ISSN 0026-2617, Microbiology, 2016, Vol. 85, No. 6, pp. 693707. Pleiades Publishing, Ltd., 2016.

Original Russian Text T.P. Tourova, D.Sh. Sokolova, E.M. Semenova, E.S. Shumkova, A.V. Korshunova, T.L. Babich, A.B. Poltaraus, T.N. Nazina, 2016, published
in Mikrobiologiya, 2016, Vol. 85, No. 6, pp. 676692.

EXPERIMENTAL ARTICLES

Detection of n-Alkane Biodegradation Genes alkB and ladA

in Thermophilic Hydrocarbon-Oxidizing Bacteria
of the Genera Aeribacillus and Geobacillus
T. P. Tourovaa, D. Sh. Sokolovaa, E. M. Semenovaa, E. S. Shumkovaa, A. V. Korshunovaa, T. L. Babicha,
A. B. Poltarausb, and T. N. Nazinaa, *
a
Winogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia
bEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

*e-mail: nazina@inmi.ru
Received June 15, 2016

AbstractAbility to degrade crude oil n-alkanes was revealed in new strains of thermophilic bacilli isolated
from petroleum reservoirs and a hot spring: Geobacillus toebii -1024, Geobacillus sp. 1017, and Aeribacillus
pallidus 8m3. The strains utilized 1030 n-alkanes (-1024), 10, C11, and 1319,22 n-alkanes (1017),
and C11C29 n-alkanes (8m3). In all three strains, PCR amplification with specific degenerate oligonucle-
otide primers revealed the alkB gene encoding rubredoxin-dependent alkane monooxygenase. In strains
-1024 and 1017, fragments of the genes homologous to the ladA gene determining flavin-dependent alkane
monooxygenase were also amplified. Nucleotide sequences of these genes were practically identical to those
of the genes ladAB23, ladAB23, and ladBB23, which were revealed previously in Geobacillus thermoleovorans
strain B23. For the latter strain, activity of respective enzymes in the oxidation of long-chain n-alkanes has
been shown. Thus, simultaneous presence of the alkB and ladA genes coding alkane monooxygenases respon-
sible for oxidation of medium-chain and long-chain n-alkanes in thermophilic bacilli was revealed for the
first time.

Keywords: alkB and ladA genes, alkane monooxygenases, Geobacillus, Aeribacillus, thermophilic bacteria, oil
degradation
DOI: 10.1134/S0026261716060199

The known data on n-alkane biodegradation genes
were mostly obtained for mesophilic bacteria (Whyte
et al., 2002; van Beilen et al., 2006). Alkane hydroxy-
lase, a three-component enzyme system with alkane
monooxygenase (AlkB) encoded by the alkB gene as
the key component, is most widespread among meso-
philic n-alkane degraders. Mesophilic bacteria are
also known to possess cytochrome P450 monooxy-
genases involved in n-alkane oxidation and belonging
to the cytochrome CYP153 family (Funhoff et al.,
2006). Similar to AlkB, for the operation of CYP153
rubredoxin and rubredoxin reductase are required.
Both monooxygenases transform medium-chain
n-alkanes (517). Mesophilic bacteria of the genera
Alcanivorax, Dietzia, Rhodococcus, Nocardia, and Gor-
donia are known to possess both AlkB and CYP153
simultaneously, which is possibly an indication of the
interaction of these enzyme systems in the course of
hydrocarbon oxidation (Whyte et al., 2002; van Beilen
et al., 2006; Wang et al., 2010; Liu et al., 2011; Lo Pic-
colo et al., 2011). A flavin-dependent dioxygenase
encoded by the almA gene and requiring copper ions
for operation, which degrades long-chain n-alkanes
(1030), is another enzyme revealed in some marine
mesophilic proteobacteria (Throne-Holst et al.,
2007).
The process of oil biodegradation by thermophilic
microorganisms and the responsible genes remain
insufficiently studied. Thermophilic bacteria of the
genus Geobacillus are common oilfield inhabitants and
can grow on crude oil, individual n-alkanes, and aro-
matic hydrocarbons (Nazina et al., 2005; Marchant
and Banat, 2010). Among thermophilic bacteria, the
fragments of the alkB gene sequences were initially
found in Geobacillus thermoleovorans 70 and G. ther-
moglucosidasius TR2 (Sharkey et al., 2004; Marchant
et al., 2006). The alkB gene sequences encoding
alkane monooxygenases of 12 other Geobacillus spe-
cies were subsequently determined (Tourova et al.,
2008; Korshunova et al., 2011). For the first time, the
presence of eight variants of the alkB gene (alkB-geo1,
alkB-geo2, alkB-geo3, alkB-geo4, alkB-geo5, alkB-
geo6, alkB-geo7, and alkB-geo8) in geobacilli was
shown (Tourova et al., 2008). Some strains were found

693
694 TOUROVA et al.
to contain three to seven homologs of the alkane
monooxygenase gene (alkB), of which only two were
common for all strains (Tourova et al., 2008; Kor-
shunova et al., 2011). High similarity between most of
the alkB homologs of Geobacillus and Rhodococcus
species was shown (Marchant et al., 2006; Tourova et
al., 2008; Liu et al., 2009; Korshunova et al., 2011).
Localization of the alkB genes in the genome of
thermophilic geobacilli remains ambiguous. Chromo-
somal localization of the alkB genes in the genome of
a hydrocarbon-degrading bacterium G. subterraneus K
was suggested based on the results of experiments with
its chromosomal and plasmid DNA (Korshunova
et al., 2011). The alkB genes were, however, not
revealed in any of completely sequenced chromo-
somes of a number of Geobacillus strains.
Another class of enzymes responsible for oxidation
of long-chain n-alkanes (15H32 and longer, up to
36H74) was recently discovered in thermophilic geo-
bacilli. The ladA functional gene determining this
novel type of alkane monooxygenase (LadA) was orig-
inally detected in the genome of a thermophilic bacte-
rium G. thermodenitrificans NG80-2 (Feng et al.,
2007). The LadA structure was subsequently deter-
mined (Li et al., 2008). Similar to AlmA, this enzyme
is a flavin-dependent monooxygenase of the family of
bacterial luciferases. The ladA gene (1323 bp) is
located on the pLW1071 plasmid (Feng et al., 2007).
The structure of LadA monooxygenase is unique: the
polypeptide is functionally segregated into three
domains, one of which acts as a monooxygenase and
two others that are responsible for NAD(P)H oxida-
tion, i.e., they act as a rubredoxin and a rubredoxin
reductase, respectively (Feng et al., 2007; Li et al.,
2008). Due to this structure, the LadA monooxygen-
ase is able to hydroxylate n-alkanes in the absence of
rubredoxin and rubredoxin reductase, which are
required for the functioning of other alkane hydroxy-
lase enzyme systems.
Three genes homologous to ladA and designated
ladAB23, ladAB23, and ladB23 were recently revealed CaCl2 2H2O, 0.02; NaCl, 2.0; MgSO4 7H2O, 0.2;
in the sequenced genome of G. thermoleovorans B23
(Boonmak et al., 2014). However, in strain B23, unlike
G. thermodenitrificans NG80-2, these three genes were
located on the chromosome within a gene cluster the
authors termed ladAB gene island. This gene cluster
contained also the gene of NADPH-dependent flavin
mononucleotide (FMN) reductase, which reduces
FMN in the active center of the LadA alkane monoo-
xygenase. Similar gene clusters were found in the
sequenced genomes of five other Geobacillus strains.
The authors also found that the ladA homologs
encode active monooxygenases, which are presumably
involved in the oxidation of a broad spectrum of
alkanes.
We have recently isolated a number of strains
(B-1024, vw3-1, 1017, and 8m3) of thermophilic
spore-forming hydrocarbon-oxidizing bacteria of the
genera Geobacillus and Aeribacillus (Tourova et al.,
2010; Shestakova et al., 2011), although the traits of
their physiology and n-alkane biodegradation remain
insufficiently studied.
The goal of the present work was to investigate bio-
degradation of oil n-alkanes by thermophilic bacteria
of the genera Geobacillus and Aeribacillus and to detect
the genes encoding the alkane hydroxylases AlkB and
LadA and the alkane hydroxylase of the cytochrome
CYP153 family.
MATERIALS AND METHODS
Bacterial strains. The strains of thermophilic
spore-forming bacteria 8m3, vw3-1, and 1017, which
were isolated from formation water of the high-tem-
perature Kongdian bed of the Dagang oilfield (Hebei
province, PRC), and strain B-1024 isolated from a
thermal spring in the Baikal rift zone (Tourova et al.,
2010; Shestakova et al., 2011) were used in the work.
Geobacillus icigianus strain G1w1T (=VKM B-2853T =
DSM 28325T), which was isolated from a Kamchatka
hot spring and kindly provided by the workers of the
Institute of Cytology and Genetics, Siberian Branch,
Russian Academy of Sciences (Bryanskaya et al., 2014,
2015), was also studied. Alkane monooxygenase genes
were also monitored in the previously studied strains
G. thermoleovorans DSM 5366T, G. jurassicus DS2,
G. subterraneus 34T, G. uzenensis UT, and G. subter-
raneus K, as well as in mesophilic Dietzia natronolim-
naea 263 (Nazina et al., 2001, 2015).
Media composition and cultivation conditions. All
strains were maintained on rich agar medium contain-
ing the following (g/L): bacto tryptone, 5.0; yeast
extract, 2.5; agar, 20.0; pH 7.0. The spectrum of sub-
strates utilized by strains 8m3, B-1024, and 1017 was
determined in the medium containing the following
(g/L): KH2PO4, 0.75; K2HPO4, 1.5; NH4Cl, 1.0;
KCl, 0.1; pH 6.87.2. The optima of temperature and
salinity were determined in the modified medium
(Adkins et al., 1991) containing the following (g/L):
K2HPO4, 2.0; NaH2PO4, 0.5; (NH4)2SO4, 1.5; NaCl,
2.0; MgSO4 7H2O, 0.1; CaCl2 2H2O, 0.132; yeast
extract, 2.0; pH 6.87.2. Bacteria were grown in sta-
tionary cultures at 60C.
For investigation of the n-alkane biodegradation
genes, bacteria were grown in the Adkins mineral
medium supplemented with oil or individual
n-alkanes (0.51% vol/vol) and yeast extract
(0.25 g/L) as a vitamin source.
Spectrophotometry. Biomass growth in liquid
medium was determined as OD600 measured on an
Ultrospec 2100 pro spectrophotometer (Amersham
MICROBIOLOGY Vol. 85 No. 6 2016
 DETECTION OF n-ALKANE BIODEGRADATION 695

