREIRA, RICARDO AMILS, and IRMA MARIN" Centro de Biologia Molecular, Universidad Aut6noma de Madrid, Cantoblanco, 28049 Madrid, Spain. Fax number: 34 1 3978344. Received July 25, 1994
Summary The nucleotide sequence of the Thiobacillus cuprinus strain DSM 5495 23S rRNA gene was determined. Comparative sequence analysis shows a high homology with the Burkholderia cepacia 23S rRNA gene. Taking into account previous sequence comparison work, this fact suggests a phylogenetic clustering of the facultative heterotrophic thiobacilli (T. cuprinus, T. intermedius and T. perometabolis). Sequence comparison suggests also that T. cuprinus could be related with some chemoorganotrophic Alcaligenaceae species.
Key words: Thiobacillus cuprinus - Chemolithotrophic bacteria - Facultative heterotrophs - Ribosomal RNA.
Bioleaching is a natural process in which metals are solubilized from ores by means of microorganisms. Among the bacteria, the most studied are those species belonging to the genus Thiobacillus. These are nonsporulating, Gram-negative, rod-shaped eubacteria that oxidize inorganic ferrous and/or sulfur compounds as electron donors, and use CO 2 as a carbon source and grow chemolithoautotrophically (Kelly and Harrison, 1989). Recently, facultative organotrophs among the Thiobacillus species have been described. The first of them was T. cuprinus, which can use not only sulfur or sulfidic ores, but also organic substrates as energy sources, and seems to have a preference for copper leaching (Huber and Stetter, 1990). Other species with similar metabolic characteristics, such as T. intermedius and T. perometabolis, were described later. Most of the genetic characteristics of the thiobacilli are still relatively unknown because their extreme growth conditions make the use of classical genetic techniques difficult. Nevertheless, sequence information is available on some cloned genes (reviewed by Tuovinen and Fry, 1993), the sequences of 5S rRNA (Lane et aI., 1985) and of 16S rRNA genes (Durand et aI., 1993, Lane et aI., 1992, " Corresponding author
Goebel and Stackebrandt, 1994). Here we present the first complete nucleotide sequence of a 23S rRNA encoding gene from a metal-mobilizing eubacterium, T. cuprinus DSM 5495 and compare it with other members of the ~1 subgroup of the Proteobacteria. We have chosen this species because of its interesting metabolism and biotechnological characteristics. Purified 23S rRNA obtained from T. cuprinus ribosomes was radioactively labelled and used as specific probe in hybridizations with blots containing SpeJ and Sail + SpeJ genomic DNA digestions. Two bands were detected, isolated and cloned (clones pEx-SL and pSU), coding for the complete 23S rRNA. Following sub cloning, the nucleotide sequence was determined using Sequenase®, and submitted to the EMBL databank under the accession number X75567. The sequenced gene has a G+C content of 52.2%, much lower than the average value for the complete genome (65 -67%) reported by Huber and Stetter. The predicted secondary structure does not exhibit any unusual feature. In the comparison with the LSU rRNA sequences of the EMBL databank, the 23S rRNA from T. cuprinus shows the highest homology (88.7%) with that from Burkholderia cepacia (Fig. 1). That confirms the affiliation of T. cuprinus to the ~1 subgroup of the purple
482
D. Moreira, R. Amils, and l. Marin Escherichia coli
I I
Aeromonas hydrophila Pseudomonas aeruginosa
Bordelella bronchiseplica & B. avium Burkholderia cepacia
---
Thiobacillus cuprinus Neisseria gonorrhoeae
Rhodobacler capsulalus Rhodobacler sphaeroides Bacil/us anthracis 0.6986
0.7370
0.7754
0.8138
0.8522
0.8906
0.9290
I~----+I------I~----~I------~I----~I~--~I
Similarity
Fig. 1. Sequence similarity dendrogram determined by weighted average linkage clustering using the numerical taxonomy program TAXAN (Information Resources Group, Maryland Biotechnology Institute). Complete 23S rRNA genes sequences were aligned by the algorithm of Needleman and Wunsch. All the sequences were obtained from the EMBL databank, accession numbers are: J01695 for E. coli, X67946 for A. hydrophila, Y00432 for P. aeruginosa, X70371 for B. bronchiseptica, X70370 for B. avium, X16368 for B. cepacia, X75567 for T. cuprinus, X67293 for N. gonorrhoeae, X06485 for R. capsulatus, and X53854 for R. sphaeroides. The Gram. positive B. anthracis (X64645) was employed as an outgroup reference.
