Phylogeny of γ-proteobacteria inferred from comparisons of 3’ end 16S rRNA gene and 5’ end 16S-23S ITS nucleotide sequences

doi:10.4236/ns.2010.26067

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Vol.2, No.6, 535-543 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.26067

Phylogeny of γ-proteobacteria inferred from

comparisons of 3’ end 16S rRNA gene and 5’

end 16S-23S ITS nucleotide sequences

Sabarimatou Yakoubou1,2, Jean-Charles Côté1*

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

Jean-Charles.Cote@agr.gc.ca

2Département des Sciences Biologiques, Université du Québec à Montréal, CP 8888, Succ. “Centre-Ville” Montréal, Québec, Canada

Received 27 January 2010; revised 25 March 2010; accepted 12 April 2010.

ABSTRACT

The phylogeny of γ-proteobacteria was inferred

from nucleotide sequence comparisons of a

short 232 nucleotide sequence marker. A total of

64 γ-proteobacterial strains from 13 Orders, 22

families, 40 genera and 59 species were analyz-

ed. The short 232 nucleotide sequence marker

used here was a combination of a 157 nucleot-

ide sequence at the 3’ end of the 16S rRNA gene

and a 75 nucleotide sequence at the 5’ end of

the 16S-23S Internal Transcribed Spacer (ITS)

sequence. Comparative analyses of the 3’ end of

the 16S rRNA gene nucleotide sequence sho-

wed that the last 157 bp were conserved among

strains from same species and less conserved

in more distantly related species. This 157 bp

sequence was selected as the first part in the

construction of our nucleotide sequence marker.

A bootstrapped neighbor-joining tree based on

the alignment of this 157 bp was constructed.

This 157 bp could distinguish γ-proteobacterial

species from different genera from same family.

Closely related species could not be distingu-

ished. Next, an alignment of the 16S-23S ITS

nucleotide sequences of alleles from same bac-

terial strain was performed. The first 75 bp at the

5’ end of the 16S-23S ITS was highly conserved

at the intra-strain level. It was selected as the

second part in the construction of our nucleotide

sequence marker. Finally, a bootstrapped neigh-

bor-joining tree based on the alignment of this

232 bp sequence was constructed. Based on the

topology of the neighbour-joining tree, four maj-

or Groups, Group I to IV, were revealed with sev-

eral sub-groups and clusters. Our results, bas-

ed on the 232 bp sequence were, in general, in

agreement with the phylogeny of γ-proteobact-

eria based on the 16S rRNA gene. The use of

this 232 bp sequence as a phylogenetic marker

presents several advantages over the use of the

entire 16S rRNA gene or the generation of exten-

sive phenotypic and genotypic data in phyloge-

netic analyses. First, this marker is not allele-de-

pendant. Second, this 232 bp marker contains

157 bp from the 3’ end of the 16S rRNA gene and

75 bp from the 5’ end of the 16S-23S ITS. The

157 bp allows discrimination among distantly

related species. Owing to its higher rate of nucl-

eotide substitutions, the 75 bp adds discrimina-

ting power among closely related species from

same genus and closely related genera from

same family. Because of its higher percentage

of nucleotide sequence divergence than the 16S

rRNA gene, the 232 bp marker can better discri-

minate among closely related γ-proteobacterial

species. Third, the method is simple, rapid, suit-

ed to large screening programs and easily acce-

ssible to most laboratories. Fourth, this marker

can also reveal γ-proteobacterial species which

may appear misassigned and for which additi-

onal characterization appear warranted.

Keywords: γ-Proteobacteria; 16S rRNA; 16S-23S

ITS; Phylogeny

1. INTRODUCTION

The phylum proteobacteria or ‘‘purple bacteria and their

relatives’’ encompasses bacteria with a wide variety of

phenotype and physiological attributes and habitats [1,2].

Proteobacteria have been classified based on the homo-

logy of 16S ribosomal RNA or by hybridization of ribo-

somal DNA with 16S and 23S ribosomal RNA [3-6].

They have been subdivided in five major classes: α-, β-,

γ-, δ- and ε- [7-9].

