| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
School of Biomedical Sciences, University of Ulster, Coleraine, County Londonderry BT52 1SA, Northern Ireland, UK
Correspondence
Ibrahim Banat
IM.Banat{at}ulster.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). These novel genera are based around separate phylogenetic groups derived from 16S rRNA gene sequence information. The original genus Bacillus contained aerobic and facultatively anaerobic, rod-shaped, Gram-positive (or Gram-variable) endospore-forming bacteria (Claus & Berkeley, 1986).
Many of the organisms previously classified in the genus Bacillus are thermophilic, aerobic, spore-forming organisms, which fall into genetic groups 1 and 5. There have been a number of taxonomic studies of the thermophilic bacilli, which retained these organisms within the genus (White et al., 1993; Rainey et al., 1994). The thermophiles in group 5 have been defined as ‘a phenotypically and phylogenetically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98·5–99·2 %)’ (Nazina et al., 2001). These observations led this group of workers to erect the novel genus Geobacillus with Geobacillus stearothermophilus as the type species. The two novel species they described (Geobacillus subterraneus and Geobacillus uzenensis) were isolated from subterranean petroleum reservoirs, geothermal locations providing an ideal source of thermophilic micro-organisms. In addition, Nazina et al. (2001) transferred some existing Bacillus species into the new genus as G. stearothermophilus, Geobacillus thermocatenulatus, Geobacillus thermoleovorans, Geobacillus kaustophilus, Geobacillus thermoglucosidasius and Geobacillus thermodenitrificans. Subsequently, other species have been added to the genus, i.e. Geobacillus caldoxylosilyticus, which was originally trivially named ‘Bacillus caldoxylolyticus’, then transferred to Saccharococcus caldoxylosilyticus (Ahmad et al., 2000), and is now regarded as G. caldoxylosilyticus (Fortina et al., 2001), and lastly Geobacillus toebii (Sung et al., 2002). Not surprisingly, most microbiologists seek to isolate thermophiles from ‘hot’ environments; however, it has recently been shown that extremely thermophilic bacilli are present in cool soil environments at population levels that preclude them being contaminants from other environments (Marchant et al., 2002a, b).
Thermophiles can be categorized as being one of three types: (i) those that are restricted biogeographically and relaxed biogeochemically, (ii) those that are relaxed biogeographically and restricted biogeochemically or (iii) those that are relaxed biogeographically and relaxed biogeochemically. The thermophilic geobacilli fall into the last group because they are widely distributed and not restricted to specialized nutritional environments. Careful examination of the soil environment for thermophilic micro-organisms has shown that the diversity is large and that there are organisms present that do not fit into the confines of Geobacillus species as recently recognized. In this paper we describe a novel species, Geobacillus debilis sp. nov., isolated from a cool soil environment in Northern Ireland, UK, belonging to a distinct Geobacillus genus subgroup. In addition, following comparison of 16S rRNA gene sequences of several geobacilli with the sequences of strains of Bacillus pallidus we suggest reassigning Bacillus pallidus to Geobacillus pallidus comb. nov.
Soil samples were collected from a site in Northern Ireland within an established mixed wet meadow area, which had been undisturbed for at least 15 years (Irish Grid reference C881 215) and from a similarly undisturbed adjacent site under mixed coniferous and deciduous trees with no ground cover (Irish Grid reference C881 216). The soil type at the sites was basalt till and the samples were taken at a depth of 50 mm below the surface.
One hundred milligrams of soil sample was suspended in 50 ml sterile Ringer's solution (Oxoid) containing 0·1 % Triton X-100 and placed in a sonicating bath (KS100; Kerry Ultrasonics Ltd) for 10 min. One millilitre of the sample was serially diluted in Ringer's solution and spread plates were prepared on nutrient broth (Oxoid) at pH 6·8–7·2 solidified with 0·8 % Gellan Gelrite gum (Sigma) and incubated at 70 °C for 24 h under aerobic conditions. Resulting colonies were isolated either onto specialized Bacillus medium (Leighton & Doi, 1971) or trypticase soya broth solidified with agar or Gelrite Gellan gum (Sigma) and further purified before being stored as stock cultures either at room temperature or at 4 °C.
Morphological and biochemical characterization of the isolates designated F10 (from the meadow site) and TfT (from the wooded site) were carried out using standard methods as described by Marchant et al. (2002b). In order to allow longer incubation times for the biochemical tests, the cultures were incubated at 60 °C rather than at the higher temperatures of 60–70 °C at which the organisms are able to grow.
DNA was extracted from pure cultures of the organisms and the 16S rRNA gene sequences were determined as described by Marchant et al. (2002b). DNA extraction from cultured bacteria was performed using a BIO 101 Inc. Soil DNA extraction kit. PCR was carried out using standard methods with the universal reverse primer 1492R with Bacteria-specific primer 27F (Bond et al., 2000) for the 16S rRNA gene. PCR amplification was carried out in reaction mixtures containing 0·2 mM each dNTP, 1·5 mM MgCl2, 25 µM each primer, 1 µl template DNA and 2·5 U Taq polymerase in a final volume of 100 µl PCR buffer. Thermocycling was carried out in a Whatman Biometra T personal thermocycler under the following conditions: initial denaturation at 95 °C for 120 s, followed by 20 cycles of denaturation at 95 °C for 60 s, primer annealing at 45 °C for 45 s, elongation at 72 °C for 90 s with a final extension at 72 °C for 600 s. PCR products were run on 1·2 or 2 % agarose gels containing ethidium bromide.
