Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA


Maugeri Dipartimento di Biologia Animale ed Ecologia Marina, Sez. Ecologia Microbica e Biotecnologie, University of Messina, Salita Sperone, 31-98166 Messina, Italy (e-mail:


Aims In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains.

Methods and Results This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans,B. caldolyticus”, B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised.

Conclusions This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species.

Significance and Impact of the Study This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.


The great diversity of thermophilic bacteria related to the genus Bacillus has been emphasized frequently. The paucity of readily identifiable phenotypic characteristics useful for an unequivocal identification of these spore-formers (Sharp et al. 1992) had led to classification methods based on phylogenetic assessments. The comparison of restriction patterns of amplified 16S rDNA (ARDRA) is a simple method for clustering organisms in genetically homogeneous clusters (Vaneechoutte et al. 1992). Recently, ARDRA has been used successfully for a rapid and reliable identification of bacilli newly isolated from hot composts, soil samples and deep-sea thermal vents (Blanc et al. 1997, 1999; Mora et al. 1998; Jeanthon et al. 1999). All Bacillus reference species described so far were isolated from soil, compost, mud, air and water of terrestrial hot springs (Sharp et al. 1992; Andersson et al. 1995; Combet-Blanc et al. 1995; Meier-Stauffer et al. 1996; Blanc et al. 1997). Until recently, few were the reports about thermophilic bacilli inhabiting marine hot springs (Hjörleifsdóttir et al. ,989; Marteinsson et al. 1996). Recent studies on shallow, marine thermal vents of the Eolian Islands, Italy allowed isolation and characterization of thermophilic spore-formers which showed a high heterogeneity with respect to known thermophilic bacilli (Maugeri et al. 2000). Genotypic studies allowed recognition of six new thermophilic Bacillus species among these isolates, one of which has been described as B. vulcani (Caccamo et al. 2000).

In this paper, we report the rapid and reliable identification of thermophilic bacilli newly isolated from shallow, marine hydrothermal vents of the Eolian Islands by comparison of their amplified 16S rDNA restriction patterns with those of thermophilic Bacillus reference species.


Sampling and isolation

Water and sediment samples were collected from seven shallow, submarine thermal vents located around Vulcano, Lipari, Panarea and Stromboli Islands (Eolian Islands, Italy). Samples temperature ranged from 40 to 95°C. Different dilutions of samples were spread onto Bacto Marine Agar 2216 (Difco) plates and incubated in aerobic conditions at 55°C for three days. After isolation and purification, strains were routinely cultivated at 55°C and maintained onto Marine Agar slopes.

Phenotypic studies

Growth optimum was determined after incubation at different temperatures, pH and salt concentrations. Metabolic tests were performed using API 20 NE and API 50 CHB strips incubated at 55°C (bioMérieux). Starch hydrolysis was tested according to White et al. (1993).

Bacterial strains

Designation and source of field strains analysed in this study are listed in Table 1.

Table 1.   Designation and source of strains used in this study Thumbnail image of

Nine well-described thermophilic Bacillus species, one of which, B. vulcani, was isolated from Vulcano Island (Eolian Islands), were used as reference strains (Table 1). Eight thermophilic Bacillus strains (later named ‘Eolian strains’), isolated previously from the Eolian Islands and identified at this laboratory, were also included for comparison (Table 1). Five of the Eolian strains represented novel thermophilic Bacillus species (Maugeri et al. 2000).

DNA extraction and amplification

Genomic DNA extraction and 16S rDNA amplification were performed according to Rainey et al. (1994).

Amplified 16S rDNA restriction analysis (ARDRA)

Approximately 1·5 μg of amplified 16S rDNA was cleaved with 3 units of the restriction enzyme AluI (Boehringer Mannheim) in a total volume of 30 μl by incubating the reaction mixtures at 37°C for 3 h. The enzyme was then inactivated by heating the mixtures at 65°C for 15 min. The reaction products were analysed by agarose gel (2·5% w/v) electrophoresis in TAE buffer (0·04 M Tris-acetate, 0·001 M EDTA) 1× with 1 μl 10 ml−1 ethidium bromide.


Phenotypic properties

Seventy-three aerobic spore-forming strains were isolated from seven sampling stations. All grew at 55–70°C with 2% NaCl and were positive for gelatin and esculin hydrolysis. Most strains fermented ribose, fructose, maltose, grew on sucrose and hydrolysed starch.