Most γ-proteobacteria are Gram-negative. This class

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

536

comprises 14 Orders and more than 200 genera. The

γ-proteobacteria exhibit a wide range of metabolic diver-

sity. Most are chemo-organotrophs, some are phototro-

phs or chemolitotrophs [1,2,10,11]. This class includes

several medically and scientifically important bacteria.

Some genera are human (Klebsiella, Shigella, Salmon-

ella, Yersina, Vibrio), animal (Pasteurella) or plant path-

ogens (Pseudomonas, Xanthomonas , Xylella). Others are

obligate endosymbionts (Buchnera, Soda lis, Wiggleswo-

rthia and Coxiella) [10-12]. Because of their biological

importance, γ-proteobacteria are extensively studied.

The 16S ribosomal RNA (rRNA) gene has been est-

ablished as the macromolecule of choice for phylogen-

etic analyses [5,13]. The current phylogeny of γ-proteo-

bacteria is based on the homology of 16S rDNA nucleo-

tide sequences [3-6,11,14].

The 16S-23S internal transcribed spacer (ITS) region

is more variable than the 16S rRNA gene. It has been

used, among others, in the study of specific γ-proteoba-

cterial diversity at the species level, including Escheri-

chia, Ha emophilus , Xanthomonas, Klebsiella and Pseud-

omonas [15-19].

Additional approaches, based on different genes, have

been used for the study of γ-proteobacterial phylogeny

[12,20-25]. Very recently, Gao et al., [26] have used a

combination of phylogenomic and comparative genomic

approaches to reconstruct the phylogeny of γ-proteob-

acteria.

In an earlier work on the bacterial Gram-positive Ba-

cillus genus and related genera [27], a short 220 bp nu-

cleotide sequence “marker” was used to reconstruct their

phylogeny. This 220 bp marker was a combination of a

150 bp sequence at the 3’ end of the 16S rRNA gene and

a 70 bp sequence at the 5’ end of the 16S-23S ITS se-

quence. Owing to its higher rate of nucleotide substitu-

tion, the 70 bp sequence at the 5’ end of the 16S-23S ITS

sequence added a greater discriminatory power among

closely related species than 16S rRNA gene nucleotide

sequences alone. They showed that the phylogeny in-

ferred from the 220 bp marker was in agreement with

then current classifications based on phenetic and mo-

lecular data. The marker also indentified species which

appeared misassigned. It also created new clusters sug-

gesting the creation of new taxa levels. In a very recent

study, we [28] have tested whether or not this marker

could reconstruct the phylogeny of the bacterial Gram-

positive Order of the Bacilla les.

In the current study, we further assess the usefulness

of a similar marker among 13 of the 14 γ-proteobacterial

Orders. The last 157 bp at the 3’ end of the 16S rRNA

gene was combined with the first 75 bp at the 5’ end of

the 16S-23S Internal Transcribed Spacer (ITS) to yield a

single 232 bp DNA marker. This marker was used to

reconstruct the phylogeny of γ-proteobacteria. A total of

64 γ-proteobacteria from 13 Orders, 22 families, 40 gen-

era and 59 species was analyzed.

2. MATERIALS AND METHODS

2.1. Bacterial Species and Strains

A total of 64 γ-proteobacterial species and strains were

analyzed. They were selected on the basis that their com-

plete genome sequences were freely available in Gen-

Bank, at the National Center for Biotechnology Informa-

tion (NCBI) completed microbial genomes database

(http://www.ncbi.nlm.nih.gov/genomes/MICROBES/Co

mplete.html). They encompassed 13 Orders, 22 families,

40 genera and 59 species. These 13 γ-proteobacterial Or-

ders included six Aeromonadales families, one Cardiob-

acteriales family, two Chromatiales families, one Enter-

obacteriales family, two Legionellales families, one Me-

thylococcales family, two Oceanospirillales families,

one Pasteurellales family, two Pseudomonadales fami-

lies, two Thiotrichales families, one Vibrionales family

and one Xanthomonada les family. All bacterial species

and strains and the GenBank accession number for their

fully sequenced genome are listed in Table 1.