bacteria (Woese et al., 1984). A previous phylogenetic study based on 55 rRNA sequences (Lane et al., 1985) shows that B. cepacia is related to thiobacilli which share metabolic features with T. cuprinus: T. intermedius (a facultative chemolithotroph) and T. perometabolis (a chemolithotrophic heterotroph). This fact could indicate a phylogenetic clustering of the facultative heterotrophic thiobacilli, and their relationships with some Pseudomonadaceae, but more 235 and 165 rRNA sequence information is needed in order to clarify this hypothesis, specifically if the high phylogenetic dispersion of the thiobacilli group is taken into consideration. Our work suggests also a possible relationship of T. cuprinus with some species of the Alcaligenaceae genus Bordetella (B. bronchiseptica and B. avium, with homology values of 87.8% and 87.6%, respectively); this is an interesting finding because the genus Bordetella contains some pathogenic species characterized by their chemoorganotrophic respirative metabolism, with requirement of organic sulfur (Pittman, 1984). These data could be suggesting a common phylogenetic origin of some of the chemoorganotrophic purple bacteria, which share several common metabolic characteristics. However, this is a premature hypothesis and it is necessary to prove it by means of the comparison of a larger number of sequences, including 165 rRNA sequences. In this sense, determination of complete sequences of different thiobacilli rRNAs will increase our knowledge of the phylogenetic adscription and structure of this interest-
ing group of bacteria and will facilitate its comparison with other microorganisms capable of metabolizing sulfur and metal-containig minerals in order to investigate its evolutionary and biotechnological implications. Acknowledgements. We wish to thank Drs. J. M. Requena and M. So to for their help in sequencing experiments. This work was supported by a grant of the CICYT (BI091-0223-C03-01) and an EEC grant (Bl02-CT93-0274 DG 12 SSMA). D.M. is a fellow of the Ministerio de Educaci6n y Ciencia.
References Durand, P., Reysenbach, A. 1., Prieur, D., Pace, N. R.: Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin. Arch. MicrobioI. 159, 39-44 (1993). Goebel, B. M., Stackebrandt, E.: Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments. Appl. Environ. MicrobioI. 60,1614-1621 (1994). Huber, H., Stetter, K. 0.: Thiobacillus cuprinus sp. nov., a novel facultatively organotrophic metal-mobilizing bacterium. Appl. Envirom. Microbiol. 56, 315-322 (1990). Kelly, P. D., Harrison, A. P.: Genus Thiobacillus, pp. 18421858. In: Bergey's manual of systematic bacteriology (Staley, J. T., Bryant, M. P., Pfenning, N. and Holt, J. G., eds.) Baltimore, Williams & Wilkins 1989.
T. cuprinus 23S rRNA primary structure Lane, D.]., Harrison, A. P., Stahl, D. , Pace, B., Giovannoni, S.]. , Olsen, G.]. Pace, N. R.: Evolutionary relationships among sulfur- and iron-oxidizing eubacteria. ]. Bacteriol. 174, 269278 (1992). Lane, D.]., Stahl, D. A., Olsen, G.]., Heller, D.]., Pace, N. R.: Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences. J. Bacteriol. 163, 75-81 (1985).
Pittman, M.: Genus Bordetella, pp. 388-393. In: Bergey's manual of systematic bacteriology (Krieg, N. R., Holt, ]. G., cds.) Baltimore, Williams & Wilkins 1984. Tuovinen, O. H., Fry, I.].: Bioleaching and mineral biotechnology. Curr. Op. Biotech. 4, 344-355 (1993). Woese, C. R., Weinsburg, W. G., Paster, B.j., Hahn, C. M., Tanner, R. S., Krieg, N. R., Koops, H. P., Harms, H., Stackebrandt, E.: The phylogeny of purple bacteria: the beta subdivision. System. Appl. Microbiol. 5, 327-336 (1984).
Irma Marin, Centro de Biologia Molecular, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Fax number: 34 1 397 8344.
