Phylogeny of the Order Bacillales inferred from 3’ 16S rDNA and 5’ 16S-23S ITS nucleotide sequences

doi:10.4236/ns.2010.29121

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Vol.2, No.9, 990-997 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.29121

Phylogeny of the Order Bacillales inferred from

3’ 16S rDNA and 5’ 16S-23S ITS nucleotide sequences

Sabarimatou Yakoubou1,2, Dong Xu1, Jean-Charles Côté1*

1Agriculture and Agri-Food Canada, Research Centre, Gouin Blvd, St-Jean-sur-Richelieu, Canada; *Corresponding Author:

Jean-Charles.Cote@agr.gc.ca

2Département des Sciences Biologiques, Université du Québec à Montréal, Succ.“Centre-Ville” Montréal, Canada

Received 16 April 2010; revised 20 May 2010; accepted 25 May 2010.

ABSTRACT

A short 220 bp sequence was used to study the

taxonomic organization of the bacterial Order

Bacillales. The nucleotide sequences of the 3’

end of the 16S rDNA and the 16S-23S Internal

transcribed spacer (ITS) were determined for 32

Bacillales species and strains. The data for 40

additional Bacillales species and strains were

retrieved directly from Genbank. Together, these

72 Bacillales species and strains encompassed

eight families and 21 genera. The 220 bp se-

quence used here covers a conserved 150 bp

sequence located at the 3’ end of the 16S rDNA

and a conserved 70 bp sequence located at the

5’ end of the 16S-23S ITS. A neighbor-joining

phylogenetic tree was inferred from compara-

tive analyses of all 72 nucleotide sequences.

Eight major Groups were revealed. Each Group

was sub-divided into sub-groups and branches.

In general, the neighbor-joining tree presented

here is in agreement with the currently accepted

phylogeny of the Order Bacillales based on

phenotypic and genotypic data. The use of this

220 bp sequence for phylogenetic analyses

presents several advantages over the use of the

entire 16S rRNA genes or the generation of ex-

tensive phenotypic and genotypic data. This 220

bp sequence contains 150 bp at the 3’ end of the

16S rDNA which allows discrimination among

distantly related species and 70 bp at the 5’ end

of the 16S-23S ITS which, owing to its higher

percentage of nucleotide sequence divergence,

adds discriminating power among closely re-

lated species from same genus and closely re-

lated genera from same family. The method is

simple, rapid, suited to large screening pro-

grams and easily accessible to most laborato-

ries.

Keywords: Bacillales; 16S rDNA; 16S-23S ITS;

Phylogeny

1. INTRODUCTION

In the 1st Edition of Bergey’s Manual of Systematic

Bacteriology (1986), the genus Bacillus, a member of

the Class Bacilli, encompassed the Gram-positive, rod-

shaped, endospore-forming, either obligate or facultative

aerobe bacteria [1]. A total of 34 species and 26 addi-

tional species incertae sedis were described. The genus

was highly heterogeneous, exhibiting a wide range of

nutritional requirements, physiological and metabolic

diversity and DNA base composition. In the following

two decades, numerical classifications [2], 16S rDNA

sequence alignments [3], characterizations at the geno-

typic and phenotypic levels of selected Bacillus species,

all led to the creation of several new genera (briefly re-

viewed in Xu and Côté [4]).

In 2003, we developed a method for the identification,

classification and phylogenetic analyses of Bacillus spe-

cies and species from closely related genera [4]. The

method was simple, rapid, suited to large screening pro-

grammes and easily accessible to most laboratories. In

summary, the method relied on comparisons of a 220 bp

nucleotide sequence marker among Bacillus species.

This 220 bp marker was a combination of a 150 bp se-

quence at the 3’ end of the 16S rRNA gene and a 70 bp

sequence at the 5’ end of the 16S-23S ITS sequence. In

our original study, a total of 40 species was analyzed. We

showed that the phylogeny inferred from the 220 bp

marker was in agreement with then current classifica-

tions based on phenetic and molecular data, with excep-

tions. It revealed species and genera which appeared

misassigned and for which additional characterization

appeared warranted.