2.2. Sequences Analysis

The 16S rRNA gene nucleotide sequences were retriev-

ed from GenBank (Table 1) for the 64 γ-proteobacteria

species and strains under study. First, all 64 sequences

were aligned using ClustalW [29] (data not shown).

Next, the 3’ end of the 16S rRNA gene nucleotide sequ-

ences of alleles from same bacterial strain, of alleles

from different strains from same species, and of alleles

from different species from same genus (data not shown)

were aligned using ClustalW [29]. The length of the

nucleotide sequence most conserved was determined at

157 bp. Likewise, the 16S-23S Internal Transcribed

Spacer (ITS) nucleotide sequences of alleles from same

bacterial strain were also aligned using ClustalW. The

length of the nucleotide sequence most conserved was

determined at 75 bp.

These two most conserved nucleotide sequences, the

157 bp at the 3’ end of 16S, and the 75 bp at the 5’ end

of 16S-23S ITS were combined into a single 232 bp seq-

uence for each bacterial species and strain under study.

This 232 bp sequence will be used here as a phylogen-

etic marker for the γ-proteobacteria under study.

2.3. Phylogenetic trees

Two neighbor-joining trees were constructed [30], a first

one based on the alignment of the last 157 bp at the 3’

end of the 16S rRNA gene described above, a second

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

537

one based on the alignment of the 232 bp sequence also

described above. Both trees were bootstrapped using

1,000 random samples of sites from the alignment, all

using CLUSTAL W [29] at the DNA Data Bank of Jap-

an (DDBJ) (http://clustalw.ddbj .nig.ac.jp/top-e.html ),

with the Kimura’s parameter method [31]. The neighbor-

joining tree was drawn using TreeView (version 1.6.6)

[32,33].

Table 1. γ-proteobacteria strains used in this study.

Orders, Families,

Genera, Species

Strain/Source

GenBank

Accession no.

Orders, Families,

Genera, Species

Strain/Source

GenBank

Accession no.

Aeromonadales

Aeromonodaceae

Aeromonas hydrophyla

Aeromonas salmonida

ATCC 7966

A449

NC_008570.1

NC_009348.1

Shigella flexineri

Sodalis glossinidus

Wigglesworthia glossinidia

Yersinia enterocolitica

Yersinia pestis

Yersinia pseudotuberculosis

2457T

morsitans

8081

CO92

IP31758

NC_004741.1

NC_007712.1

NC_004344.2

NC_008800.1

NC_003143.1

NC_009708.1

Legionellales

Coxiellaceae

Coxiella burneti

Coxiella burnetii

Legionellaceae

Legionella pneumophila

Dugway 5j108-111

RSA 493

Lens

Corby

NC_009727.1

NC_002971.3

NC_006369.1

NC_009494.1

Methylococcales

Methylococcaceae

Methylococcus capsulatus

Bath

NC_002977.6

Alteromonadales

Alteromonadaceae

Marinobacter aquaeolei

Saccharophagus degradans

Colwelliaceae

Colwellia psychrerythrea

Idiomarinaceae

Idiomarina ihoihiensis

Pseudoalteromonadaceae

Pseudoalteromonas atlantica

Pseudoalteromonas haloplanktis

Shewanellaceae

Shewanella amazonensis

Shewanella denitrificans

Shewanella frigidimarina

Shewanella oneidensis

VT8

2-40

34H

L2TR

T6c

TAC125

SB2B

OS217

NCIMB 400

MR-1

NC_008740.1

NC_007912.1

NC_003910.7

NC_006512.1

NC_008228.1

NC_007481.1

NC_008700.1

NC_007954.1

NC_008345.1

NC_004347.1

Cardiobacteriales

Cardiobacteriaceae

Dichelobacter nodosus

VCS1703A

NC_009446.1

Pseudomonadales

Moraxellaceae

Acinetobacter baumannii

Acinetobacter sp.