Table 1. Conditions for amplification of alkane monooxygenase genes and the primers used
Reaction protocol (stage,
Gene Primers (name and sequence) Reference
temperature, and duration)
alkB Alk-BFB 5'-GGTACGGSCAYTTCTACRTCGA-3' Round 1: 194, 5 min; Tourova
Alk-BRB 5'-CGGRTTCGCGTGRTGRT-3' 294, 30 s; 60, 30 s; et al., 2008
72, 30 s; 30 cycles;
372, 8 min
Round 2: 194, 5 min;
294, 30 s; 60, 30 s;
72, 30 s; 25 cycles;
372, 8 min
ladAB23 Alpha-F 5'-AATTCCATATGGATCAACCCCTATTATTC-3' Round 1: 195, 60 s; Boonmak
Alpha-R 5'-TAGGGCCCGGGCTAGCCATCTGTTCGTGAGC-3' 295, 30 s; 65, 90 s; et al., 2014
ladAB23 Beta-F 5'-AATGGCATATGAGTGTGCGGCAAATG-3' 72, 60 s; 30 cycles;
Beta-R 5'-GATTCCCGGGTCACACGCGGATCGACTT-3' 372, 8 min
Round 2: 195, 60 s;
ladB23 BF 5'-GGAAACATATGGTTGAATTTATTACGATGG-3' 295, 30 s; 65, 90 s;
BR 5'-CGACCCGGGTTAGATCCATACTTCCGTTTGT-3' 72, 60 s; 25 cycles;
372, 8 min
cyp153A P450F 5'-TGTCGGTTGAAATGTTCATYGCNMTGGAYCC-3' 194, 3 min; Wang
P450R 5'-TGCAGTTCGGCAAGGCGGTTDCCSRYRCAVCKRTG-3' 294, 30 s; 54, 30 s; et al., 2010
72, 80 s; 35 cycles;
3 2, 8 min

Biosciences, United Kingdom). Uninoculated
medium was used as the control.
Gas chromatography. Bacterial growth in the
medium with crude oil was determined by measuring
the content of n-alkanes and iso-alkanes of the ali-
phatic fraction of degraded oil compared to the con-
trol (%) (Borzenkov et al., 2006). The cultures were
grown in mineral medium with 5 g/L oil and 0.25 g/L
yeast extract at 60 for 1421 days. The uninocu-
lated control with oil was incubated under the same
conditions. Residual oil was extracted with chloro-
form, the extract was dehydrated with Na2SO4 and fil-
tered, and the solvent was removed in a rotary evapo-
rator. Oil was then transferred into a glass vial and
dried for 24 h at room temperature. The decrease in
the n-alkane content was determined by gasliquid
chromatography on a Kristall 5000.1 chromatograph
(Khromatek, Russia) with a flame ionization detector
and a ZB-FFAP 15-m capillary column. Helium was
used as a carrier gas. The initial column temperature
was 100, the final temperature was 320, and the
heating rate was 5/min. Quantification of the chro-
matographic data was carried out using the method of
internal normalization. The internal standard used for
analysis of the chromatograms of the crude oil ali-
phatic fraction was the total height of phytane and
pristane peaks (iso-C19 + iso-C20). In some experi-
ments, 2,2,4,4,6,8,8-heptamethylnonane was added
as an internal standard.
DNA isolation. DNA was isolated from the biomass
using the DIAtomTMDNA Prep100 kit (Biokom, Rus-
sia) according to the manufacturers recommenda-
tions. The purified DNA preparation was dissolved in
50 L of deionized water (MQ) and used as a template
for PCR.
Amplification of alkane monooxygenase genes. PCR
conditions and the primers used for amplification of
alkane monooxygenase genes alkB, ladA, ladA,
ladB, and cyp153A are listed in Table 1. Amplification
of the alkB, ladA, ladA, and ladB genes was per-
formed in two stages. The first stage was carried out in
10 L of the mixture, with DNA (525 ng) as a tem-
plate. The PCR product obtained in the first round
was used as a template for the second PCR round. The
optimal annealing temperature was determined exper-
imentally. To remove the primers, PCR product was
precipitated with ammonium acetate and ethanol
(final concentrations of 1.125 M and 65%, respec-
tively), incubated for 20 min at room temperature, and
centrifuged for 20 min at 13000 g. The pellet was
washed with 70% ethanol. The purified PCR product
was dissolved in 10 L illi-Q. Visualization was
achieved by electrophoresis in 1% agarose gel in 0.5
TBE buffer. The gel was supplemented with ethidium
bromide (0.5 g/mL).
Sequencing of PCR products. DNA was sequenced
according to Sanger on an ABI 3730 DNA Analyzer
automatic sequencer using the ABI PRISM BigDyeTM
Terminator v. 3.1 kit (Applied Biosystems, United