In the 2nd Edition of Bergey’s Manual of Systematic

Bacteriology [5], a new taxonomy of the Class Bacilli is

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

991

presented. It is the result of phylogenetic and princi-

pal-component analyses of comprehensive datasets of

16S rDNA sequences [5,6]. The Bacillus genus belongs

to the Order Bacillales. The Order Bacillales contains

nine families: Alicyclobacillaceae, Ba cillaceae, Listeri-

aceae, Paenibacillaceae, Pasteuriaceae, Planococcaceae,

Sporolactobacillaceae, Staphylococcaceae and Thermo-

actinomycetaceae. All nine families contain a total of 51

genera.

Today, we further assess the usefulness of the 220 bp

marker by extending its analyses beyond the genus Ba-

cillus, to a higher taxa level, the Order Ba cillales. Wher-

eas our first study focused on species from the genus

Bacillus and species from closely related genera [4], we

report here the phylogenetic analyses of 72 species and

strains from eight Bacillales families and 21 genera.

2. MATERIALS AND METHODS

2.1. Bacterial Strains and Culture

Conditions

A total of 72 strains of the Order Bacillales were used in

this study. These encompassed eight families, 21 genera

and 67 species. A total of 31 Bacillales strains were ob-

tained from the “Deutsche Sammlung von Mikroorgan-

ismen und Zellkulturen” (DSMZ) GmbH, Braunschweig,

Germany and were grown according to the DSMZ guide-

lines (http://www.dsmz.de/microorganisms/media_list.

php). Jeotgalib acillus alimentarius was obtained from

the “Czech Collection of Microorganisms” (CCM), Ma-

saryk University, Brno, Czech Republic and grown ac-

cording to the CCM guidelines (http://www.sci.muni.

cz/ccm/index.html). The nucleotide sequences of the 40

remaining strains were retrieved directly from GenBank.

All bacterial strains and their sources are listed in Table

Escherichia coli strain TOP10 (Invitrogen Inc., Bur-

lington, ON, Canada) was used for cloning PCR frag-

ments. Strain TOP10 was cultured on Luria-Bertani (LB)

agar plates to select transformants or in LB broth to mul-

tiply the cells, with shaking at 180-200 rpm at 37℃, 1 h.

2.2. DNA Extraction

Bacterial cells were washed with TESS buffer [10 mM

Tris/HCl (pH 8.0), 1 mM Na2EDTA, 0.1 M NaCl and

0.1% Sarkosyl (N-lauroylsarcosine)] and resuspended in

TE buffer [10 mM Tris/HCl (pH 8.0), 1 mM Na2EDTA].

Cells were lysed with 10 mg/ml lysozyme and 0.1% SDS.

The subsequent phenol/chloroform extractions and etha-

nol precipitation were carried out as described by Sam-

brook and Russel [7].

Recombinant plasmid from E. coli strain TOP10 was

isolated using the alkaline-lysis method [8] with some

modifications as described elsewhere [4].

2.3. Amplification of the 3’ End 16S rDNA

and the 16S-23S ITS Region

A pair of primers: L516SF (5’-TCGCTAGTAATCGCGG

ATCAGC-3’) and L523SR (5’-GCATATCGGTGTTAG

TCCCGTCC-3’), Reference [4] was used for the ampli-

fication of the 3’ end of 16S rDNA, the 16S-23S ITS

region and the 5’ end of 23S rDNA. Amplification was

performed in a Thermal Cycler 9600 (Perkin Elmer,

Waltham, MA, USA) and the reaction mixtures contained

50 ng template DNA, 0.25 µM each primer, 200 µM

dNTP, 1.5 mM MgCl2 and 1.25 U Taq DNA polymerase

(QIAGEN Inc. Mississauga, ON, Canada) in a final

volume of 50 µl. PCR was performed under the follow-

ing conditions: 45 s at 95℃ and then 30 cycles of 15 s at

94℃, 30 s at 53℃ and 90 s at 72℃. Amplification

products were visualized on agarose gels.

2.4. Cloning and Sequencing Methods

The amplified DNAs were cloned into a pCRII-TOPO

cloning vector using the TOPO TA cloning kit (Invitro-

gen, Inc.), following the manufacturer’s instructions.