32
System. Appl. Microbiol. Vol. 17/4
483
Complete Primary Structure of the 23S rRNA Coding Gene from Thiobacillus cuprinus and its Similarity with that of Burkholderia cepacia DAVID MOREIRA, RICARDO AMILS, and IRMA MARIN" Centro de Biologia Molecular, Universidad Aut6noma de Madrid, Cantoblanco, 28049 Madrid, Spain. Fax number: 34 1 3978344. Received July 25, 1994
Summary The nucleotide sequence of the Thiobacillus cuprinus strain DSM 5495 23S rRNA gene was determined. Comparative sequence analysis shows a high homology with the Burkholderia cepacia 23S rRNA gene. Taking into account previous sequence comparison work, this fact suggests a phylogenetic clustering of the facultative heterotrophic thiobacilli (T. cuprinus, T. intermedius and T. perometabolis). Sequence comparison suggests also that T. cuprinus could be related with some chemoorganotrophic Alcaligenaceae species.
Key words: Thiobacillus cuprinus - Chemolithotrophic bacteria - Facultative heterotrophs - Ribosomal RNA.
Bioleaching is a natural process in which metals are solubilized from ores by means of microorganisms. Among the bacteria, the most studied are those species belonging to the genus Thiobacillus. These are nonsporulating, Gram-negative, rod-shaped eubacteria that oxidize inorganic ferrous and/or sulfur compounds as electron donors, and use CO 2 as a carbon source and grow chemolithoautotrophically (Kelly and Harrison, 1989). Recently, facultative organotrophs among the Thiobacillus species have been described. The first of them was T. cuprinus, which can use not only sulfur or sulfidic ores, but also organic substrates as energy sources, and seems to have a preference for copper leaching (Huber and Stetter, 1990). Other species with similar metabolic characteristics, such as T. intermedius and T. perometabolis, were described later. Most of the genetic characteristics of the thiobacilli are still relatively unknown because their extreme growth conditions make the use of classical genetic techniques difficult. Nevertheless, sequence information is available on some cloned genes (reviewed by Tuovinen and Fry, 1993), the sequences of 5S rRNA (Lane et aI., 1985) and of 16S rRNA genes (Durand et aI., 1993, Lane et aI., 1992, " Corresponding author
Goebel and Stackebrandt, 1994). Here we present the first complete nucleotide sequence of a 23S rRNA encoding gene from a metal-mobilizing eubacterium, T. cuprinus DSM 5495 and compare it with other members of the ~1 subgroup of the Proteobacteria. We have chosen this species because of its interesting metabolism and biotechnological characteristics. Purified 23S rRNA obtained from T. cuprinus ribosomes was radioactively labelled and used as specific probe in hybridizations with blots containing SpeJ and Sail + SpeJ genomic DNA digestions. Two bands were detected, isolated and cloned (clones pEx-SL and pSU), coding for the complete 23S rRNA. Following sub cloning, the nucleotide sequence was determined using Sequenase®, and submitted to the EMBL databank under the accession number X75567. The sequenced gene has a G+C content of 52.2%, much lower than the average value for the complete genome (65 -67%) reported by Huber and Stetter. The predicted secondary structure does not exhibit any unusual feature. In the comparison with the LSU rRNA sequences of the EMBL databank, the 23S rRNA from T. cuprinus shows the highest homology (88.7%) with that from Burkholderia cepacia (Fig. 1). That confirms the affiliation of T. cuprinus to the ~1 subgroup of the purple
482
D. Moreira, R. Amils, and l. Marin Escherichia coli
I I
Aeromonas hydrophila Pseudomonas aeruginosa
Bordelella bronchiseplica & B. avium Burkholderia cepacia
---
Thiobacillus cuprinus Neisseria gonorrhoeae
Rhodobacler capsulalus Rhodobacler sphaeroides Bacil/us anthracis 0.6986
0.7370
0.7754
0.8138
0.8522
0.8906
0.9290
I~----+I------I~----~I------~I----~I~--~I
Similarity
Fig. 1. Sequence similarity dendrogram determined by weighted average linkage clustering using the numerical taxonomy program TAXAN (Information Resources Group, Maryland Biotechnology Institute). Complete 23S rRNA genes sequences were aligned by the algorithm of Needleman and Wunsch. All the sequences were obtained from the EMBL databank, accession numbers are: J01695 for E. coli, X67946 for A. hydrophila, Y00432 for P. aeruginosa, X70371 for B. bronchiseptica, X70370 for B. avium, X16368 for B. cepacia, X75567 for T. cuprinus, X67293 for N. gonorrhoeae, X06485 for R. capsulatus, and X53854 for R. sphaeroides. The Gram. positive B. anthracis (X64645) was employed as an outgroup reference.