MICROBIOLOGY Vol. 85 No. 6 2016

696 TOUROVA et al.
States). The primers listed above were used for
sequencing of alkane monooxygenase genes.
Analysis of the nucleotide sequences. Assembly,
editing, and translation of the nucleotide sequences
were carried out using Bioedit (http://jwbrown.
mbio.ncsu.edu/BioEdit/bioedit.html). Preliminary
analysis of the sequences was performed using the
BLAST algorithm (NCBI server, www.ncbi.
nlm.nih.gov/blast/). Phylogenetic trees were con-
structed using the TREECONW software package
(Van de Peer and De Wachter, 1994).
Nucleotide sequences of the ladA, ladA, and
ladB genes of bacteria G. toebii -1024, Geobacillus sp.
1017, and A. pallidus 8m3 were deposited into Gen-
Bank under accession nos: KX579924KX579932.
RESULTS
Phylogenetic Analysis of New Geobacillus Strains
Based on analysis of the genes of 16S rRNA, gyrase
(gyrB), and topoisomerase IV (r), the isolates were
previously assigned to the species Geobacillus toebii
(strains B-1024 and vw3-1), Geobacillus kaustophilus
(strain 1017), and Geobacillus pallidus (strain 8m3)
(Tourova et al., 2010; Shestakova et al., 2011). Subse-
quently, however, a number of new species were
described and changes in taxonomy of the genus Geo-
bacillus were proposed. As a result, some species were
abrogated, and the species G. pallidus and G. debilis
were assigned to new genera, Aeribacillus and Caldiba-
cillus, respectively (Miana-Galbis et al., 2010; Coor-
evits et al., 2012). Moreover, application of new data
on genome sequencing for a number of Geobacillus
species and members of related genera for phyloge-
netic analysis facilitated more accurate identification
of the studied Geobacillus strains. A new phylogenetic
analysis of the genes of 16S rRNA, gyrase (gyrB), and
topoisomerase IV (r) was therefore carried out
using available data on the type strains of Geobacillus,
Aeribacillus, Caldibacillus, and Anoxybacillus species.
For phylogenetic analysis, we used 21 Geobacillus
strains and 1 Anoxybacillus strainin the genomes of
which BLAST analysis of the GenBank data revealed
homologs of the ladAB23, ladAB23, and ladB23
genesas well as G. thermodenitrificans strain
NG80-2, which was found to contain the ladA gene in
the plasmid part of its genome (Feng et al., 2007).
Topology of the phylogenetic tree obtained by
analysis of the 16S rRNA genes (Fig. 1) was similar to
that revealed by previous analysis (Tourova et al.,
2010). Identification of strains ww3 and B-1024 as
G. toebii was confirmed; the new strain 8m3 was iden-
tified as A. pallidus. However, position of strain 1017 in
cluster I, the largest cluster of closely related Geobacil-
lus species, remained ambiguous, indicating its possi-
ble identification as a new species.
According to analysis of the 16S rRNA gene
sequences, most Geobacillus strains which contained
the genes homologous to ladAB23, ladAB23, and lad-
B23 in their sequenced genomes belonged to cluster I
(17 strains including the G. icigianus type strain).
Among them six strains were tentatively identified at
the species level, while the position of others remained
ambiguous. Moreover, initial identification of strain
PSS2 as a G. subterraneus strain was doubtful, since it
fell into the same subcluster with the type strain of
G. icigianus. The remaining four Geobacillus strains,
including the G. thermoglucosidasius type strain,
formed a compact subcluster within cluster II, which
confirmed their identification. Genus identification of
Anoxybacillus sp. strain ATCC BBA-2555 was also
confirmed; it fell into the cluster of Anoxybacillus
strains as a separate lineage.
Divergence between the sequences of the house-
keeping genes gyrB and r in most Geobacillus spe-
cies was previously shown to exceed the level deter-
mined by comparison of the 16S rRNA sequences.
Topologies of the relevant trees, while correlating with
that of the 16S rRNA gene tree, were considerably
more complex, making it possible to determine the
individual branches (subclusters), which to a signifi-
cant degree correspond to the species structure of the
genus Geobacillus (Nazina et al., 2001; Tourova et al.,
2010). This was especially pronounced in the case of
closely related Geobacillus species of cluster I, which
were not easily differentiated using the 16S rDNA tree.
On the relevant GyrB and ParE trees, the species
G. thermodenitrificans, G. subterraneus, G. vulcani,
G. icigianus, and G. stearothermophilus of cluster I
formed individual lineages (subclusters) and were eas-
ily differentiated (Figs. 1b, 1c). The species G. thermo-
catenulatus was well differentiated only on the ParE
tree, while the species pairs G. uzenensisG. jurassicus
and G. thermoleovoransG. kaustophilus remained
indistinguishable on both trees.
The new strain 1017 also formed an individual lin-
eage of the GyrB and ParE trees, which probably indi-
cated its species status and will require in-depth phe-
notypic analysis. Only the gyrB gene was previously
amplified for the new strain 8m3 (Tourova et al.,
2010); the relevant gene of the type strain A. pallidus is
not present in available databases. On the GyrB tree
strain 8m3 formed an individual deep-branching lin-
eage, which confirmed its identification as a strain of
Aeribacillus, rather than of Geobacillus. While only the
parE gene was previously amplified for strains -1024
and vw3-l (Tourova et al., 2010), on the relevant tree
they formed a subcluster together with G. toebii, con-
firming their identification as strains of this species.
According to the results of gyrB and parE analysis,
the Geobacillus strains containing homologs of the
MICROBIOLOGY Vol. 85 No. 6 2016
 DETECTION OF n-ALKANE BIODEGRADATION 697

(a)
Geobacillus thermoleovorans CCB_US3_UF5, NR_074931
0.02 Geobacillus thermoleovorans B23, BATY01000041
Geobacillus sp. A8, KM192159
Geobacillus sp. CAMR5420_1482739.4, JHUS01000002
Geobacillus sp. FW23, JF 683583
Geobacillus kaustophilus NBRS 102445T, X60618
Geobacillus sp. ZGt-1, KT026965
Geobacillus thermoleovorans DSM 5366T, Z26923
Geobacillus sp. WSUCF1, ATCO01000178
Geobacillus lituanicus N-3T, AY044055
Geobacillus vulcani PSS 1, JPOI01000001
99 Geobacillus vulcani 3S-1T, AJ293805
Geobacillus sp. GHH01, CP004008
Geobacillus thermocatenulatus GS-1, JFHZ01000108
89
Geobacillus sp. BCO2, LJAJ01000125 I
Geobacillus sp. T6, GCA_001025095.1
92
Geobacillus thermocatenulatus BGSC 93A1T, AY608935
Geobacillus sp. 1017, GU459251
Geobacillus stearothermophilus R-35646T, FN428694
99 Geobacillus uzenensis UT, AF276304
Geobacillus jurassicus DS1T, AY312404
99 Geobacillus icigianus G1w1T, KF631430
Geobacillus sp. B4113_201601, NZ_LQYX01000047
99 Geobacillus subterraneus PSS2, JQMN01000001
100
Geobacillus sp. LC300, CP008903
Geobacillus subterraneus 34T, AF276306
Geobacillus sp. G11MC16, NZ_ABVH01000028
86
100 Geobacillus thermodenitricans NG80-2, NR_074976
80 81 Geobacillus thermodenitricans R-35647T, FN538993
92 Geobacillus sp. Y4.1MC1_581103.5, NC_014650
Geobacillus thermoglucosidasius DSM2542T, FN428685
100 Geobacillus thermoglucosidasius TNO-09.020, CM001483
Geobacillus thermoglucosidasius C56-YS93, NR102795
98 Geobacillus thermantarctius DSM 9572T, FR749957
98 II
Geobacillus caldoxylosilyticus S1812T, AF067651
Geobacillus galactosidasius CF1BT, AM408559
Geobacillus toebii R-35642T, FN428690
91
Geobacillus toebii B-1024, GU994003
100 Geobacillus toebii vw3-1, GU994004
Coldibacillus debilis R-35653T, FN428699
81 Anoxybacillus tepidamans R-35643T, FN428691
Anoxybacillus sp. ATCC BBA-2555, KJ722458
Anoxybacillus amylolyticus DSM 15939T, CP015438
100 Aeribacillus pallidus 8m3, GU459252
Aeribacillus pallidus DSM3670T, Z26930

Fig. 1. Phylogenetic trees of bacteria of the genera Geobacillus, Aeribacillus, and closely related genera of thermophilic bacilli con-
structed based on the 16S rRNA gene sequences (a) and on the sequences of the housekeeping genes gyrB (b) and r (c). The
trees were constructed using the neighbor-joining algorithm, with the . subtilis sequence as an outgroup. The organisms studied
in this work are indicated in boldface, type strains of the reference species are marked by gray shading, and G. thermoleovorans
B23, the strain in which the ladAB genes were initially detected, is indicated by a frame. White and black arrows mark the organ-
isms possessing the alkB and ladAB genes, respectively. The clusters are numbered according to Korshunova et al. (2011). The
numerals indicate the branching order determined by bootstrap analysis (values over 80% were considered significant). Scale bar
shows the evolutionary distance corresponding to 2 (Fig. 2a) and 5 replacements (Figs. 2b, 2c) per 100 nucleotides.

MICROBIOLOGY Vol. 85 No. 6 2016

698 TOUROVA et al.

(b)
Geobacillus thermoleovorans B23,BATY01000041
0.05 Geobacillus thermoleovorans CCB_US3_UF5,NC_016593
Geobacillus sp. FW23, NZ_JGCJ01000013
Geobacillus kaustophilus NBRS 102445T, NZ_BBJV01000018

Geobacillus thermoleovorans DSM 5366T, NZ_CP014335

Geobacillus sp. WSUCF1, NZ_ATCO01000182
Geobacillus sp. GHH01, NC_020210
Geobacillus sp. CAMR5420_1482739.4, NZ_JHUS01000002 IA
Geobacillus sp. ZGt-1, NZ_LDPD01000028
Geobacillus sp. A8, NZ_AUXP01000012
100
Geobacillus sp. BCO2, LJAJ01000051
Geobacillus sp. T6, NZ_LDNZ01000060
100 Geobacillus thermocatenulatus GS-1, NZ_JFHZ01000043
94
T
100 100 Geobacillus thermocatenulatus BGSC 93A1 , GU323952
Geobacillus vulcani PSS 1, JPOI01000001
Geobacillus sp. 1017, GU459239
100 Geobacillus uzenensis UT, GU459233
Geobacillus jurassicus DS1T, BCQG01000011
IC

100 Geobacillus icigianus G1w1T, NZ_JPYA01000010

82 Geobacillus sp. B4113_201601, LQYX01000115
100 Geobacillus subterraneus PSS 2, NZ_JQMN01000001
Geobacillus stearothermophilus R-35646T, NZ_JQCS01000193
100
100 Geobacillus sp. LC300, NZ_CP008903 IB
Anoxybacillus sp. ATCC BBA-2555, gvrB-2, JYCG01000149