Escherichia coli strain TOP10 transformants were se-

lected on LB agar plates containing kanamycin (50 µg/ml),

5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside

(X-Gal) (40 µg/ml). Multiple clones were submitted for

futher analyses for each Bacillales species. The recom-

binant plasmids were isolated using the modified alka-

line-lysis method, digested with EcoRI and visualized on

agarose gels to confirm the presence of an inserted frag-

ment.

The nucleotide sequences of cloned fragments were

determined by the dideoxynucleotide chain termination

method [9], using a capillary array automated DNA se-

quencer (ABI 3730xl DNA analyzer; Applied Biosys-

tems, Foster City, CA, USA). The sequences of both

strands were determined.

2.5. Sequence Analysis

The 3’ end of the 16S rDNA and the 16S-23S ITS of the

32 Bacillales species and strains sequenced in this study

were used for analysis. Forty sequences publicly avail-

able from GenBank were added for comparison purposes

to cover a wider range of Bacillales families and genera,

for a total of 72 Bacillales species and strains. A

neighbor-joining tree was constructed [10] based on the

alignment of the 72 3’ end of the 16S rDNA and the 5’

end of the 16S-23S ITS sequences.

The tree was bootstrapped using 1,000 random sam-

ples of sites from the alignment, all using CLUSTAL W

software [11] at the DNA Data Bank of Japan (DDBJ)

(http://clustalw.ddbj.nig.ac.jp/top- e.html), with the Ki-

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

992

Table 1. Bacillale families, genera, species and strains used to construct the bootstrapped neighbor-joining tree inferred from the 220

bp sequence, and shown in Figure 3.

Families, genera and species Source/Strain GenBank accession no.

This study* Retrieved** Groups Sub-groups

Alicyclobacillaceae

Alicyclobacillus

acidocaldarius subsp.acidocaldarius

acidocaldarius subsp. rittmannii

acidoterrestris

cycloheptanicus

herbarius

hesperidum

DSM 446

DSM 11297

DSM 3922

DSM 4006

DSM 13609

DSM 12489

EU723605

EU723607

EU723608

EU723610

EU723613

EU723615

Ungrouped

Bacillaceae

Amphibacillus

fermentum

tropicus

xylanus

Anoxybacillus

flavithermus

Bacillus

clausii

subtilis

thuringiensis

thuringiensis var. konkukian

weihenstephanensis

Filobacillus

milensis

Geobacillus

caldoxylosilyticus

kaustophilus

stearothermophilus

subterraneus

thermocatenulatus

thermodenitrificans

thermoglucosidasius

thermoleovorans

uzenensis

Gracilibacillus

dipsosauri

halotolerans

Halobacillus

sp.

Oceanobacillus

iheyensis

Virgibacillus

marismortui

pantothenticus

proomii

salexigens

DSM 13869

DSM 13870

DSM 6626

WK1

168

Al Hakam

97-27

KBAB4

DSM 13259

DSM 12041

HTA426

DSM 7263

DSM 22

DSM 13552

DSM 730

NG80-2

DSM 465

DSM 2542

DSM 5366

DSM 13551

DSM 11125

DSM 11805

SA-Hb6

HTE831

DSM 12325

DSM 26

DSM 13055

DSM 11483

EU723600

EU723602

EU723603

EU723621

EU723625

EU723629

EU723631

EU723636

EU723638

EU723643

EU723644

EU723648

EU723651

EU723655

EU723657

EU723664

EU723672

EU723675

EU723666

CP000922

AP006627

AL009126

CP000485

AE017355

CP000903

BA000043

CP000557

AB367166

BA000028

VIII Ungrouped

VIII iii

VIII Ungrouped

VI i

VI ii

Ungrouped

VIII

VIII ii

VIII i

VIII ii

Listeriaceae

Listeria

inoccua

monocytogenes

elshimeri

Clip11262

EGD-e

SLCC5334

AL592022

AL591824

AM263198

VI iii

Paenibacillaceae

Aneurinibacillus

aneurinilyticus

migulanus

thermoaerophilus

Brevibacillus

agri

borstelensis

brevis

DSM 5562

DSM 2895

DSM 10154

ATCC 51360

ATCC 51668

ATCC8246

EU723616

EU723617

EU723618

AF478091

AF478093

AY478094

III i

III ii

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

993

Table 1. Continued.