DNA sequencing was carried out directly on purified PCR products. Three forward and three reverse sequencing primers were used (Marchant et al., 2002b). The sequencing reactions were carried out and analysed using the ABI Prism 3100 Genetic Analyzer system as specified by the manufacturer. The genomic G+C ratio was determined using HPLC by DSMZ, Braunschweig, Germany.
Obligately thermophilic bacilli were easily isolated in large numbers from the cool soil environments examined (Marchant et al., 2002a, b). The strains isolated showed temperature ranges for visible growth of 40–75 °C and had extremely high growth rates with minimum generation times of less than 30 min at 70 °C. Many of the isolates could be readily assigned to existing Geobacillus species (Nazina et al., 2001; Fortina et al., 2001) on the basis of morphology, metabolic characteristics and 16S rRNA gene sequences. Two isolates, F10 and TfT, showed low 16S rRNA gene sequence similarity with any recognized Geobacillus species, showing 93 % similarity to a sequence for G. stearothermophilus (AY297092) and thus indicating a more distant phylogenetic relationship. It has been proposed that sequence similarity below 95 % indicates a novel species (Fogel et al., 1999), although Stackebrandt & Goebel (1994) set the threshold at 97 %; this suggests that strains F10 and TfT represent a novel species of the genus Geobacillus. A higher sequence similarity of 99 % was given for a strain given the name ‘Bacillus thermozeamaize’ (unpublished; accession no. Y288912) isolated from corn steep liquor and that was presumably a thermophile. Construction of phylogenetic trees using neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981, 1988, 1996) and parsimony (Felsenstein, 1996) methods indicated that strains F10 and TfT represent a novel species, for which we propose the name Geobacillus debilis sp. nov. However, G. debilis shows close affinities to Bacillus pallidus, which we propose should become Geobacillus pallidus comb. nov. (Fig. 1). G. debilis strains F10 and TfT each show 91 % 16S rRNA gene sequence similarity to the sequence of G. pallidus. Comparison of the mean distance analysis tree in Fig. 1 with the tree in Nazina et al. (2001) shows similar relationships. In both cases, G. kaustophilus, G. thermoleovorans, G. thermocatenulatus, G. subterraneus, G. uzenensis and G. thermodenitrificans form a closely related cluster. In addition, G. caldoxylosilyticus and Bacillus (Geobacillus) pallidus occupy similar relative positions in both trees, with subsequently described G. toebii and G. debilis fitted close by in our tree. We have been able to confirm these relationships in a second phylogenetic tree constructed by mean distance analysis (data not shown).
|
gave species as rod-shaped cells of varying dimensions but less than 10 µm long. Although there are references to these thermophilic bacilli producing chains of cells, some isolates produce enormously long, undivided flexuous cells (Marchant et al., 2002b). In the case of G. debilis, no elongated cells have been observed and the rods were 0·5–1 µm wide by 1–14 µm long. Spore formation is also a defining character of Bacillus and Geobacillus and in many strains sporulation is copious and readily initiated. We have only been able to show the production of spores in G. debilis following prolonged storage of plate cultures. The spores are produced singly and are not swollen beyond the size of the cells. Cells are motile and produce flat, cream-coloured colonies with smooth margins. Cells stain Gram-negative, but variable Gram-staining reactions are the norm for this group of Gram-positive bacteria. G. debilis shows a temperature range for growth of 50–70 °C, which is a rather narrower range than for other species in the genus. The organism is a strict aerobe and does not use nitrate or sulphate as a terminal electron acceptor.
Detailed characteristics of the two strains of G. debilis used in this study are shown in Table 1. Morphological and biochemical characterizations, including alkane utilization (pentane, hexane, heptane, dodecane, hexadecane, octadecane and nonadecane), were carried out using standard methods as described by Marchant et al. (2002b). Characteristics of all recognized Geobacillus species are shown in Table 2.
|
|
), several showed close 16S rRNA gene sequence similarity to the thermophilic B. pallidus, which was originally isolated from thermophilic treated wastes and was described by Scholz et al. (1987). B. pallidus has subsequently been employed in studies of thermostable enzymes and biodegradation studies including breakdown of 2-propanol (Bustard et al., 2002). A close examination of the published characteristics of B. pallidus (Table 2
) together with the morphological description (Scholz et al., 1987) and with the sequence similarity for the 16S rRNA gene available on the European Bioinformatics Institute database and with these of our Geobacillus isolates obtained from soil strongly suggests that B. pallidus should be transferred and renamed G. pallidus. A phylogenetic tree for Geobacillus species including G. pallidus presented in Fig. 1 indicates the affinity of G. pallidus with G. caldoxylosilyticus, G. toebii and G. debilis.
Description of Geobacillus debilis sp. nov.
