International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1985–1989
NOTE
1
School of Biological and
Biomedical Sciences,
Glasgow Caledonian
University, Cowcaddens
Road, Glasgow G4 0BA, UK
2
Vakgroep BFM WE10V,
Laboratorium voor
Microbiologie,
Universiteit Gent, K. L.
Ledeganckstraat 35,
B-9000 Gent, Belgium
DOI : 10.1099/ijs.0.02282-0
Bacillus luciferensis sp. nov., from volcanic soil
on Candlemas Island, South Sandwich
archipelago
Niall A. Logan,1 Liesbeth Lebbe,2 An Verhelst,2 Johan Goris,2
Gillian Forsyth,1 Marina Rodriguez-Diaz,1 Marc Heyndrickx2†
and Paul De Vos2
Author for correspondence : Niall A. Logan. Tel : j44 141 331 3207. Fax : j44 141 331 3208.
e-mail : N.A.Logan!gcal.ac.uk
Aerobic, endospore-forming bacteria were found in soil taken from an active
fumarole on Lucifer Hill, Candlemas Island, South Sandwich archipelago.
Amplified rDNA restriction analysis, SDS-PAGE, repetitive element primed-PCR
(rep-PCR) and routine phenotypic tests suggested that six of the isolates
represent a novel taxon, and 16S rDNA sequence comparisons support the
proposal of a novel species, Bacillus luciferensis sp. nov., the type strain of
which is strain LMG 18422T (l CIP 107105T).
Keywords : Bacillus luciferensis, Candlemas Island, South Sandwich Islands,
maritime Antarctica, volcanic soil
Continental and maritime Antarctica possesses a small
number of sites where volcanic activity warms the soil
and the steam rising from fumaroles condenses. Such
sites are of special biological interest because of their
relatively steady water supplies and elevated temperatures. The South Sandwich archipelago comprises
11 islands, all of which exhibit recent or continuing
volcanic activity. They lie on the Scotia Ridge, between
latitude 56m 18h and 59m 28h S and longitude 26m 14h
and 28m 11h W ; this volcanic arc has a deep-sea trench
on its convex (eastern) side, descending to over 7000 m.
Captain James Cook discovered Candlemas Island on
Candlemas Day (February 2) 1775 during the voyage
of HMS Resolution, but weather and ice conditions
prevented landing. It was visited by sealing vessels
during the 19th century, whaling expeditions worked
around it in the early 20th century and the first landing
for scientific purposes was made by Larsen in 1908
(Kemp & Nelson, 1931). Other brief scientific visits
were made by the RRS Shackleton in 1961 and the
Royal Navy ice patrol ship HMS Protector in 1962
.................................................................................................................................................
Details of the FAME compositions of B. luciferensis strains are available as
supplementary material in IJSEM Online (http ://ijs.sgmjournals.org/).
† Present address : Government Dairy Research Station, Brusselsesteenweg 370, B-9090 Melle, Belgium.
Abbreviations : ARDRA, amplified rDNA restriction analysis ; FAME, fatty
acid methyl ester ; rep-PCR, repetitive element-primed PCR ; TSA, trypticase
soy agar.
The EMBL accession number for the 16S rRNA gene sequence of Bacillus
luciferensis LMG 18422T is AJ419629.
(Holdgate & Baker, 1979). Protector made a longer
visit in 1964, supporting a British Antarctic Survey
(BAS) expedition (Holdgate & Baker, 1979), so that
Candlemas Island became studied more extensively
by biologists and geologists than any other member
of the archipelago. Apart from sealing activities and
these scientific visits, there has been little human
disturbance.
Candlemas Island (see figures and plates in Tomblin,
1979 ; Fig. 1 in Logan et al., 2000) is irregular in shape
and has a diverse topography. Its fauna and flora are
more extensive than other islands in the arc and
include penguins, petrels, skuas and seals, grass,
bryophytes and lichens. The southern massif, which
forms the largest part of the island, is the remnant of a
long-extinct volcano ; it rises to 550 m and is permanently ice-capped. The northern part is actively
volcanic and younger than the southern part and
comprises a complex of scoria cones, called Lucifer
Hill, surrounded by a roughly circular mass of five
main lava flows. The oldest of these bear ash mantles,
while the youngest, northern, lava field is very recent
(Tomblin, 1979). The northern and southern parts of
the island are linked by an area of low, flat sand
containing two large lagoons. Lucifer Hill (57m 04h S,
26m 42h W) reaches an altitude of 232 m and bears