Families, genera and species Source/Strain GenBank accession no.

This study* Retrieved** Groups Sub-groups

brevis

chosinensis

parabrevis

formosus

Paenibacillaceae

Paenibacillus

alginolyticus

alvei

chondroitinus

larvae

lautus

lentimorbus

macerans

pabuli

popilliae

validus

NBRC10059

ATCC 51359

ATCC 8186

ATCC51669

ATCC 51185

ATCC 6344

ATCC 51184

ATCC 9545

ATCC 43898

ATCC 8244

ATCC 43899

ATCC 14707

KLN 3

JDR-2

ATCC 43897

AP008955

AF478095

AF478097

AF478096

AF478104

AF478098

AF478105

AF487106

AF487100

AY763503

AF478101

AF478102

CP001656

DQ062684

AF478103

III ii

Pasteuriaceae

Pasteuria

ramosa

penetrans

CJ-1

AY762091

AY123968

Planococcaceae

Jeotgalibacillus

alimentarius

Marinibacillus

marinus

Ureibacillus

terrenus

thermosphaericus

CCM 7134

DSM 1297

DSM 12654

DSM 10633

EU723660

EU723661

EU723667

EU723670

VI iv

Sporolactobacillaceae

Sporolactobacillus

terrae

M-116

D16289.1

Ungrouped

Staphylococcaceae

Staphylococcus

aureus

arnosus

epidermidis

haemolyticus

aprophyticus

Macrococcus

caseolyticus

RF122

TM300

RP62A

JCSC1435

ATCC 15305

JCSC5402

AJ938182

AM295250

CP000029

AP006716

AP008934

AP009484

VII

*Sequences generated in this study; **Sequences publicly available in GenBank at the onset of this work and retrieved directly.

mura’s parameter method [12]. The neighbor-joining

phylogenetic tree was drawn using TreeView (version

1.6.6) [13,14].

3. RESULTS

Two primers, one located about 200 nt upstream from

the 3’ end of the 16S rRNA gene, the other about 80 nt

downstream from the 5’ end of the 23S rRNA gene (Fig-

ure 1), were used to amplify the last 200 bp of the 16S

rRNA gene and the entire 16S-23S Internal transcribed

spacer (ITS) region from 32 Bacillales species and

strains (Table 1). The amplicons ranged in length from

450 to 1,200 bp. The number of amplicons per strain

ranged from 1 to 6. A subset of these results is shown in

Figure 2. Each amplicon was cloned and its nucleotide

sequence determined. The homologous DNA sequences

from 40 more Bacillales species and strains were re-

trieved directly from GenBank and added in the study

(Table 1). Together, these 72 Bacillales species and

strains belong to eight Bacillales families and 21 genera.

The last 150 bp located at the 3’ end of 16S rDNA and

the first 70 bp located at the 5’ end of 16S-23S ITS, were

combined into a 220 bp sequence. A multiple alignment

of these nucleotide sequences from the 72 Bacillale spe-

cies and strains was performed (supplementary data) and

a bootstrapped neighbor-joining tree was constructed

(Figure 3).

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

994

L516SF

L523SR

5’ 3’

16SrRNAgene

23SrRNAgene

ITS

Figure 1. Schematic representation of the 16S and 23S rRNA

genes separated by an Internal transcribed spacer (ITS). Orien-

tations and positions of the primers used for amplification,

L516SF and L523SR, are shown. The contiguous small grey

and black boxes, indicated by the letters “a” and “b” corre-

spond to the last 150 bp at the 3’ end of the 16S rRNA gene

and the first 70 bp at the 5’ end of the ITS, respectively. To-

gether, these boxes correspond to the 220 bp marker used in

this study.

800 —

100 —

1600 —

M1234 56 7891011

Figure 2. Agarose gel electrophoresis of the amplification

products in selected species in the Order Bacillales using the

L516SF/L523SR primer pair. Lane 1, 100 bp DNA marker;

lane 2, Alicyclobacillus acidocaldarius subsp. acidocaldarius;

lane 3, Alicyclobacillus herbarius; lane 4, Geobacillus uzenen-

sis; lane 5, Gracilibacillus halodurans; lane 6, Geobacillus

kaustophilus; lane 7, Amphibacillus tropicus; lane 8, Virgiba-

cillus proomii; lane 9, Virgibacillus salexigens; lane 10,

Marinibacillus marinus; lane 11, Aneurinibacillus aneur-

inilyticus ; lane 12, Filobacillus milensis.