Geobacillus debilis (de'bil.is. L. masc. adj. debilis weak or feeble, referring to the restricted substrate range for this species).
Gram-negative rods, 0·5–1·0 µm wide by 1·0–14·0 µm long, motile, spores sparsely produced terminally, sporangium not swollen. Colonies flat, cream-coloured with smooth margins. Growth occurs at 50–70 °C, with an optimum above 60 °C. Obligate aerobe. The DNA G+C content is 49·9 mol%. Production of acid from raffinose and trehalose, and also from ribose, sorbitol and arabinose in some strains. No utilization of starch but casein and gelatin used in some strains; very limited use of alkanes. Habitat, soil.
The type strain is TfT (=DSM 16016T=NCIMB 13995T); strains have been isolated from 50 mm undisturbed subsurface soil samples from Northern Ireland.
Description of Geobacillus pallidus (Scholz et al. 1988) Banat, Marchant and Rahman comb. nov.
Basonym: Bacillus pallidus Scholz et al. 1988.
The description is identical to that given for the genus Geobacillus by Nazina et al. (2001) and the species description given by Scholz et al. (1987). The type strain is H12T (=ATCC 51176T=DSM 3670T=LMG 19006T).
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bond, P. L., Smriga, S. P. & Banfield, J. F. (2000). Phylogeny of microorganisms populating a thick, subaerial predominantly lithotrophic biofilm at an extreme acid mine drainage site. Appl Environ Microbiol 66, 3842–3849.
Bustard, M. T., Whiting, S., Cowan, D. A. & Wright, P. C. (2002). Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus. Extremophiles 6, 319–323.[Medline]
Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872, 174AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1105–1139. Edited by P. H. A. Sneath. Baltimore: Williams & Wilkins.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (1988). Phylogenies from molecular sequences: inference and reliability. Annu Rev Genet 22, 521–565.[CrossRef][Medline]
Felsenstein, J. (1996). Inferring phylogeny from protein sequences by parsimony, distance and likelihood methods. Methods Enzymol 266, 368–382.[Medline]
Fogel, G. B., Collins, C. R., Li, J. & Brunk, C. F. (1999). Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microb Ecol 38, 93–113.[CrossRef][Medline]
Fortina, M. G., Mora, D., Schumann, P., Parini, C., Manachini, P. L. & Stackebrandt, E. (2001). Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmed et al. 2000) comb. nov. Int J Syst Evol Microbiol 51, 2063–2071.[Abstract]
Leighton, T. J. & Doi, R. H. (1971). The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem 246, 3189–3195.
Marchant, R., Banat, I. M., Rahman, T. J. & Berzano, M. (2002a). What are high temperature bacteria doing in cold environments? Trends Microbiol 10, 120–121.[Medline]
Marchant, R., Banat, I. M., Rahman, T. J. & Berzano, M. (2002b). The frequency and characteristics of highly thermophilic bacteria in cool soil environments. Environ Microbiol 4, 595–602.[CrossRef][Medline]
Nazina, T. N., Tourova, T. P., Poltaraus, A. B. & 8 other authors (2001). Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51, 433–446.[Abstract]
Rahman, T. J., Marchant, R. & Banat, I. M. (2004). Distribution and molecular investigation of highly thermophilic bacteria associated with cool soil environments. Biochem Soc Trans 32, 209–213.[CrossRef][Medline]
Rainey, F. A., Fritze, D. & Stackebrandt, E. (1994). The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis. FEMS Microbiol Lett 115, 205–212.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Scholz, T., Demharter, W., Hensel, R. & Kandler, O. (1987). Bacillus pallidus sp. nov., a new thermophilic species from sewage. Syst Appl Microbiol 9, 91–96.
Stackebrandt, E. & Goebel, B. M. (1994). A place for DNA–DNA reassociation and 16S ribosomal RNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Sung, M.-H., Kim, H., Bae, J.-W. & 9 other authors (2002). Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from hay compost. Int J Syst Evol Microbiol 52, 2251–2255.[Abstract]
Sunna, A., Tokajian, S., Burghardt, J., Rainey, F., Antranikian, G. & Hashwa, F. (1997). Identification of Bacillus kaustophilus, Bacillus thermocatenulatus and Bacillus strain HSR as members of Bacillus thermoleovorans. Syst Appl Microbiol 20, 232–237.
Suzuki, Y., Kishigami, T., Inoue, K., Mizoguchi, Y., Eto, N., Takagi, M. & Abe, S. (1983). Bacillus thermoglucosidasius sp. nov., a new species of obligately thermophilic bacilli. Syst Appl Microbiol 4, 487–495.
White, D., Sharp, R. J. & Priest, F. G. (1993). A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area. Antonie van Leeuwenhoek 64, 357–386.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  Y. Zhou, J. Xu, L. Xu, and B. J. Tindall Falsibacillus pallidus to replace the homonym Bacillus pallidus Zhou et al. 2008 Int J Syst Evol Microbiol, December 1, 2009; 59(12): 3176 - 3180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Zeigler Application of a recN sequence similarity analysis to the identification of species within the bacterial genus Geobacillus Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1171 - 1179. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