bacteria (Woese et al., 1984). A previous phylogenetic study based on 55 rRNA sequences (Lane et al., 1985) shows that B. cepacia is related to thiobacilli which share metabolic features with T. cuprinus: T. intermedius (a facultative chemolithotroph) and T. perometabolis (a chemolithotrophic heterotroph). This fact could indicate a phylogenetic clustering of the facultative heterotrophic thiobacilli, and their relationships with some Pseudomonadaceae, but more 235 and 165 rRNA sequence information is needed in order to clarify this hypothesis, specifically if the high phylogenetic dispersion of the thiobacilli group is taken into consideration. Our work suggests also a possible relationship of T. cuprinus with some species of the Alcaligenaceae genus Bordetella (B. bronchiseptica and B. avium, with homology values of 87.8% and 87.6%, respectively); this is an interesting finding because the genus Bordetella contains some pathogenic species characterized by their chemoorganotrophic respirative metabolism, with requirement of organic sulfur (Pittman, 1984). These data could be suggesting a common phylogenetic origin of some of the chemoorganotrophic purple bacteria, which share several common metabolic characteristics. However, this is a premature hypothesis and it is necessary to prove it by means of the comparison of a larger number of sequences, including 165 rRNA sequences. In this sense, determination of complete sequences of different thiobacilli rRNAs will increase our knowledge of the phylogenetic adscription and structure of this interest-
ing group of bacteria and will facilitate its comparison with other microorganisms capable of metabolizing sulfur and metal-containig minerals in order to investigate its evolutionary and biotechnological implications. Acknowledgements. We wish to thank Drs. J. M. Requena and M. So to for their help in sequencing experiments. This work was supported by a grant of the CICYT (BI091-0223-C03-01) and an EEC grant (Bl02-CT93-0274 DG 12 SSMA). D.M. is a fellow of the Ministerio de Educaci6n y Ciencia.
References Durand, P., Reysenbach, A. 1., Prieur, D., Pace, N. R.: Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin. Arch. MicrobioI. 159, 39-44 (1993). Goebel, B. M., Stackebrandt, E.: Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments. Appl. Environ. MicrobioI. 60,1614-1621 (1994). Huber, H., Stetter, K. 0.: Thiobacillus cuprinus sp. nov., a novel facultatively organotrophic metal-mobilizing bacterium. Appl. Envirom. Microbiol. 56, 315-322 (1990). Kelly, P. D., Harrison, A. P.: Genus Thiobacillus, pp. 18421858. In: Bergey's manual of systematic bacteriology (Staley, J. T., Bryant, M. P., Pfenning, N. and Holt, J. G., eds.) Baltimore, Williams & Wilkins 1989.
T. cuprinus 23S rRNA primary structure Lane, D.]., Harrison, A. P., Stahl, D. , Pace, B., Giovannoni, S.]. , Olsen, G.]. Pace, N. R.: Evolutionary relationships among sulfur- and iron-oxidizing eubacteria. ]. Bacteriol. 174, 269278 (1992). Lane, D.]., Stahl, D. A., Olsen, G.]., Heller, D.]., Pace, N. R.: Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences. J. Bacteriol. 163, 75-81 (1985).
Pittman, M.: Genus Bordetella, pp. 388-393. In: Bergey's manual of systematic bacteriology (Krieg, N. R., Holt, ]. G., cds.) Baltimore, Williams & Wilkins 1984. Tuovinen, O. H., Fry, I.].: Bioleaching and mineral biotechnology. Curr. Op. Biotech. 4, 344-355 (1993). Woese, C. R., Weinsburg, W. G., Paster, B.j., Hahn, C. M., Tanner, R. S., Krieg, N. R., Koops, H. P., Harms, H., Stackebrandt, E.: The phylogeny of purple bacteria: the beta subdivision. System. Appl. Microbiol. 5, 327-336 (1984).
Irma Marin, Centro de Biologia Molecular, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Fax number: 34 1 397 8344.
32
System. Appl. Microbiol. Vol. 17/4
483