At first sight, eight major Groups are revealed at the

80% nucleotide sequence identities, Group I to VIII.

Group I contains all eleven Geobacillus species and

strains. Alicyclobacillus herbarius forms a single branch

between Groups I and II. Group II contains both Pasteu-

riaceae species. Group III contains ten species and

strains from two genera of the Paenibacillaceae family.

Two sub-groups can be revealed. Sub-group i encom-

passes the three Aneurinibacillus species. They share at

least 82% nucleotide sequence identities. Sub-group ii

encompasses the seven Brevibacillu s species and strains.

They share at least 80% nucleotide sequence identities.

Group IV contains all 11 Paeniba cillus species. Group V

contains five of the six species and strains in the Alicy-

clobacillaceae family. Alicyclobacillus herbarius is the

sixth species, and forms a single branch as explained

above, because it shares less than 80% nucleotide se-

quence identities with members of Group V. Group VI is

more heterogenous. It contains 12 species and strains

from five genera from three families: the Planococca-

ceae, Bacillaceae and Listeriaceae. Four sub-groups and

two branches are revealed. Jeotgalibacillus alimentarus

forms the first branch of Group VI, and belongs to the

Planococcaceae family. Sub-group i contains two Ba-

cillus species of the Bacillaceae family, B. clausii and B.

subtilis. Marinibacillus marinus forms the second branch

of Group VI. It is the second genus of the Planococca-

ceae family. Sub-group ii contains three highly related

Bacillus species and strains, the two B. thuringiensis

strains and B. weihenstephanensis. Sub-group iii con-

tains all three Listeria species of the Listeriaceae family.

Sub-group iv contains both species of the third Plano-

coccaceae genus, Ureiba cillus. Next, a branch is formed

by Sporolactobacillus terrae. This genus is the only one

known in the Sporolactobacillaceae family. This is fol-

lowed by a branch formed by Filobacillus milensis, a

member of the Bacilla ceae family. Group VII contains

five Staphylococcus and one Macrococcus species. Both

genera belong to the Staphylococcaceae family. Group

VIII contains 12 species from six genera, all in the Ba-

cillaceae family. Three subgroups and thee branches can

be revealed. Sub-group i contains Oceanobacillus and

Halobacillus. They share 87% nucleotide sequence iden-

tities. The grouping of Oceanobacillus and Ha lobacillus

species in sub-group i is in agreement with the work of

Lu et al., properties, and genetic data [15]. Sub-group ii

contains five species from two genera. It can be further

divided into two clusters. The first one contains Virgiba-

cillus (V.) pantothenticus, V. proomii and Gracilibacillus

halotolerans, the second cluster contains V. marismortui

and V. salexigens. The subdivision of all four Virgibacil-

lus species into two sub-groups is in agreement with the

work of Heyrman et al., based on genetic, chemotax-

onomic and phenotypic data [16]. Sub-group iii contains

two of the three Amphibacillus (Am.) species, Am. xy-

lanus and Am. tropicus. Group VIII is completed by

three branches: Gracilibacillus dipsosauri, Anoxybacil-

lus flavithermus and Amphiba cillus fermentum. All

members of Group VIII are halophilic and alkaliphilic

bacilli. This grouping is in agreement with the one pro-

posed by Zhilina et al., based on physiology and genetic

data [17].