Geobacillus subterraneus 34T, GU459230 IE

95 100 Geobacillus sp. G11MC16, NZ_ABVH01000007

Geobacillus thermodenitricans NG80-2, NC_009328 ID
Geobacillus thermodenitricans R-35647T, NZ_AYKT01000007
Coldibacillus debilis R-35653T, NZ_KB912886
Geobacillus caldoxylosilyticus S1812T, NZ_BAWO01000004
100 Geobacillus toebii R-35642T, NZ_BDAQ01000005 IIB
99 Geobacillus thermoglucosidasius TNO-09.020, AJJN01000011
99 100 Geobacillus thermoglucosidasius C56-YS93, NZ_ADNQ01000024
IIA
Geobacillus sp. Y4.1MC1_581103.5, NC_014650
96 Geobacillus thermoglucosidasius DSM2542T, NZ_CP012712

Anoxybacillus amylolyticus DSM 15939T, CP015438

Anoxybacillus sp. ATCC BBA-2555, gvrB-1, NZ_JYCG01000277
86
Anoxybacillus tepidamans R-35643T, NZ_JHVN01000001
Aeribacillus pallidus 8m3, GU459236

Fig. 1. (Contd.)

ladAB23, ladAB23, and ladB23 genes in their (one strain), G. thermodenitrificans (one strain), and
sequenced chromosomes fell into subclusters G. ther- G. thermoglucosidasius (three strains). One strain pre-
moleovoransG. kaustophilus (seven strains), G. ther- viously identified at the genus level, as well as strain
mocatenulatus (two strains), G. stearothermophilus PSS2 (which was initially identified as G. subter-

MICROBIOLOGY Vol. 85 No. 6 2016

 DETECTION OF n-ALKANE BIODEGRADATION 699

(c)
0.05 Geobacillus sp. FW23, JGCJ01000050
Geobacillus sp. GHH01, NC_020210
Geobacillus sp. ZGt-1, LDPD01000018
Geobacillus sp. A8, AUXP01000035
Geobacillus thermoleovorans B23, BATY01000109
98
Geobacillus kaustophilus NBRS 102445T, BBJV01000017
IA-2
Geobacillus thermoleovorans DSM 5366T, CP014335
94 Geobacillus sp. CAMR5420_1482739.4, JHUS01000039
Geobacillus sp. WSUCF1, ATCO01000182

80 Geobacillus thermoleovorans CCB_US3_UF5, NC_016593

Geobacillus sp. 1017, GU459248
Geobacillus sp. BCO2, LJAJ01000019
Geobacillus sp. T6, LDNZ001000155
100 IA-1
Geobacillus thermocatenulatus BGSC 93A1T, GU323953
94
Geobacillus thermocatenulatus GS-1, JFHZ01000098
T
100 Geobacillus uzenensis U , GU459245 IC
Geobacillus jurassicus DS1T, BCQG01000021
Geobacillus stearothermophilus R-35646T, JQCS01000147
Geobacillus sp. LC300, NZ_CP008903 IB
100
100 Anoxybacillus sp. ATCC BBA-2555, gvrB-2, JYCG01000043
Geobacillus vulcani PSS 1, JPOI01000001
98
Geobacillus sp. B4113_201601, LQYX01000032
100
Geobacillus icigianus G1w1T, JPYA01000088
98
100 Geobacillus subterraneus PSS2, JQMN01000001
Geobacillus subterraneus 34T, GU459241 IE
Geobacillus sp. G11MC16, ABVH01000019
99
T
100 Geobacillus thermodenitricans R-35647 , AYKT01000008 ID
Geobacillus thermodenitricans NG80-2, CP000557
Geobacillus caldoxylosilyticus S1812T, BAWO01000014
97 Geobacillus toebii B-1024, GU323954
100 Geobacillus toebii vw3-1, GU459350 IIB
81
Geobacillus toebii R-35642T, BDAQ01000004
Geobacillus thermoglucosidasius TNO-09.020, AJJN01000013
Geobacillus thermoglucosidasius DSM 2542T, BAWP01000021
100 IIA
Geobacillus sp. Y4.1MC1_581103.5, NC_014650
Geobacillus thermoglucosidasius C56-YS93, NC_015660
Anoxybacillus tepidamans R-35643T, JHVN01000010

90 Anoxybacillus amylolyticus DSM 15939T, CP015438

Anoxybacillus sp. ATCC BBA-2555, gvrB-1, JYCG01000102
Caldibacillus debilis R-35653T, ARVR01000013

Fig. 1. (Contd.)

raneus), belonged to the G. icigianus subcluster.
According to these findings, the taxonomic position of
strain PSS2 should be reconsidered.
Analysis of the gyrB and r genes from the
sequenced genome of Anoxybacillus sp. (Filippidou
et al., 2015) yielded unexpected results. Its shotgun
assembly contained two sets of these genes, which is
unusual for bacterial housekeeping genes. The genes
of one set, similar to the 16S rRNA gene of this strain,
belonged to the Anoxybacillus species cluster, while the
genes of the other set fell into the subcluster of the rel-
evant genes of G. stearothermophilus. Since this result

MICROBIOLOGY Vol. 85 No. 6 2016

700 TOUROVA et al.
caused doubts concerning the purity of the culture,
this strain was excluded from further phylogenetic
analysis of the homologs of the ladAB23, ladAB23,
and ladB23 genes.
Morphological and Physiological Characterization
of Thermophilic Bacilli and Degradation
of Oil n-Alkanes
Phenotypic characteristics of the strains 1017,
-1024, and 8m3, which were chosen for in-depth
analysis, are listed in Table 2. The strains were spore-
forming rods exhibiting positive Gram reaction. The
cells of strains 1017 and B-1024 were nonmotile, while
those of strain 8m3 were motile with peritrichous flag-
ellation. Endospore location was subterminal in
A. pallidus 8m3 and Geobacillus sp. 1017 and terminal
in G. toebii -1024. The colonies of all strains grown at
60 on agar medium with tryptone and yeast extract
were opaque, with light beige pigmentation, mucous,
homogeneous structure, convex profile, rounded
shape, and even edges; they differed, however, in size
and in surface type (Table 2). All strains were freshwa-
ter bacteria with growth maximum in the absence of
NaCl. The strains Geobacillus sp. 1017 and A. pallidus
8m3 grew within the range from 0 to 2% NaCl, while
G. toebii -1024 grew weakly in the medium with 5%
NaCl. Growth occurred within the temperature range
from 38 to 6870, with the maximum at 60, 2014). Such PCR products were not detected in the
which agreed with the temperature in the habitats from
which bacteria were isolated. A broad range of organic
substrates (sugars, organic acids, amino acids, indi-
vidual n-alkanes, and crude oil) was used as carbon
and energy sources for aerobic growth. All phenotypic
features of A. pallidus strain 8m3 were confirmed by
genome analysis, which revealed the relevant genetic
determinants (Poltaraus et al., 2016).
Degradation of the n-alkane fraction of crude oil
was studied for the new and known strains A. pallidus
8m3, G. toebii -1024, Geobacillus sp. 1017, G. thermo-
leovorans 5366T, and G. icigianus G1w1T. While G. ici-
gianus G1w1T is one of the few validly described mem-
bers of the genus, for which the ladAB were revealed in
the genome and description of this species mentioned
hydrocarbon utilization, but not their spectrum. Oil
from the Uzen oilfield contained a broad spectrum of
saturated n-alkanes (Fig. 2f). Residual n-alkane
levels in degraded oil presented on Figs. 2a2e show
that n-alkanes with different chain lengths were uti-
lized. G. icigianus G1w1T (Fig. 2a; 1230), G. toebii
-1024 (Fig. 2b; 1030), and A. pallidus 8m3 (Fig. 4) the sequences of each of the genes formed sep-
(Fig. 2c; 1228) utilized the broadest range of
n-alkanes. Geobacillus sp. 1017 (Fig. 2d) grew on 10,11
and 1319,22 n-alkanes, while G. thermoleovorans
5366T and strain G1w1T used a broad spectrum of
n-alkanes, including long-chain ones (Fig. 2e, 22
29, 3234).
Detection and Analysis of Alkane Monooxygenase Genes
of Thermophilic Bacilli
Search for the genes of n-alkane biodegradation
(ladA, ladA, ladB, cyp153A, and alkB) in new
strains of thermophilic bacilli 8m3, B-1024, vw3-l,
1017, and G. icigianus G1w1T was carried out by PCR
with the primers listed in Table 1. The known Geoba-
cillus strains G. thermoleovorans DSM 5366T, G. juras-
sicus DS2, G. subterraneus strains 34T and K, and
G. uzenensis UT, which are capable of degrading a
broad spectrum of n-alkanes (Nazina et al., 2001,
2005), were also investigated. The expected length of
PCR products was ~1500 bp for ladA and ladA,
~1000 bp for ladB, 800 bp for cyp153A, and 500 bp for
alkB.
The ladAB genes. Primers for the genes encoding
flavin-dependent alkane monooxygenase involved in
the degradation of long-chain alkanes (ladA, ladA,
and ladB) were applied to obtain specific PCR prod-
ucts (~1500 bp for ladA and ladA, ~1000 bp for
ladB), using as templates the DNA of new strains 1017
and B-1024, and of the type strain G. icigianus G1w1T
(Figs. 3a3c), which was previously shown to possess
the ladA, ladA, and ladB genes (Braynskaya et al.,
DNA of strains vw3-l, G. jurassicus DS2, G. thermole-
ovorans DSM 5366T, G. subterraneus strains 34T and
K, G uzenensis UT, and A. pallidus 8m3 (data not
shown).
The sequenced fragments of PCR products from
strains 1017, B-1024, and G1w1T exhibited high
homology with the relevant ladAB23, ladAB23, and
ladB23 genes of G. thermoleovorans B23 (98.699.9%
by nucleotides and 98.399.8% by amino acid resi-
dues). At the same time, the sequences of the ladA,
ladA, and ladB gene fragments of G. icigianus G1w1T
obtained by PCR were identical to the corresponding
sequences of these genes present in the shotgun
assembly (Braynskaya et al., 2014).
For phylogenetic analysis of obtained ladA,
ladA, and ladB gene fragments, translated amino acid
sequences of the relevant genes revealed by BLAST
analysis in the total genomes presently available in
GenBank were used for reference. Homologs of all
three genes were reliably detected only in the genomes
of Geobacillus strains (6 complete genomes and
15 shotgun assemblies). On the phylogenetic tree
arate clusters, with the clusters of the ladA and ladA
genes exhibiting higher similarity to each other (~40
45% homology) than to the ladB gene sequences
(~2530%). The ladB gene was more closely related
MICROBIOLOGY Vol. 85 No. 6 2016
 DETECTION OF n-ALKANE BIODEGRADATION 701