patches of moss around active fumaroles high on the
hill, around inactive fumaroles at Clinker Gulch and
on lava soils at the base of the volcanic cone, with
temperatures ranging from 85 mC down to 0 mC. Mossy
02282 # 2002 IUMS Printed in Great Britain
1985
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 14:28:18
N. A. Logan and others
isolates, we propose the novel species Bacillus luciferensis sp. nov.
.................................................................................................................................................
Fig. 1. Photomicrograph of sporangia and vegetative cells of
Bacillus luciferensis sp. nov. LMG 18422T viewed by phasecontrast microscopy ; ellipsoidal spores lie subterminally and
occasionally terminally in unswollen or slightly swollen
sporangia. Bar, 2 µm.
soil samples were collected from the summit and base
of Lucifer Hill by BAS personnel during the 1996–97
austral summer, with a view to investigating the
aerobic, endospore-forming bacterial flora. The present note describes the isolation and characterization of
a group of novel Bacillus strains found in warm soil
taken from a mossy area on an active fumarole at the
summit of Lucifer Hill. A representative strain (LMG
18422T) was reported by Logan et al. (2000) as being
unidentifiable and, following the study of five further
Strains were isolated and maintained on trypticase soy
agar (TSA) containing 5 mg MnSO l−" to enhance
sporulation, as described by Logan et %al. (2000). Strain
LMG 18422T and the further strains LMG 21400
(l B1762), R-11670 (l B1763), R-14109 (l B1840),
R-14110 (l B1841) and R-14111 (l B1842) were
isolated from a sample of soil by moss on a warm,
active fumarole (temperature range 30–60 mC ; altitude
232 m ; site 1 in Logan et al., 2000) near the southern
crater of Lucifer Hill, at the top of the scoria cone. The
strains were characterized phenotypically by the
methods of Logan & Berkeley (1984) ; other characters
were determined, and the data analysed numerically,
as described by Logan et al. (2000). For amplified
rDNA restriction analysis (ARDRA), enzymically
amplified 16S rDNA of strain LMG 18422T was
obtained by the PCR and analysed by restriction
digestion with five restriction enzymes (HaeIII, DpnII,
RsaI, BfaI and Tru9I) as described previously
(Heyndrickx et al., 1996). A 16S rDNA sequence was
obtained for the same strain and aligned with sequences in the EMBL database and the DNA base
composition of this strain was also determined, both as
described by Logan et al. (2000). Repetitive element
primed-PCR (rep-PCR) patterns with (GTG)5 primers
were obtained according to the method of Versalovic
et al. (1991), as adapted by Gevers et al. (2001). Cells
of the six strains were obtained as described by
Heyndrickx et al. (1998) and subjected to SDS-PAGE
Similarity (%)
60
70
80
90
100
Bacillus luciferensis LMG 21400
Bacillus luciferensis R-11670
Bacillus luciferensis R-14110
Bacillus luciferensis R-14111
Bacillus luciferensis LMG 18422T
Bacillus luciferensis R-14109
Bacillus halmapalus LMG 17950T
Bacillus cohnii LMG 16678T
Bacillus subtilis LMG 7135T
Bacillus sporothermodurans LMG 17894T
.................................................................................................................................................................................................................................................................................................................
Fig. 2. Dendrogram of ARDRA banding patterns of B. luciferensis strains, representatives of closely related taxa and
Bacillus subtilis using the Dice coefficient.
Similarity (%)
90
100
R-14109
R-14110
R-14111
LMG 18422T
LMG 21400
R-11670
.................................................................................................................................................................................................................................................................................................................
Fig. 3. Normalized computer profiles from SDS-PAGE analyses of whole-cell proteins of B. luciferensis strains. The
dendrogram is based on UPGMA clustering of the correlation coefficient (r) of the total-protein profiles. The zone used
for clustering was between point 30 and point 330 (a complete lane contains 400 points).
1986
International Journal of Systematic and Evolutionary Microbiology 52