Psychrobacter arcticus

Psychrobacter cryohalolentis

Pseudomonadaceae

Pseudomonas fluorescens

Pseudomonas syringea

ATCC 17978

ADP1

273-4

Pf5

DC3000

NC_009085.1

NC_005966.1

NC_007204.1

NC_007969.1

NC_004129.6

NC_004578.1

Chromatiales

Chromatiaceae

Nitrosococcus oceani

Ectothiorhodospiraceae

Alkalilimnicola ehrlichei

Halorhodospira halophila

ATCC 19707

MLHE-1

SL1

NC_007484.1

NC_008453.1

NC_008789.1

Thiotrichales

Francisellaceae

Francisella philomiragia

Francisella tularensis

Piscirickettsiaceae

Thiomicrospira crunogena

philomiragia

horlatica

XCL-2

NC_010336.1

NC_007880.1

NC_007520.2

Vibrionales

Vibrionaceae

Photobacterium profundum

Vibrio cholerae

Vibrio parahaemolyticus

Vibrio vulnificus

SS9

N16961

RIMD 2210633

CMCP6

NC_006370.1

NC_002505.1

NC_004603.1

NC_004459.2

Enterobacteriales

Enterobacteriaceae

Buchnera aphidicola

Citrobacter koseri

Enterobacter sakazakii

Enterobacter sp

Escherichia coli

Klebsiella pneumoniae

Photorhabdus luminescens

Salmonella enterica

Shigella boydii

Shigella dysenteriae

APS

ATCC BAA-895

ATCC BAA-894

638

CFT073

O157:H7 Sakai

342

NTUH-K2044

TT01

Ty2

arizonae

Sb227

Sd197

NC_002528.1

NC_009792.1

NC_009778.1

NC_009436.1

NC_004431.1

NC_002695.1

NC_011283.1

NC_012731.1

NC_005126.1

NC_004631.1

NC_010067.1

NC_007613.1

NC_007606.1

Xanthomonadales

Xanthomonadaceae

Xanthomonas axonopodis

Xanthomonas campestris

Xylella fastidiosa

306

8004

9a5c

NC_003919.1

NC_007086.1

NC_002488.3

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

538

3. RESULTS AND DISCUSSION

In a previous study [27], on the bacterial genus Bacillus

and closely-related genera, we reported the development

of a short DNA marker that could be used to reconstruct

their phylogeny. This marker was a combination of 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 internal

transcribed spacer (ITS) into a single 220 bp “marker”. It

could cluster Bacillus species and species from closely

related genera into taxa akin to genera and could also

distinguish closely related species. The 3’ end of the 16S

rRNA gene contained three regions that were known to

be highly conserved among bacteria [34]. The 5’ end of

the 16S-23S rRNA ITS was conserved among alleles

from same strains [27].

In the current study on γ-proteobacteria, we further

assessed the usefulness of this marker. The sizes of the

3’ end of the 16S rRNA gene and the 5’ end of the

16S-23S rRNA ITS retained here for the construction of

our phylogenetic marker for γ-proteobacteria are slightly

different at 157 and 75 bp, respectively, for a total

marker size of 232 bp. These sizes were selected as fol-

lows: first, an alignment of the 16S rRNA gene nucleo-

tide sequence of alleles from same strain showed that

these sequences were highly conserved. The intra-strain

alleles shared 99% nucleotide sequence identities. Al-

leles from species from same genus, however, covered a

wider spectrum of nucleotide sequence identities. Where-

as alleles from Pseudomonas (Ps.) fluorescens Pf-5 and

from Ps. syringae pv. tomato strain DC3000 share 98%

nucleotide sequence identities, alleles from Vibrio (V.)

cholerae and from V. parahaemolyticus RIMD 2210633

share 92% nucleotide sequence identities. Comparative

analyses of the 3’ end of the 16S rRNA gene nucleotide

sequence showed that the last 157 bp were in many cases

highly conserved among strains from same species. This

is exemplified by Salmonella (Sal.) enterica arizonae

and Sal. enterica Ty2 which share 99% nucleotide se-

quence identities over the last 157 bp at the 3’ end of the

16S rRNA gene. Species from same genus share lower

nucleotide sequence identities. This is exemplified by

Shewanella (She) amazonensis and She. denetrificans

which share 94% nucleotide sequence identities over the

last 157 bp at the 3’ end of the 16S rRNA gene.