Table 2. Morphological and physiological characteristics and taxonomic position of the studied aerobic thermophilic bacilli
Strain
Feature
B-1024 1017 8m3
16S rRNA gene similarity, %, phylogeneti- 99.4; G. toebii SK-1 99.5; G. kaustophilus R-35646 99.8; A. pallidus DSM 3670T
cally related strain (acc. number) (NR_025143) (FN428694) (NR_026515)
gyrB gene similarity, %, phylogenetically ND** 96.0; G. kaustophilus NBRC 74.0; G. toebii R-35642T,
related strain (acc. number) 102445T, (DAQ01000005)
(NZ_BBJV01000018)
r gene similarity, %, phylogenetically 99.2; G. toebii 97.0; G. kaustophilus NBRC ND**
related strain (acc. number) R-35642T, 102445T, (BBJV01000017)
(BDAQ01000004)
Colonies:
Size, mm 1.06.4 1.04.0 1.04.5
Surface Smooth Rough Smooth
Motility +
Growth on*:
Propionate +
Lactate +
Succinate + + +
Malate + + +
Fumarate + + +
Pyruvate + + +
Alanine +
Acid produced from sugars:
Sucrose +/ + +
Fructose + + +
Trehalose + +
Xylose + +
Cellobiose +/ + +
Raffinose + + +
Utilization of n-alkanes:
n-octane (8H18)
n-undecane (11H24) + + +
n-dodecane (12H26) + +
n-tridecane (13H28) + + +
n-hexadecane (16H34) + + +
Squalane (30H62) +
Crude oil + + +
Catalase + + +
Temperature range for growth, 3870 3868 3870
* Butyrate, isobutyrate, isopropanol, oxalate, methanol, ethanol, formate, and arabinose were not used for growth by all strains.
** ND stands for no data. + and designate the presence or absence of the feature, respectively.

MICROBIOLOGY Vol. 85 No. 6 2016

702 TOUROVA et al.

() (b) (c)
100 100 100
90 90 90
80 80 80
70 70 70
60 60 60
50 50 50
40 40 40
30 30 30
20 20 20
10 10 10
Residual n-alkanes, %

0 0 0
11
12
13
i16
14
iC17
C15
C16
iC18
C17
iC19
C18
iC20
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30

10
11
12
i14
iC15
13
i16
14
iC17
C15
C16
iC18
C17
iC19
C18
iC20
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30

10
11
12
i14
iC15
13
i16
14
iC17
C15
C16
iC18
C17
iC19
C18
iC20
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
Oil n-alkanes Oil n-alkanes Oil n-alkanes
(d) (e) (f)
FID, 103 mV
FID-1 Component
100 100

iC20
iC18 C17C16
90 90

C15
6

14
13
80 80

12

C19
11
5

iC19
C18
70 70

C20
iC17
60 60

C21
4

10

i16
50 50

C22
40 40

C23
3

C24
30 30

C25
C27C26

C30
C31
20 20 2

C28
C29
10 10
0 0 1
10
11
12
i14
i15
13
i16
14
iC17
C15
C16
iC18
C17
iC19
C18
iC20
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31

11
12
i14
iC15
13
FID
i16
14
iC17
C15
C16
iC18
C17
iC19
C18
iC20
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34

Oil n-alkanes Oil n-alkanes 10 20 30 40 50

min

Fig. 2. Residual content of n-alkanes, % (ae) in oil degraded by strains G. icigianus G1w1T (a), G. toebii -1024 (b), A. pallidus
8m3 (c), Geobacillus sp. 1017 (d), and G. thermoleovorans 5366T (e), and the chromatogram of the saturated hydrocarbon fraction
of the sterile oil used (f). Residual n-alkane concentrations were calculated using the method of internal normalization (ad) and
with 2,2,4,4,6,8,8-heptamethylnonane as an internal control (e). The strains were incubated for 21 days with 1% crude oil
(vol/vol) under stationary conditions at 60C.

(~4045%) to the cluster of the sequences of the ssuD
gene, which determines alkane sulfate monooxygen-
ase, the enzyme which, together with flavin alkane
monooxygenase, belongs to the family of bacterial
luciferases.
The ladAB gene sequences inside the clusters
formed five separate branches (subclusters) (Fig. 4),
with almost identical sequences within each cluster
(98100% homology). The composition of subclus-
ters corresponded only partially to the species compo-
sition of the genus Geobacillus. The obtained ladA
sequences of the new strains belonged to the largest
subcluster 1, comprising the genome sequences of
12 strains, including the ladA genes of 5 strains of the
G. kaustophilusG. thermoleovorans group, 3 strains
identified as G. icigianus (including the type strain
G1w1T), 1 strain identified as G. vulcani, and the genes
of new strains G. toebii B-1024 and strain 1017 (the
taxonomic position of the latter is presently undeter-
mined). Subcluster 2 comprised the genes of three
strains identified as G. thermocatenulatus. Subcluster 3
comprised the ladA genes of three strains of the
G. kaustophilusG. thermoleovorans group and of one
strain identified as G. stearothermophilus. Subcluster 4
contained the ladA genes of four strains identified as
G. thermoglucosidasius, including the type strain of the
species. The last subcluster 5 was represented by the
genome of a single strain identified as G. thermode-
nitrificans.
The internal structure of the clusters formed by
translated amino acid sequences of the ladA and ladB
genes was identical to the structure of the ladA clus-
ter (Fig. 4).
The cyp153 genes. The cyp153A gene is known to
be present in a number of Dietzia strains (Alonso-
Gutierrez et al., 2011). In the positive control repre-
sented by the DNA of Dietzia natronolimnaea 263
(Nazina et al., 2015), electrophoresis of amplification
products obtained with the primers to the cyp153A
gene revealed a band of ~1000 bp, confirming the
specificity of cyp153A detection (Fig. 3e). Such spe-

MICROBIOLOGY Vol. 85 No. 6 2016

 DETECTION OF n-ALKANE BIODEGRADATION 703

Alfa Beta B Con Alfa Beta B Con Alfa Beta B Con 1024 1017 8m3 Glwl Con

1500 1500

1500 500

() (b) (c) (d)

DS2 8m3 5366T 34T UT vw3-1 strK 1017 1024 263 Con DS2 8m3 5366T 34T UT vw3-1 strK 1017 Con

5000

1500 1500
1000

500
500

(e) (f)