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 14:28:18
Bacillus luciferensis sp. nov.
Table 1. Some characters that distinguish B. luciferensis
sp. nov. from related species
.................................................................................................................................................
.................................................................................................................................................
Fig. 4. Neighbour-joining clustering of 16S rDNA sequences
(rooted with Filobacillus milosensis as reference) based on a
selection of 16S rDNA sequences from the EMBL database
(accession numbers in parentheses) and B. luciferensis LMG
18422T. Bootstrap analysis shows the robustness of the
branching. Bar, 5 % sequence dissimilarity.
analysis according to Pot et al. (1994) and the data
were collected and interpreted as described by Vauterin
& Vauterin (1992). For GC analysis of methylated
fatty acids (FAME), cells were grown and analysed as
described by Heyndrickx et al. (1998) and Vauterin et
al. (1991) and according to the Microbial Identification
System (MIDI Inc.).
All the strains were found to be Gram-positive,
facultatively anaerobic, motile rods that form ellipsoidal spores that lie subterminally and sometimes
terminally in unswollen or slightly swollen sporangia
(Fig. 1). They had similar patterns of results for
phenotypic characters and clustered together at 95 %
similarity in a UPGMA cluster analysis (not shown)
based upon these characters ; this cluster merged with
strains of Bacillus sporothermodurans at only 85 %
similarity, indicating that the former group is phenotypically distinct. Although the six strains were isolated
from the same sample of soil, they showed sufficient
phenotypic variation to suggest that they are not
repeated isolations of the same strain. It is of interest
that, although the temperature of the soil in which the
organisms were found ranged from 30 to 60 mC, our
strains did not grow at above 35–45 mC in the laboratory. The ARDRA patterns of the six strains were
all very similar, showing more than 93 % similarity
(Fig. 2). Comparison of the ARDRA pattern of strain
LMG 18422T with a database of over 1200 authentic
strains of species of aerobic endospore-forming bacteria did not yield an identification ; the highest
similarity found was 89n8 %, with the type strain of
Bacillus horikoshii. In the SDS-PAGE analysis, strains
showed at least 87 % similarity, reflecting limited
intraspecies variation (Fig. 3). Two subgroups with
over 95 % internal similarity can be recognized
(R-14109, R-14110 and R-14111, and LMG 21400 and
R-11670), while the type strain, LMG 18422T, holds
a central position in the dendrogram. In 16S rDNA
sequence comparisons with entries in the EMBL
database, the closest matches achieved for LMG
18422T were of only 95 %, with Bacillus halmapalus
and Bacillus cohnii (Fig. 4). Phenotypic characters that
distinguish the isolates from the phylogenetically
Species : 1, B. luciferensis sp. nov. ; 2, B. sporothermodurans ;
3, B. halmapalus ; 4, B. horikoshii ; 5, B. cereus. With the
exception of microscopic observations, optimum pH for
growth, anaerobic growth and casein hydrolysis, all
characters were determined using tests in the API 20E and 50
CHB systems. Characters are scored as : j, 85 % positive ;
(j), 75–84 % positive ; , variable (26–74 % positive) ; (k),
16–25 % positive ; k, 0–15 % positive ; , weak positive
reaction ; (), very weak positive reaction. All of the species
are positive for aesculin hydrolysis. B. cohnii is an obligate
alkaliphile, with optimum pH for growth of " 9n7 (whereas
the species included are all neutrophilic), so data for acid
production from carbohydrates are not available. For some
of the other characters included in the table, strains of this
species were reported to give the following results : terminal,
ellipsoidal spores swelling the sporangia ; gelatin and casein
hydrolysed ; nitrate reduced (Spanka & Fritze, 1993).
Character
Sporangia :
Spore shape*
Spore position†
Sporangia swollen
Anaerobic growth
Gelatin hydrolysis
Casein hydrolysis
Nitrate reduction
Voges–Proskauer
Acid from :
N-Acetyl -glucosamine
Amygdalin
Arbutin
Cellobiose
Galactose
β-Gentiobiose
-Glucose
-Fructose
Glycerol
Glycogen
Maltose
Mannitol
-Mannose
-Melezitose
Salicin
Starch
Sucrose
-Trehalose
-Turanose
1
2
3
4
5