When each of the eight major Groups of this neighbor-

joining tree are analyzed separately, strains from same

species, species from same genus and genera from same

family are grouped together. Each Group corresponds to

a single Bacillales family, exclusive of other families,

with one exception, Group VI which contains three fami-

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

995

Anoxybacillus flavithermus

Amphibacillus fermentum

Gracilibacillus dipsosauri

Virgibacillus pantothenticus

Virgibacillusproomii

Gracilibacillus halotolerans

Virgibacillus marismortui

Virgibacillus salexigens

Geobacillus thermodenitrificans NG80-2

Geobacillus kaustophilus

Geobacillus thermoleovorans

Geobacillus kaustophilus HTA426

Geobacillus thermodenitrificans

Geobacillus thermotecatenulatus

Geobacillus subterraneus

Geobacillus uzenensis

Geobacillus stearothermophilus

Geobacillus caldoxylosilyticus

Geobacillus thermoglucosidasius

Alicyclobacillus herbarius

Pasteuria ramosa

Pasteuria penetrans

Aneurinibacillus thermoaerophilus

Aneurinibacillus aneurinilyticus

Aneurinibacillus migulanus

Brevibacillus borstelensis

Brevibacillus parabrevis

Brevibacillus agri

Brevibacillus brevis

Brevibacillus formosus

Brevibacillus brevis NBRC100599

Brevibacillus choshinensis

Paenibacillus larvae

Paenibacillus macerans

Paenibacillus validus

Paenibacillus sp.

Paenibacillus alvei

Paenibacillus lentimorbus

Paenibacillus popilliae

Paenibacillus pabuli

Paenibacillus chondroitinus

Paenibacillus lautus

Paenibacillus alginolyticus

Alicyclobacillus acidocaldarius acidocaldarius

Alicyclobacillus acidocaldarius rittmannii

Alicyclobacillus hesperidum

Alicyclobacillus acidoterrestris

Alicyclobacillus cycloheptanicus

Jeotgalibacillus alimentarius

Bacillus clausii

Bacillus subtilis

Marinibacillus marinus

Bacillus thuringiensis konkukian

Bacillus thuringiensis Al-Hakam

Bacillus weihenstephanensis

Listeria welshimeri

Listeria monocytongenes

Listeria innocua

Ureibacillus terrenus

Ureibacillus thermosphaericus

Sporolactobacillus terrae

Filobacillus milensis

Staphylococcus saprophyticus

Staphylococcus carnosus

Macrococcus caseolyticus

Staphylococcus aureus

Staphylococcus epidermidis

Staphylococcus haemolyticus

Oceanobacillus iheyensis

Halobacillus sp

Amphibacillus xylanus

Amphibacillus tropicus

0.01

U ng rouped

Ung rouped

Ungrouped

Ung rouped

Ungrouped

Gro upsSub- Groups

Clusters

III

VII

VIII

B acillaceae

iii

P asteuriaceae

B acillaceae

P lanococcaceae

Listeriace ae

P aenibacillaceae

Alicy clobacillaceae

B acillaceae

S porolactobacil laceae

S taphyloc occaceae

Families

Figure 3. Bootstrapped neighbor-joining tree of 72 Bacillales species and strains inferred from the align-

ment of the 220 bp marker. Major Groups are indicated in capital roman numerals. Sub-groups are indi-

cated in lower case riman numerals. Bootstrap values higher than 50% are indicated (expressed as per-

centage of 1000 replication). The horizontal bar represents 1% nt difference.

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

996

lies. In some cases, Groups were divided into sub-groups

which corresponded to genera, with one exception, Group

VIII where sub-groups encompassed different genera.

The family Sporolactobacillaceae forms a branch be-

tween Groups VI and VII. Three Bacillales families:

Bacillaceae, Pa enibacilla ceae and Alicyclobacillaceae,

show some level of heterogeneity. Genera of the Bacil-

laceae family are found into three Groups, Groups I, VI

and VIII, and in a branch between Groups VI and VII.

Genera of the Paenibacillaceae family are found into

two Groups, Groups III and IV. Analysis of the Alicy-

clobacillaceae family reveals a different story. Five of

the six species and strains are clustered together in

Group V. The sixth, Alicyclobacillus herbarius, is more

distant. It forms a branch between Groups I and II. This

is in agreement with the work of Goto et al. [18]. Al-

though distinct from all other Alicyclobacillaceae based

on genomic data, including 16S rDNA sequences, Ali-

cyclobacillus herbarius was grouped with the family

based on the presence of ω-cycloheptane fatty acids [18].

4. DISCUSSION

In a 2003 study [4], on Bacillus and closely-related gen-

era, a multiple alignment of the 3’ end of the 16S rRDA

sequence showed that the last 157 nucleotides shared

extensive identities among closely related species from

same genus. This 157 nucleotide sequence, however,

was not conserved among species from different genera.