A bootstrapped neighbor-joining tree based on the align-

ment of this 157 bp located at the 3’ end of the 16S

rRNA gene was constructed (Figure 1). Although, in

most cases, this 157 bp could distinguish species from

different genera from same family, in some cases, close-

ly related species from different genera from same fam-

ily appeared undistinguishable. This is the case for Mann-

heimia succiniciproducens and both Actinobacillus (Act),

species, Act. pleuropneumonia and Act. succinogenes.

This is also true for Xylella (Xy.) fastidiosa and both

Xanthomonas (

X.) species, X. axonopodis pv. citri str.

306 and X. campestris pv. campestris str.8004. And this

is true for the human and animal pathogenic Entero-

bacteriaceae: the Yersinia, Shigella, Kleb siella, Escheri-

chia, Enterobacter and Citrobacter species. In some

cases, this 157 bp could distinguish species from same

genus as exemplified by the Psychrobacter, Pseudomo-

nas, Shewan ella and Vibrio species. In other cases,

closely related species from same genus could not be

distinguished as exemplified by the Francisella , Aero-

monas, Actinobacillus, Haemophilus, Acinetobacter and

Xanthomonas species.

In all cases, this 157 bp could distinguish species from

different families, with one exception: She. amazonensis

and Photobacterium (Ph.) profundum, members of the

Shewanellacea and Vibrionacea family, respectively.

Both appear undistinguishable. Clearly, this 157 bp seq

uence cannot distinguish closely-related species. An ad-

ditional DNA sequence appears necessary to better disti-

nguish closely-related species.

Next, an alignment of the 16S-23S ITS nucleotide se-

quences of alleles from same bacterial strain was carried

with a subset of the bacteria under study: Xanthomonas

campestris pv. campestris str. 8004, Ps. syringae pv.

tomato str. DC3000, Act. succinogenes 130Z, E. coli

K12, V. parahaemolyticus RIMD 2210633 and Shigella

flexneri 2a str. 301 (Figure 2). The total number of al-

leles vary from two to ten for X. campestris pv. campes-

tris str. 8004 and V. parahaemolyticus RIMD 2210633,

respectively. The allelic sequences were highly ho-

mologous for some species and highly hererologous for

others. Xanthomonas campestris pv. campestris str. 8004

and Ps. syringae pv. tomato str. DC3000 carry two and

five identical alleles, respectively. Actinoba cillus suc-

cinogenes 130Z, E. coli K12, V. parahaemolyticus

RIMD 2210633 and Sh igella flexneri 2a str. 301 carry

five, eight, ten and six alleles respectively, with varying

level of heterogeneity, where highly homologous alleles

are grouped together and can be distinguised from dif-

ferent alleles in same strain (Figure 2). Alleles carry

from zero to four tRNA genes. An alignment of the nu-

cleotide sequences among alleles at the intra-strain level

required the introduction of several gaps. The first 75 bp

at the 5’ end of the 16S-23S ITS, however, was highly

conserved at the intra-strain level. It was retained here

for the construction of our phylogenetic marker. The two

conserved nucleotide sequences identified above, the

157 bp at the 3’ end of 16S rRNA gene and the 75 bp at

the 5’ end of 16S-23S ITS, were combined into a single

232 bp sequence. This will be used here as a phyloge-

netic marker for the γ-proteobacteria under study.