Amplified ribosomal DNA restriction analysis (ARDRA)

Distinctive patterns were obtained after restriction with Alu I of the amplified 16S rDNA of the thermophilic Bacillus reference strains, except for ‘B. caldotenax’ that showed the same profile as B. kaustophilus (not shown). The comparison of restriction profiles of field and reference strains allowed us to identify 57 from 73 isolates (78%). Nineteen isolates formed four clusters (A, B, C, D) with distinctive restriction patterns identical to the profiles of four from the nine well-described Bacillus species used in this study (Fig. 1a). The cluster composition was as follows: cluster A included 13 isolates and the species B. thermodenitrificans DSM 466; cluster B included three isolates and the species ‘B. caldolyticus’ DSM 405; cluster C included two isolates and the new species B. vulcani DSM 13174T isolated from Vulcano Island (Caccamo et al. 2000); cluster D included one isolate and the species B. stearothermophilus DSM 22T.

Figure 1.

 (a,b) Representation of distinctive restriction patterns of the 13 different clusters (from A to M) of field and reference strains

Thirty-eight isolates exhibited restriction patterns identical to the profiles of seven Eolian Bacillus reference strains except B. pallidus strain 5s. Nine different profiles (E, F, G, H, I, J, K, L, M) were recognized among this large group of isolates, allowing us to group them in as many clusters (Fig. 1a,b). Nine isolates in cluster E exhibited the same pattern of Bacillus strain 1bw (Fig. 1a). Eight isolates in cluster F showed the same restriction profile of B. thermodenitrificans strain A2 (Fig. 1a). Eight isolates exhibiting the pattern of B. licheniformis strain B3–15 (Fig. 1b) constituted the cluster G. Four isolates exhibiting the profile of Bacillus strain 4–1 (Fig. 1b) were grouped together in cluster J. Four isolates, exhibiting the profile of Bacillus strain 1as (Fig. 1b), formed the cluster K. Three isolates, showing the pattern of Bacillus strain 5–2 (Fig. 1b), were included in cluster L. Two isolates, having the restriction pattern of Bacillus strain 10–1 (Fig. 1b), were grouped in cluster M.

Sixteen isolates exhibited restriction patterns different from all reference species. Six of them showed the profile H and were clustered together, as well as five isolates having the same pattern grouped in cluster I (Fig. 1b). However, these profiles were more similar to those of Eolian reference strains than to those of described Bacillus species. The profiles of the remaining five isolates (7%), PL21, PL22, PL30, PC5, Z1 (not shown) were different from each other and could not be related to any restriction pattern available for all isolates and reference strains.


The molecular technique reported here (ARDRA) and the use of a significant number of Bacillus reference strains allowed the identification of 78% of the isolates analysed in this study. The restriction patterns of field strains were related mainly to those of Eolian Bacillus strains, including the novel Eolian species described as B. vulcani DSM 13174T. Only B. thermodenitrificans DSM 466, B. stearothermophilus DSM 22 and ‘B. caldolyticus’ DSM 405, from well-described reference species isolated from other sites, found some representatives among the group of field strains. The remaining 22% of isolates, exhibiting seven different 16S rDNA restriction patterns, remained unidentified, and only the determination of the 16S rRNA gene sequence will allow us to assign these strains to known or novel species.

Therefore, the application of DNA-based fingerprinting methods, as a preliminary screening of a group of isolates, could be a useful alternative to more laborious techniques currently used for microbial identification, such as morphological and physiological analyses.

This study revealed a high taxonomic diversity among the thermophilic bacilli isolated from the Eolian Islands. The results indicated a distinct distribution of the species within the Eolian hydrothermal vent system. B. thermodenitrificans was the most dominant species among the thermophilic isolates, accounting for 30%, and was found only at Vulcano Island. Seven species instead, namely ‘B. caldolyticus’ and the novel species represented by Bacillus strain 1bw, Bacillus strain 4–1, Bacillus strain 1as, Bacillus strain 5–2 and Bacillus strain 10–1, were demonstrated to be ubiquitous within the hydrothermal system of the Eolian archipelago. The species B. vulcani, the few representatives of which came only from the sediment of La Roya at Vulcano Island, was demonstrated not to be ubiquitous.