 ()

j
j
j‡
k
j


k
k


k
k
 ()

j
k
j
j
k
k


j
k
j
j
k
k


k
j
j
j
(j)
j
j
j
j
j
k
j
j
j
k
k
j
k
k
j
j

j
j
j
k
k
j
j
k
k
j
j
j
k
j
j
k
j
j
k
j
j
j
()
k
k
()
()
()
()
()
k
()
()
()
()
k
()
()
()
()
()
()
k
k
()
k
()
()
()
k
()
()
()
k
k
k
()
()
()
()
j
k
j
(j)
k
(k)
j
j
j
j
j
k
k
k
j
j

j
k
* , Ellipsoidal ; , spherical.
† , Central\paracentral ; , subterminal ; , terminal. Positions
observed infrequently are shown in parentheses.
‡ B. luciferensis sp. nov. grows poorly on skim milk medium.
related species Bacillus cereus, B. cohnii, B. halmapalus
and B. horikoshii (see Fig. 4) and the phenotypically
related B. sporothermodurans are shown in Table 1.
http://ijs.sgmjournals.org
1987
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 14:28:18
N. A. Logan and others
Similarity (%)
96
97
98
99 100
LMG 18422T
R-11670
R-14109
.....................................................................................................
R-14110
Fig. 5. Dendrogram of rep-PCR patterns of
B. luciferensis strains based on Pearson’s
correlation coefficient.
R-14111
LMG 21400
The rep-PCR patterns of the six strains (Fig. 5) show
similarities of more than 95 % by Pearson’s productmoment correlation coefficient ; this indicates the very
high genetic homogeneity of this group of isolates and
supports their allocation to the same species. Indeed,
De Bruijn et al. (1996) showed that rep-PCR even
allows discrimination between strains of the same
species, so confirming that organisms with nearly
identical rep patterns must have very close relationships. Our failure to identify the Candlemas Island
strains by the genotypic and phenotypic methods tried
and the high similarities of the strains to each other in
the phenotypic analyses and rep-PCR patterns support
the proposal of a novel species, the description of
which follows.
Description of Bacillus luciferensis sp. nov.
Bacillus luciferensis (lu.cif.er.enhsis. N.L. adj. luciferensis referring to Lucifer Hill, a volcano on Candlemas
Island, South Sandwich Islands, the soil of which
yielded the organism).
Isolated from geothermal soil taken from an active
fumarole on Lucifer Hill, a volcano on Candlemas
Island, South Sandwich archipelago. Cells are motile,
round-ended rods (0n4–0n8i3–6 µm), occurring singly
and in pairs and showing pleomorphism (narrow and
broad cells, the latter showing swellings) on TSA
MnSO . Gram-positive, but becoming Gram-negative
within % 24 h of culture at 30 mC. Endospores are
apparent in overnight cultures incubated at 30 mC on
TSA MnSO and are formed in large numbers within
% incubation ; they are ellipsoidal, lie
2–3 days of
subterminally and occasionally terminally and may
swell the sporangia slightly (Fig. 1). After 1 day on
TSA, colonies are 1–5 mm in diameter, raised, with
irregular margins and surfaces, and show some spiking
along streak lines ; the larger colonies appear to
comprise several smaller colonies that have coalesced.
Colonies are moist and loosely butyrous, creamy-grey
and translucent with a glossy appearance ; by 10i
magnification, they show cream and whitish streaking
giving an appearance reminiscent of cut glass. Minimum temperature for growth lies between 15 and
20 mC, the optimum temperature for growth is 30 mC
and the maximum growth temperature lies between 35
and 45 mC. Minimum pH for growth lies between 5n5
and 6n0, the optimum pH for growth is 7n0 and the
maximum pH for growth lies between 8n0 and 8n5.
Facultatively anaerobic and weakly catalase-positive.
1988
Casein is hydrolysed, but growth is poor on skim milk
medium. In the API 20E strip, Voges–Proskauer
reaction is positive and gelatin is hydrolysed ; reactions
for o-nitrophenyl β--galactopyranoside hydrolysis,
arginine dihydrolase, lysine decarboxylase, ornithine
decarboxylase, citrate utilization, hydrogen sulphide
production, urease, tryptophan deaminase, indole
production and nitrate reduction are negative. Hydrolysis of aesculin is positive. Acid without gas is
produced from the following carbohydrates in the API
50 CH gallery using the CHB suspension medium :
amygdalin, arbutin, cellobiose, -fructose, β-gentiobiose, -glucose, maltose, -melezitose, N-acetyl
-glucosamine, salicin, starch (weak), sucrose, trehalose and -turanose. Acid is not produced from
the following carbohydrates : adonitol, - or arabinose, - or -arabitol, dulcitol, erythritol, or -fucose, galactose, gluconate, glycerol, glycogen,
meso-inositol, inulin, 2-keto--gluconate, 5-keto-gluconate, lactose, -lyxose, -mannitol, -mannose,
melibiose, methyl α--glucoside, methyl α--mannoside, methyl xyloside, -raffinose, ribose, rhamnose,
sorbitol, -sorbose, -tagatose, xylitol or - or xylose. Sensitive to disks of the following antibiotics :