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

539

Buchnera aphidicola

Thiomicrospira crunogena

Pseudoalteromonas atlantica Pseudoalteromonas haloplanktis

Francisella philomiragia

Francisella tularensis

eromonas salmonicida

eromonas hydrophilaMannheimia succiniciproducens

ctinobacillus pleuropneumonia

ctinobacillus succinogenes

Haemophilus ducrey

Haemophilus influenzae

Pasteurella multocida

Shewanella amazonensis

Photobacterium profundum

Methylococcus capsulatu

lcanivorax borkumensis

Psychrobacter cryohalolentis

Psychrobacter arcticu

Idiomarina ioihiensis

Chromohalobacter salexigens

Coxiella burnetii RSA493

Coxiella burnetii Dugway 5J108-111

100

cinetobacter baumanni

cinetobacter sp ADP1

Dichelobacter nodosu

ylella fastidiosa

anthomonas axonopodis

anthomonas campestris

Nitrosococcus oceani

lkalilimnicola ehrliche

Halorhodospira halophila

Marinobacter aquaeole

Legionella pneumophila str.Corby

Legionella pneumophila str.Lens

Pseudomonas fluorescen

Pseudomonas syringae

Saccharophagus degradans

Salmonella enterica

Photorhabdus luminescens

Wigglesworthia glossinidia

Shewanella oneidensis

Colwellia psychrerythraea

Shewanella denitrificans

Shewanella frigidimarina

Vibrio vulnificus

Vibrio cholerae

Vibrio parahaemolyticus

Enterobacter sakazaki

Citrobacter koser

Enterobacter sp

Escherichia coli.

Klebsiella pneumoniae

Shigella boydi

Shigella dysenteriae

Shigella flexneri

Yersinia enterocolitica

Yersinia pestis

Sodalis glossinidius

Yersinia pseudotuberculosis

0.01

Figure 1. Bootstrapped neighbor-joining tree of γ –proteobacteria species inferred from the alignment of a 157 nucleotide sequence

at the 3’ end of the 16S rRNA gene. Bootstrap values higher than 50% are indicated (expressed as percentage of 1000 replication).

The horizontal bar represents 1% nucleotide difference.

A bootstrapped neighbor-joining tree based on the

alignment of a 232 bp sequence was constructed (Figure

3). Based on the topology of the neighbor-joining tree,

four major Groups, Group I to IV are revealed (Figure

3). Based on nucleotide sequence identities, sub-groups

and clusters can be formed. Group I contains seven Ord-

ers and nine families. Group II contains eight Orders and

eleven families. Group III contains three Orders and five

families. Group IV contains one Order and one family.

Of the 13 Orders under study, four are present in more

than Group one. The Thio trichales are present in Groups

I and III. The Alteromonadales are present in Groups I,

II and III. The Legionellales are present in Groups I and

II. The Vibrionales are present in Groups I and III. All

other nine Orders are present in a single Group. Of the

22 families under study, four are present in more than

one Group. The Alteromon adaceae are present in

Groups I and II. The Pseudoalteromonadaceae (Pse) are

present in Groups I and III. The Vibrionaceae are pre-

sent in Groups I and III. The Shewanellaceae are present

in Groups I and III. All other 18 families are present in a

single Group. All species from same genus are present in

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

540

same Group with the exception of Pse. haloplanktis and

Pse. atlantica present in Group I and III, respectively.

Group I can be sub-divided into six sub-groups, sub-

group I-1 to I-6. Sub-group I-1 contains both species of

the Francisellaceae family. Sub-group I-2 contains three

species, members of two families: Pseudomonadacea e

and Alteromonadaceae. Sub-group I-3 comprises Pse.

haloplanktis and both Legionella pneumophila strains.

Although in the same sub-group, Pse. haloplanktis sh-

ows 20% nucleotide sequence divergence with the two

Legionella strains. Both Legionella strains are tightly

grouped together and form cluster I-3-1. Sub-group I-4

contains Ph. profundum and She. amazonensis, two spe-

cies from two different families, Vibrionaceae and She-

wanellaceae, respectively. Although they appear very si-

milar on the neighbor-joining tree, both sequences show

15% nucleotide divergence. Sub-group I-5 contains both

Aeromonas species, tightly grouped together. Sub-group

I-6 contains all six Pasteurellaceae species. The Mann-

heimia species is tightly grouped with the two Actino-

bacillus species and form cluster I-6-1. Both Haemop-

hilus species form cluster I-6-2. In Group I, clusters co-

mprised species from same genus or closely related spe-

cies from different genera from same family. All other

branches corresponded to families.