In the same study, a multiple alignment of the 16S-23S

Internal transcribed spacer (ITS) sequence showed that

the first 70 nucleotides were conserved between alleles

of the same strain and between alleles of different strains

from same species. This sequence, however, was not

conserved among alleles of different species of the same

genus. These two sequences, the last 150 bp at the 3’ end

of the 16S rRNA gene and the first 70 bp at the 5’ end of

the 16S-23S rRNA ITS, were combined into a single 220

bp marker. This marker was used to infer the phylogeny

of Bacillus species and species from closely related gen-

era. It could cluster Bacillus species and species from

closely related genera into taxa akin to genera and could

also distinguish closely related species. In this 2003

study, a total of 40 species was analyzed.

In the current study, we further assessed the usefulness

of the 220 bp marker at a higher taxonomic level, the

Order Bacillales. A total of 72 Bacillales species and

strains from eight Bacillales families and 21 genera were

covered. The number of Bacillus species included in this

current study on Bacillales was deliberately kept low

since this genus had already been covered extensively in

our earlier study [4]. The neighbor-joining tree presented

here was compared with the revised road map of the

Order Bacillales shown in the 2nd Edition of Bergey’s

Manual of Systematic Bacteriology [5]. This revised

road map [5] is a consensus phylogenetic tree of the Or-

der Bacillales. It is the consensus tree inferred from nu-

merous phylogenetic and principal-component analyses

of comprehensive datasets of 16S rDNA sequences [5,6].

Our phylogenetic tree presented here is in agreement

with the currently accepted phylogeny of the Order Ba-

cillales, based on phenotypic and genotypic data. It is, in

general, in agreement with the revised road map of the

Order Bacillales [5]. In addition, some bacterial species

that were not grouped at the genus level in our neighbor-

joining tree, exemplified by Alicyclobacillus herbarius,

were also confirmed by others to be different based on

phenotypic and genotypic data [18].

The main discrepancy between our results, obtained

with the 220 bp marker, and the revised road map shown

in the 2nd Edition of Bergey’s Manual of Systematic

Bacteriology [5], rests on the grouping of the Bacilla-

ceae family. In our study, members of the Bacillaceae

family are present in three of the eight Groups. In the

revised road map [5], two major Bacillaceae groups are

presented. It is recognized, however, that some species

have been misassigned to the Bacillaceae family [5].

The revised road map is constructed based on 16S rDNA

sequences [5]. Our 220 bp marker contains 150 bp from

16S rDNA and 70 bp from ITS. Owing to its higher rate

of nucleotide substitutions, this 70 bp adds discriminat-

ing power among species from same genera and genera

from same family. As indicated by Ludwig et al. [5], and

as shown here, the reorganization of the Bacillaceae

family is still a work in progress.

The use of this 220 bp marker presents several advan-

tages over the use of the entire 16S rRNA gene or the

generation of extensive phenotypic and genotypic data in

phylogenetic analyses. As shown in an earlier study [4],

the method is simple, rapid, suited to large screening

programmes and easily accessible to most laboratories.

We have shown here that it can group Bacillales families

and genera in accordance with established phylogenies.

Because the 220 bp marker shows a higher percentage of

nucleotide sequence divergence than the 16S rRNA gene,

it can better discriminate among closely related Ba-

cillales species. It can also reveal Bacillales species

which may appear misassigned and for which additional

characterization appear warranted.

In conclusion, in an earlier study [4], a 220 bp marker,

based on 3’ end of 16S rRDA and 5’ end of 16S-23S

rRNA ITS, was developed and used to classify species in

the Ba cillus genus and in closely related genera. Here,

we showed that this 220 bp marker could be used to re-

construct the phylogeny at a higher taxa level: the Order

Bacillales. We are planning to follow-up this work by

S. Yakoubou et al. / Natural Science 2 (2010) 990-997

997

assessing the resolving power of this marker in recon-

structing the phylogeny at a lower taxa level: the Bacil-

lus cereus group, sensu lato.

Recently, in parallel, a similar maker was tested and

shown to be able to reconstruct the phylogeny of the

Class γ-proteobacteria [19].

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