Fig. 3. Electrophoresis of the PCR products of the ladA (designated Alfa), ladA (Beta), and ladB (B) genes in genomic DNA
of thermophilic bacilli G. icigianus G1w1T (a), Geobacillus sp. 1017 (b), and G. toebii B-1024 (c), and of the genes cyp153A (e) and
alkB (d, f) of the above bacilli and of the strains A. pallidus 8m3 (8m3), G. jurassicus DS2 (DS2), G. thermoleovorans DSM 5366T
(5366T), G. subterraneus 34T (34T), G. uzenensis UT (UT), G. toebii vw3-1 (vw3-1), G. subterraneus K (strK), Geobacillus sp. 1017
(1017), G. toebii -1024 (1024), and of the mesophilic bacterium D. natronolimnaea 263 (263, panel e). The first lane on the panels
(d, e) and (f) shows the results of electrophoresis of the M27 DNA marker (100 bp + 1.5 Kb + 3 Kb, Sibenzim, Novosibirsk, Rus-
sia). The first lane on the panels (ac) shows the results of electrophoresis of the GeneRulerTM 1 kb Plus DNA Ladder (Fermen-
tas, United States). Con designates the negative controls.

cific amplification was not observed, however, for any
of the studied strains of thermophilic bacilli.
The alkB genes. Detection of the alkB genes by
amplification with degenerate oligonucleotide primers
(Table 1) was carried out for new strains B-1024, 1017,
8m3, and G1w1T, which have not been studied in this
respect. In all strains, application of the Alk-BFB for-
ward primer and the Alk-BRB reverse primer revealed
a fragment (~500 bp) corresponding to the target
product, indicating the presence of alkB (Figs. 3d, 3f).
Further investigation of the composition of the alkB
homologs in these strains is required.
Thus, for the first time in any strains of thermo-
philic bacilli, the simultaneous presence of the alkB
and ladAB genes encoding two different alkane mono-
oxygenases was revealed by PCR amplification.
DISCUSSION
Abundant information concerning the genes
responsible for n-alkane degradation by aerobic bacte-
ria is presently available. Mesophilic bacteria of the
genera Alcanivorax, Dietzia, Rhodococcus, Nocardia,
and Gordonia were found to possess two different
genes encoding the AlkB and CYP153 enzymes. Avail-
able information on the genes of hydrocarbon degra-
dation in thermophilic bacilli is scarcer and requires
experimental confirmation for a broader spectrum of
strains. In the present work, physiological characteris-
tics, growth on oil, and taxonomy of new strains of
thermophilic bacilli are reported, as are the results of
detection of the ladA, ladA, ladB, cyp153A, and
alkB genes in the new and known Aeribacillus and
Geobacillus strains.
Investigation of morphology and physiology of the
isolates, together with analysis of their 16S rRNA,
gyrase (gyrB), and topoisomerase IV (r) genes,
resulted in their identification as A. pallidus strain
8m3, Geobacillus sp. strain 1017, and G. toebii strains
-1024 and vw3-1. The strains grew on crude oil uti-
lizing n-alkanes with different chain lengths, which
made it possible to carry out the search for different

MICROBIOLOGY Vol. 85 No. 6 2016

704 TOUROVA et al.

10 Geobacillus sp. FW23, NZ_JGCJ01000024

Geobacillus thermoleovorans CCB_US3_UF5,NC_016593
Geobacillus sp. ZGt-1, LDPD01000140
Geobacillus sp. A8, AUXP01000165
Geobacillus thermoleovorans B23,BATY01000072 1
Geobacillus toebii B-1024, EX519923
99 Geobacillus sp. 1017, KX579924
Geobacillus sp. B4113_201601, LQYX01000026
Geobacillus subterraneus PSS2, JQMN01000001
Geobacillus vulcani PSS1, JPOI01000001
Geobacillus icigianus G1w1T, JPYA01000034
98 Geobacillus sp. G11MC16, ABVH01000005 5
LadA
99 Geobacillus sp. T6, LDNZ01000080
Geobacillus sp. BCO2, LJAJ01000023 2
Geobacillus thermocatenulatus GS-1, JFHZ01000039
95 Geobacillus sp. WSUCF1, ATCO01000099
Geobacillus sp. LC300, NZ_CP008903 3
99 Geobacillus sp. GHH01, NC_020210
Geobacillus sp. CAMR5420_1482739.4, JHUS01000068
Geobacillus thermoglucosidasius DSM 2542T, CP012712
Geobacillus sp. Y4.1MC1_581103.5, NC_014650 4
98 99 Geobacillus thermoglucosidasius TNO-09.020, AJJN01000031
Geobacillus thermoglucosidasius C56-YS93, NR_102795
Geobacillus thermodenitricans NG80-2, NC_009329
100 Amycolicicoccus subavus DQS3-9A1, NC_015564
99
4
5
99 99 2
99 3
99 LadA

1
99

99 1
84
99 LadB
90 2
3
99 99
5
99 4
T
84 Geobacillus thermoglucosidasius DSM 2542 , NZ_CP012712
99 Geobacillus sp. Y4.1MC1_581103.5, NC_014650
99 Geobacillus thermoglucosidasius TNO-09.020, NC_015660
99 Geobacillus thermoglucosidasius C56-YS93, NR_102795
Geobacillus caldoxylosilyticus S1812T, NZ_BAWO01000002
97 Geobacillus icigianus G1w1T, JPYA01000105
99 Geobacillus subterraneus PSS2, JQMN01000001
Geobacillus sp. B4113_201601, LQYX01000074
99 Geobacillus vulcani PSS1, JPOI01000001
Geobacillus sp. GHH01, NC_020210
95 Geobacillus sp. WSUCF1, ATCO01000100
99 Geobacillus sp. CAMR5420_1482739.4, JHUS01000055
99 Geobacillus sp. T6, LDNZ01000106 SsuD
Geobacillus sp. BCO2, LJAJ01000001
99 Geobacillus thermocatenulatus GS-1, JFHZ01000045
Geobacillus thermodenitricans NG80-2, NC_009328
89 92
Geobacillus thermodenitricans R-35647T, AYKT01000002
Geobacillus sp. G11MC16, ABVH01000009
T
99 Geobacillus kaustophilus NBRS 102445 , BBJV01000006
Geobacillus sp. FW23, JGCJ01000015
85 Geobacillus sp. ZGt-1, LDPD01000023
Geobacillus sp. A8, AJXP01000077
Geobacillus thermoleovorans B23, AB898656
Geobacillus thermoleovorans CCB_US3_UF5, NC_016593

Fig. 4. Phylogenetic tree of bacteria of the genera Geobacillus and Aeribacillus constructed based on translated amino acid
sequences of the ladA, ladA, ladB, and ssuD genes using the neighbor-joining algorithm. The organisms studied in this work
are indicated in boldface, type strains of the reference species are marked by gray shading, and G. thermoleovorans B23, the strain
in which the ladAB genes were initially detected, is indicated by a frame. The numerals indicated the branching order determined
by bootstrap analysis (values over 80% were considered significant). Scale bar shows the evolutionary distance corresponding to
10 replacements per 100 amino acid residues.