Group II can be sub-divided into four sub-groups,

sub-group II-1 to II-4, and one ungrouped species, Me-

thylococcus capsulatus. Sub-group II-1 comprises mem-

bers of four families from four Orders. Members of

sub-group II-1 share up to 30% nucleotide sequence di-

vergences. Closely related species can be further gro-

uped together. This is the case for the two Acinetobacter

species and all three Xanthomonadaceae species which

form cluster II-1-1 and II-1-2, respectively. Sub-group

II-2 contains Nitrosoccocus oceani and Halorhodospira

halophila, member of the Chromatiaceae and Ectothior-

hodospira cea family respectively. Both species show

42% nucleotide sequence divergences. Both families

belong to the Chromatiales Order. Sub-group II-3 con-

tains six species, from five families and three Orders.

Members of sub-group II-3 share up to 24% nucleotide

sequence divergences. Two closely related species, Psy-

chrobacter (Psy) cryohalolentis and Psy. arcticus form

cluster II-3-1. Sub-group II-4 comprises both strains of

Coxiella burnetti, members of the Coxelliacea e family. In

Group II, clusters comprised species from same genus or

closely related species from different genera from same

family. All other branches corresponded to families.

Pseudomonas syringae pv. tomato str. DC3000

PSTPTO _R01

PSTPTO _R04

PSTPTO _R07

PSTPTO _R02

PSTPTO _R015

Xanthomonas campestris pv. campestris str. 8004

XC_4386

XC_4393

rrsA

rrsH

rrrsC

rrsE

100 200 300400 500 600 700800 850

Asuc_R 0 01 7

Asuc_R 0 04 9

Asuc_R0019

Asuc_R0035

Asuc_R0054

Actinobacillus succinogenes 130Z

Shigella flexneri 2astr. 301

Vibrio paraheaemolyticus RIMD 2210633

Vpr003

Vpr006

Vpr001

Vpr007

Vpr005

Vpr004

Vpr002

Vpr022

Vpr025

Vpr019

rrsA

rrsB

rrsC

rrsE

rrrsH

rrrsG

rrrsB

rrrsD

rrrsG

Escherichia coli K12 substr. MG 1655

tRNAIle

tRNA AlatRNAGlutRNA LystRNA Val

Figure 2. Schematic representation of allelic 16S-23S rDNA Internal Transcribed Spacer of γ –prot-

eobacteria.The non filed boxes represent regions of homologus nucleotide sequences between allelic

ITS of the same bacteria. Filed boxes represent tRNA. The blank spaces between boxes represent non

conservation regions between allelic ITS of the same bacteria.

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

541

Francisella philomiragia

Francisella tularensis

100

Pseudomonas fluorescens

Pseudomonas syringae

Saccharophagusdegradans

Pseudoalteromonashaloplanktis

Legionella pneumophila str.Corby

Legionella pneumophila.str.Lens

Photobacterium profundum

Shewanella amazonensis

971

Aeromonashydrophila

Aeromonassalmonicida

Mannheimia succiniciproducens

Actinobacillus pleuropneumonia

Actinobacillus succinogenes

Haemophilusducreyi

Haemophilusinfluenzae

Pasteurella multocida

Methylococcus capsulatus

Dichelobacter nodosus

Acinetobacter baumannii

Acinetobacter sp.

Alkalilimnicolaehrlichei

Xylella fastidiosa

Xanthomonas axonopodis

Xanthomonas campestris

100

Nitrosococcus oceani

Halorhodospirahalophila

Marinobacter aquaeolei

Alcanivoraxborkumensis

Psychrobactercryohalolentis

Psychrobacter arcticus

Chromohalobacter salexigen

Idiomarina loihiensis

Coxiella burnetii RSA 493

100

Thiomicrospira crunogena

Shewanella oneidensis

Shewanella denitrificans

Shewanella frigidimarina

Vibrio vulnificus

Vibrio cholerae

Vibrio parahaemolyticus

Pseudoalteromonasatlantica

Colwellia psychrerythraea

Buchnera aphidicola

Wigglesworthia glossinidia

Salmonella enterica Typhi

Photorhabdusluminescens

Klebsiella pneumoniae NTUH-K2044

Escherichia coli CFT073

Salmonella enterica Arizonae

Klebsiella pneumoniae

Yersinia pseudotuberculosis

Yersinia enterocolitica

Yersinia pestis

Sodalis glossinidius

Enterobactersakazakii

Shigella flexneri

Shigella dysenteriae

Shigella boydii

Eschecherichia coli O157H7Sakai

Enterobactersp.