ampicillin (25 µg), chloramphenicol (50 µg), colistin
sulphate (100 µg), kanamycin (30 µg), nalidixic acid
(30 µg), nitrofurantoin (50 µg), streptomycin (25 µg)
and tetracycline (100 µg). The major cellular fatty acids
are anteiso C : and iso C : (respectively repre"& total
!
senting about "&25! and 50 % of
fatty acid). The
following fatty acids are present in smaller amounts
(between about 1n5 and 4n5 %) : iso C : , C : , C : ω c
"% ! "% C
! "' and
" (
alcohol, iso C : , C : ω c, C : , anteiso
:!
!
"'
"
""
!
"(
"'
"'
C : ω t, while trace but significant amounts of iso C :
#! "of* summed feature 5 (C
"( !
and
: " iso I and\or C"( : "
"(
anteiso B) are also present. Details of the FAME
composition are available as supplementary material
in IJSEM Online (http :\\ijs.sgmjournals.org\).
The GjC content is 33n0 mol % for the type strain,
strain LMG 18422T (l CIP 107105T l Logan collection number B1761T l isolate SSI061) ; the 16S
rDNA sequence of this strain is deposited at EMBL
under accession number AJ419629.
Acknowledgements
We dedicate this paper to the fond memory of David WynnWilliams, an inspirational Antarctic microbiologist.
We are very grateful to P. Convey of the British Antarctic
Survey for collecting soil samples from Candlemas Island
International Journal of Systematic and Evolutionary Microbiology 52
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 14:28:18
Bacillus luciferensis sp. nov.
and to H. G. Tru$ per for advice on nomenclatural etymology.
N. A. L. thanks Wilma Dodd for technical support. We are
most grateful to bioMe! rieux SA for providing API materials
and for supporting G. F. and M. R.-D. P. D. V. is indebted to
the Fund for Scientific Research – Flanders for research
grant G.0156.02.
References
De Bruijn, F. J., Rademaker, J., Schneider, M., Rossbach, U. &
Louws, F. J. (1996). Rep-PCR genomic fingerprinting of plantassociated bacteria and computer-assisted phylogenetic analyses. In
Biology of Plant–Microbe Interaction ; Proceedings of the 8th International Congress of Molecular Plant–Microbe Interactions, pp. 497–
502. Edited by G. Stacey, B. Mullin & P. Gresshoff. Saint Paul, MN :
American Phytopathological Society Press.
Gevers, D., Huys, G. & Swings, J. (2001). Applicability of rep-PCR
fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 205, 31–36.
Heyndrickx, M., Vauterin, L., Vandamme, P., Kersters, K. & De
Vos, P. (1996). Applicability of combined amplified ribosomal DNA
restriction analysis (ARDRA) patterns in bacterial phylogeny and
taxonomy. J Microbiol Methods 26, 247–259.
Heyndrickx, M., Lebbe, L., Kersters, K., De Vos, P., Forsyth, G. &
Logan, N. A. (1998). Virgibacillus : a new genus to accommodate
Bacillus pantothenticus (Proom and Knight 1950). Emended description
of Virgibacillus pantothenticus. Int J Syst Bacteriol 48, 99–106.
Holdgate, M. W. & Baker, P. E. (1979). The South Sandwich Islands.
I. General description. Br Antarct Surv Sci Rep 91, 1–76.
Kemp, S. & Nelson, A. L. (1931). The South Sandwich Islands. Discov
Rep 3, 133–198.
Logan, N. A. & Berkeley, R. C. W. (1984). Identification of Bacillus
strains using the API system. J Gen Microbiol 130, 1871–1882.
Logan, N. A., Lebbe, L., Hoste, B. & 7 other authors (2000). Aerobic
endospore-forming bacteria from geothermal environments in northern
Victoria Land, Antarctica, and Candlemas Island, South Sandwich
archipelago, with the proposal of Bacillus fumarioli sp. nov. Int J Syst
Evol Microbiol 50, 1741–1753.
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Chemical Methods in
Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow &
A. G. O’Donnell. Chichester : Wiley.
Spanka, R. & Fritze, D. (1993). Bacillus cohnii sp. nov., a new,
obligately alkaliphilic, oval-spore-forming Bacillus species with ornithine and aspartic acid instead of diaminopimelic acid in the cell wall.
Int J Syst Bacteriol 43, 150–156.
Tomblin, J. F. (1979). The South Sandwich Islands. II. The geology of
Candlemas Island. Br Antarct Surv Sci Rep 92, 1–33.
Vauterin, L. & Vauterin, P. (1992). Computer aided objective
comparison of electrophoretic patterns for grouping and identification
of microoganisms. Eur Microbiol 1, 37–41.
Vauterin, L., Yang, P., Hoste, B., Vancanneyt, M., Civerolo, E. L.,
Swings, J. & Kersters, K. (1991). Differentiation of Xanthomonas
campestris pv. citri strains by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of proteins, fatty acid analysis, and DNA-DNA
hybridization. Int J Syst Bacteriol 41, 535–542.
Versalovic, J., Koeuth, T. & Lupski, J. R. (1991). Distribution of
repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 19, 6823–6831.
http://ijs.sgmjournals.org
1989
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 14:28:18