MICROBIOLOGY Vol. 85 No. 6 2016

 DETECTION OF n-ALKANE BIODEGRADATION
genes responsible for n-alkane biodegradation. Type
strains of known species G. icigianus, which possesses
the ladA genes, and G. thermoleovorans, in which these
genes were not revealed, were used for comparison.
The absence of positive results of PCR amplifica-
tion of the cyp153A gene fragments of the studied ther-
mophilic bacilli was not unexpected, since earlier
analysis of several genomic and metagenomic data-
bases revealed the presence of this type of alkane
monooxygenases only in the members of Proteobacte-
ria and Actinobacteria (Nie et al., 2014). At the same
time, PCR with degenerate primers Alk-BFB and
Alk-BRB revealed the alkB gene fragments in the
DNA of all studied strains. Interestingly, the alkB gene
was not revealed in the sequenced chromosomes of
thermophilic bacilli.
According to experimental data and BLAST analy-
sis of the sequenced genomes, homologs of the
ladAB23, ladAB23, and ladB23 genes are relatively ously analyzed in G. thermoleovorans B23 and G. ther-
common among Geobacillus strains, while they have
not been reliably detected outside this genus.
On the phylogenetic tree of translated amino acid
sequences, homologs of the ladAB23, ladAB23, and
ladB23 genes formed separate clusters for each gene
(Fig. 4), with homology between them not exceeding
45%. The almost identical topology of the clusters of
each gene indicated their collateral evolution. This
finding confirms the result of the work in which the
ladA, ladA, and ladB genes were first revealed and
their localization on the chromosome of G. thermole-
ovorans strain B23 was determined (Boonmak et al.,
2014). Location of these genes on the chromosome
was not random. They were components of a 11873-bp
fragment consisting of 10 genes and termed the ladAB
gene island. The authors also revealed LadAB gene
islands of similar gene composition and location in
complete genomes of five Geobacillus strains. FMN
reductases were essential components of these frag-
ments. Each of the LadAB islands found was
located on the chromosome between the genes of per-
mease and fructokinase, which are located alongside
in the genomes of members of this genus lacking the
LadAB gene island. Our analysis revealed that the
fragments homologous to the LadAB gene island in
G. thermoleovorans B23 occur in all complete genomes
and most shotgun assemblies, in which the homologs
of the ladAB23, ladAB23, and ladB23 genes were
revealed.
On the phylogenetic tree of translated amino acid
sequences of the genes ladA, ladA, and ladB, topol-
ogy of each cluster differed significantly from that of
the ssuD gene cluster (Fig. 4). In general, phylogeny of
the ssuD genes in the studied strains corresponded to
their species structure and correlated well with phy-
logeny determined using the 16S rRNA gene and the
housekeeping genes gyrB and par, indicating coevo-
MICROBIOLOGY Vol. 85 No. 6 2016
705
lution of these genes. At the same time, the ladA,
ladA, and ladB gene clusters separated into
branches/subclusters consisting of almost identical
sequences. Their composition and relationships cor-
related only partially with the species composition of
the relevant strains. This finding implies higher prob-
ability of horizontal gene transfer in the evolution of
the ladA, ladA, and ladB genes than in the case of
the ssuD genes.
Moreover, comparative analysis of complete
LadAB gene islands available in the GenBank data-
base revealed the same patterns as those found by
analysis of its constituent genes, ladA, ladA, and
ladB: LadAB gene islands formed five groups
according to their structure and similarity of the
nucleotide sequences, with 97.6100% within each
group. The largest group, group 1, comprised the
LadAB gene islands homologous to those of previ-
moleovorans CCB_US3_UF5 (Boonmak et al., 2014),
group 2 and related group 3 contained those homolo-
gous to the ones of Geobacillus sp. CHH01, and
group 4 contained those homologous to the ones
found in the genomes of G. thermoglucosidasius C56-
YS93 and Geobacillus sp. Y4.1MC1. The LadAB
island of Geobacillus sp. G11MC16, identified as a
G. thermodenitrificans strain, formed group 5.
Sequences of the genes ladA, ladA, and ladB of
strain G11MC16 occupy a special position on the phy-
logenetic tree. The LadAB island of its genome is
similar in composition to LadAB gene islands of
most other strains. It is, however, not flanked by the
permease and fructokinase genes, but is located in
another chromosome region, although within the
same contig as these genes. The permease and fruc-
tokinase genes are located alongside, similar to their
position on the chromosome of Geobacillus strains
lacking the LadAB gene islands.
Analysis of the structure and sequences of the
LadAB gene islands suggests a considerable role of
horizontal gene transfer in the evolution and propaga-
tion of LadAB islands in thermophilic bacilli. Since
occurrence of LadAB islands is presently known
only for geobacilli, they probably evolved in the ances-
tor/ancestors of this genus, unlike the alkB, for which
rhodococci and related microorganisms could proba-
bly act as donors in the horizontal gene transfer (Tou-
rova et al., 2008). The subsequent divergence of the
nucleotide sequences of the LadAB islands resulted
in formation of five (or probably more) groups. Inside
the groups, however, the genes of flavin-dependent
alkane monooxygenase were propagated via gene
transfer, making it possible for receiving strains to uti-
lize a broader n-alkane range. Transfer of group 1
LadAB island was probably the most efficient, since
LadAB gene islands of this type are most wide-
spread among Geobacillus species. Gene transfer of
706 TOUROVA et al.
the only LadAB gene island in group 5 was probably
hindered by its unusual localization of the chromo-
some.
The possibility of horizontal transfer of the ladA,
which was originally found on a plasmid from the
genome of G. thermodenitrificans NG80-2 (Feng et al.,
2007), and of the ladA homolog in the genome of Amy-
colicicoccus subavus DQS-9A1 (Nie et al., 2013) has
been suggested previously (Boonmak et al., 2014).
Transfer of complete LadAB gene islands in the
conjugation plasmids probably resulted in their reduc-
tion to a single ladA, which subsequently preserved
only insignificant homology to the ladA genes of the
donor.
Thus, simultaneous presence of the alkB and ladAB
genes was shown for the first time for a number of Geo-
bacillus strains oxidizing crude oil n-alkanes. Further
research on the role of the relevant enzymes in degra-
dation of the n-alkanes with different chain length is
required.
ACKNOWLEDGMENTS
The authors are grateful to A.V. Bryanskaya,
A.S. Rozanov, and S.E. Peltek (Institute of Cytology
and Genetics, Siberian Branch of the Russian Acad-
emy of Sciences) for the kindly provided strain Geoba-
cillus icigianus G1w1T. The work was supported by the
Russian Foundation for Basic Research, project
no. 15-04-02622; the work of n-alkane biodegradation
genes of G. icigianus G1w1T was supported by the Rus-
sian Science Foundation, project no. 16-14-00028.
REFERENCES
Adkins, J.P., Cornell, L.A., and Tanner, R.S., Microbial
composition of carbonate petroleum reservoir fluids,
Geomicrobiol. J., 1992, vol. 10, pp. 8797.
Alonso-Gutierrez, J., Teramoto, M., Yamazoe, A.,
Harayama, S., Figueras, A., and Novoa, B., Alkane-
degrading properties of Dietzia sp. H0B, a key player in the
Prestige oil spill biodegradation (NW Spain), J. Appl.
Microbiol., 2011, vol. 111, pp. 800810.
Boonmak, C., Takahashi, Y., and Morikawa, M., Cloning
and expression of three ladA-type alkane monooxygenase
genes from an extremely thermophilic alkane-degrading
bacterium Geobacillus thermoleovorans B23, Extremophiles,
2014, vol. 18, pp. 515523.
Borzenkov, I.A., Milekhina, E.I., Gotoeva, M.T.,
Rozanova, E.P., and Belyaev, S.S., The properties of
hydrocarbon-oxidizing bacteria isolated from the oilfields
of Tatarstan, Western Siberia, and Vietnam, Microbiology
(Moscow), vol. 75, no. 1, pp. 6672.
Bryanskaya, A.V., Rozanov, A.S., Logacheva, M.D.,
Kotenko, A.V., and Peltek, S.E., Draft genome sequence of
Geobacillus icigianus strain G1w1T isolated from hot springs
in the Valley of Geysers, Kamchatka (Russian Federation),
Genome Announc., 2014, vol. 2, no. 5. e01098-14. doi
10.1128/genomeA.01098-14
Bryanskaya, A.V., Rozanov, A.S., Slynko, N.M., Shek-
hovtsov, S.V., and Peltek, S.E., Geobacillus icigianus sp.
nov., a thermophilic bacterium isolated from a hot spring,
Int. J. Syst. Evol. Microbiol., 2015, vol. 65, pp. 864869.
Coorevits, A., Dinsdale, A.N., Halket, G., Lebbe, L.,
Vos, P., Landschoot, A., and Logan, N.A., Taxonomic
revision of the genus Geobacillus: emendation of Geobacil-
lus, G. stearothermophilus, G. jurassicus, G. toebii, G. ther-
modenitrificans and G. thermoglucosidans (nom. corrig., for-
merly thermoglucosidasius); transfer of Bacillus thermant-
arcticus to the genus as G. thermantarcticus comb. nov.;
proposal of Caldibacillus debilis gen. nov., comb. nov.;
transfer of G. tepidamans to Anoxybacillus as A. tepidamans
comb. nov.; and proposal of Anoxybacillus caldiproteolyticus
sp. nov., Int. J. Syst. Evol. Microbiol., 2012, vol. 62,
pp. 14701485.
Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C.,
Tang, Y., Liu, X., Han, W., Peng, X, Liu, R., and Wang, L.,
Genome and proteome of long-chain alkane degrading
Geobacillus thermodenitrificans NG80-2 isolated from a
deep-subsurface oil reservoir, Proc. Natl. Acad. Sci. U. S. A.,
2007, vol. 104, pp. 56025607.
Filippidou, S., Jaussi, M., Junier, T., Wunderlin, T., Rous-
sel-Delif, L., Jeanneret, N., Vieth-Hillebrand, A.,
Vetter, A., Regenspurg, S., Johnson, S.L., McMurry, K.,
Gleasner, C.D., Lo, C.-C., Li, P., Vuyisich, M.,
Chain, P.S., and Junier, P., Genome sequence of Anoxyba-
cillus geothermalis strain GSsed3, a novel thermophilic
endospore-forming species, Genome Announc., 2015, vol. 3,
no. 3. e00575-15.
Funhoff, E.G., Bauer, U., Garcia-Rubio, I., Witholt, B.,
and van Beilen, J.B., CYP153A6, a soluble P450 oxygenase
catalyzing terminal-alkane hydroxylation, J. Bacteriol.,
2006, vol. 188, pp. 52205227.
Korshunova, A.V., Tourova, T.P., Shestakova, N.M.,
Mikhailova, E.M., Poltaraus, A.B., and Nazina, T.N.,
Detection and transcription of n-alkane biodegradation
genes (alkB) in the genome of a hydrocarbon-oxidizing
bacterium Geobacillus subterraneus K, Microbiology (Mos-
cow), 2011, vol. 80, no. 5, pp. 682691.
Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L.,
Bartlam, M., Wang, L., and Rao, Z., Crystal structure of
long-chain alkane monooxygenase (LadA) in complex with
coenzyme FMN: unveiling the long-chain alkane hydroxy-
lase, J. Mol. Biol., 2008, vol. 376, no. 2, pp. 453465.
Liu, C., Wang, W., Wu, Y., Zhou, Z., Lai, Q., and Shao, Z.,
Multiple alkane hydroxylase systems in a marine alkane
degrader, Alcanivorax dieselolei B-5, Environ. Microbiol.,
2011, vol. 13, no. 5, pp. 11681178.
Liu, Y., Zhou, T., Zhang, J., Xu, L., Zhang, Z., Shen, Q.,
and Shen, B., Molecular characterization of the alkB gene
in the thermophilic Geobacillus sp. strain MH-1, Res.
Microbiol., 2009, vol. 160, no. 8, pp. 560566.
Lo Piccolo, L., De Pasquale, C., Fodale, R., Puglia, A.M.,
and Quatrini, P., Involvement of an alkane hydroxylase
system of Gordonia sp. strain SoCg in degradation of solid
n-alkanes, Appl. Environ. Microbiol., 2011, vol. 77, no. 4,
pp. 12041213.
Marchant, R. and Banat, I.M., The Genus Geobacillus and
hydrocarbon utilization, in Handbook of Hydrocarbon and
Lipid Microbiology, Timmis, K.N., Ed., Berlin: Springer,
2010, pp. 18871896.
MICROBIOLOGY Vol. 85 No. 6 2016
 DETECTION OF n-ALKANE BIODEGRADATION
Marchant, R., Sharkey, F.H., Banat, I.M., Rahman, T.J.,
and Perfumo, A., The degradation of n-hexadecane in soil
by thermophilic geobacilli, FEMS Microbiol. Ecol., 2006,
vol. 56, pp. 4454.
Miana-Galbis, D., Pinzn, D.L., Lorn, J.G.,
Manresa, A., and Oliart-Ros, R.M., Reclassification of
Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as
Aeribacillus pallidus gen. nov., comb. nov., Int. J. Syst. Evol.
Microbiol., 2010, vol. 60, pp. 16001604.
Nazina, T.N., Shumkova, E.S., Sokolova, D.Sh.,
Babich, T.L., Zhurina, M.V., Xue, Y.-F., Osipov, G.A.,
Poltaraus, A.B., and Tourova, T.P., Identification of hydro-
carbon-oxidizing Dietzia bacteria from petroleum reser-
voirs based on phenotypic properties and analysis of the 16S
rRNA and gyrB genes, Microbiology (Moscow), 2015,
vol. 84, no. 3, pp. 377388.
Nazina, T.N., Sokolova, D.Sh., Grigoryan, A.A., Shesta-
kova, N.M., Mikhailova, E.M., Poltaraus, A.B.,
Tourova, T.P., Lysenko, A.M., Osipov, G.A., and
Belyaev, S.S., Geobacillus jurassicus sp. nov., a new thermo-
philic bacterium isolated from a high-temperature petro-
leum reservoir, and the validation of the Geobacillus species,
Syst. Appl. Microbiol., 2005, vol. 28, pp. 4353.
Nazina, T.N., Tourova, T.P., Poltaraus, A.B., Novi-
kova, E.V., Grigoryan, A.A., Ivanova, A.E., Lysen-
ko, A.M., Petrunyaka, V.V., Osipov, G.A., Belyaev, S.S.,
and Ivanov, M.V., Taxonomic study of aerobic thermo-
philic bacilli: descriptions of Geobacillus subterraneus gen.
nov., sp. nov. and Geobacillus uzenensis sp. nov. from petro-
leum reservoirs and transfer of Bacillus stearothermophilus,
Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacil-
lus 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.,
2001, vol. 51, pp. 433446.
Nie, Y., Chi, C-Q., Fang, H., Liang, J.-L., Lu, S.-L.,
Lai, G.-L., Tang, Y.-Q., and Wu, X.-L., Diverse alkane
hydroxylase genes in microorganisms and environments,
Sci. Rep., 2014, vol. 4. Rep. 4968. doi 10.1038/srep04968
Nie, Y., Fang, H., Li, Y., Chi, C.-Q., Tang, Y.-Q., and
Wu, X.-L., The genome of the moderate halophile Amycol-
icicoccus subflavus DQS3-9A1T reveals four alkane hydrox-
ylation systems and provides some clues on the genetic basis
for its adaptation to a petroleum environment, PLoS One,
2013, vol. 8. e70986.
Poltaraus, A.B., Sokolova, D.S., Grouzdev, D.S.,
Ivanov, T.M., Malakho S.G., Korshunova, A.V.,
Rozanov, A.S., Tourova, T.P., and Nazina, T.N., Draft
genome sequence of Aeribacillus pallidus strain 8m3, a ther-
mophilic hydrocarbon-oxidizing bacterium isolated from
MICROBIOLOGY Vol. 85 No. 6 2016
707
the Dagang oil field (China), Genome Announc., 2016,
vol. 4, no. 3. e00500-16. doi 10.1128/genomeA.00500-16
Sharkey, F.H., Banat, I.M., and Marchant, R., A rapid and
effective method of extracting fully intact RNA from ther-
mophilic geobacilli that is suitable for gene expression anal-
ysis, Extremophiles, 2004, vol. 8, pp. 7377.
Shestakova, N.M., Korshunova, A.V., Mikhailova, E.M.,
Sokolova, D.Sh., Tourova, T.P., Belyaev, S.S.,
Poltaraus, A.B., and Nazina, T.N., Characterization of the
aerobic hydrocarbon-oxidizing enrichments from a high-
temperature petroleum reservoir by comparative analysis of
DNA- and RNA-derived clone libraries, Microbiology
(Moscow), 2011, vol. 80, no. 1, pp. 6373.
Throne-Holst, M., Wentzel, A., Ellingsen, T., Kotlar, H.,
and Zotchev, S., Identification of novel genes involved in
long-chain n-alkane degradation by Acinetobacter sp. strain
DSM17874, Appl. Environ. Microbiol., 2007, vol. 73,
pp. 33273332.
Tourova, T.P., Nazina, T.N., Mikhailova, E.M., Rodi-
onova, T.A., Ekimov, A.N., Mashukova, A.V., and Pol-
taraus, A.B., alkB homologs in thermophilic bacteria of the
genus Geobacillus, Mol. Biol., 2008, vol. 42, no. 2, pp. 217
226.
Tourova, T.P., Korshunova, A.V., Mikhailova, E.M.,
Sokolova, D.S., Poltaraus, A.B., and Nazina, T.N., Appli-
cation of gyrB and parE sequence similarity analyses for dif-
ferentiation of species within the genus Geobacillus, Micro-
biology (Moscow), 2010, vol. 79, no. 3, pp. 356369.
van Beilen, J.B., Funhoff, E.G., van Loon, A., Just, A.,
Kaysser, L., Bouza, M., Holtackers, R., Rthlisberger, M.,
Li, Z., and Witholt, B., Cytochrome P450 alkane hydroxy-
lases of the CYP153 family are common in alkane-degrad-
ing eubacteria lacking integral membrane alkane hydroxy-
lases, Appl. Environ. Microbiol., 2006, vol. 72, no. 1, pp. 59
65.
Van de Peer, Y. and De Wachter, R., TREECON for Win-
dows: a software package for the construction and drawing
of evolutionary trees for the Microsoft Windows environ-
ment, Comput. Appl. Biosci., 1994, vol. 10, pp. 569570.
Wang, L., Wang, W., Lai, Q., and Shao, Z., Gene diversity
of CYP153A and AlkB alkane hydroxylases in oil-degrading
bacteria isolated from the Atlantic Ocean, Environ. Micro-
biol., 2010, vol. 12, no. 5, pp. 12301242.
Whyte, L.G., Smits, T.H., Labbe, D., Witholt, B.,
Greer, C.W., and van Beilen, J.B., Gene cloning and char-
acterization of multiple alkane hydroxylase systems in
Rhodococcus strains Q15 and NRRL B-16531, Appl. Envi-
ron. Microbiol., 2002, vol. 68, pp. 59335942.
Translated by P. Sigalevich