Citrobacter koseri

Sub-groups Families Groups

Clusters Orders

Francisellaceae

Pseudomonadaceae

+ Alteromonadaceae

Pseudomonadaceae

+ Alteromonadaceae

Pseudoalteromonadaceae

+ Legionellaceae

Vibrionaceae

+ Shewanellaceae

Aeromonadaceae

Pasteurellaceae

Cardiobacteriaceae

Methyloccocaceae

Moraxellaceae

Ectothiorhodospiraceae

Xanthomonadaceae

Chromatiaceae

+ Ectothiorhodospiraceae

Alteromonadaceae

Alcanivoraceae

Moraxellaceae

Halomonadaceae

Idiomarinaceae

Coxiellaceae

Piscirickettsiaceae

Shewanellaceae

Vibrionaceae

Pseudoalteromonadaceae

+ Colwelliaceae

Enterobacteriaceae

Thiothricales

Pseudomonadales

Alteromonadales

Legionellales

Vibrionales

Alteromonadales

Aeromonadales

Pasteurellales

Meth yloc coc al es

Cardiobacteriales

Pseudomonadales

Chromatiales

Xanthomonadales

Chromatiales

Alteromonadales

Oceanospirillales

Pseudomonadales

Oceanospirillales

Alteromonadales

Legionellales

Alteromonadales

Thiothricales

Vibrionales

Enterobacteriales

Alteromonadales

III

IIIIII

IVIV

I-1

I-2

I-3

I-4

I-5

I-6

Ungrouped

II-1

II-2

II-3

II-4

Ungrouped

III-1

III-2

IV-2

IV-1

IV-3

I-1

I-2

I-3

I-4

I-5

I-6

Ungrouped

II-1

II-2

II-3

II-4

Ungrouped

III-1

III-2

IV-2

IV-1

IV-3

I-6-1

I-6-2

II-1-1

II-1-2

II-3-1

III-1-1

III-1-2

II-3-2

I-3-1

I-6-1

I-6-2

II-1-1

II-1-2

II-3-1

III-1-1

III-1-2

II-3-2

I-3-1

Coxiella burnetti Dugway5j108-111

Figure 3. Bootstrapped neighbor-joining tree of γ-proteobacteria species inferred from the alignment of 232

nucleotide sequence marker. This 232 nucleotide sequence marker is a combination of a 157 nucleotide se-

quence at the 3’ end of the 16S rRNA gene and a 75 nucleotide sequence at the 5’ end of the 16S-23S Internal

Transcribed Spacer (ITS) sequence. Major Groups are indicated in capital roman numerals. Sub-groups and

clusters are indicated in arabic numbers. Bootstrap values higher than 50% are indicated (expressed as per-

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

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

542

Group III can be sub-divided into two sub-groups,

sub-groups III-1 and III-2, and one ungrouped species,

Thiomicrospira crunogena. Sub-group III-1 contains

three Shewallenaceae and three Vibrionaceae species.

She. denitrificans and She. frigidimarina, and the three

Vibrio species form two clusters, III-1-1 and III-1-2,

respectively. Sub-group III-2 contains two genera from

two families of the same Order. Both species, on two

separate branches, show 23% nucleotide sequence di-

vergence. In Group III, both clusters comprised species

from same genus. All other branches corresponded to

families.

Group IV contains all the Enterobacteriaceae species

under study. Three sub-groups can be revealed: sub-

group IV-1 to IV-3. Sub-group IV-1 contains two insect

obligate endosymbionts. Sub-group IV-2 contains Salm-

onella enterica Ty2 and Photorhabtus luminescens. All

other Enterobacteriaceae species are in sub-group IV-3.

The latter are closely related to each others.

Our results, based on the 232 bp phylogenetic marker

described here are, in general, in agreement with the

phylogeny of γ-proteobacteria based on the 16S rRNA

gene with some exceptions. In the neighbor-joining tree,

clusters comprised species from same genus or closely

related species from different genera from same family.

All other branches corresponded to families. As indi-

cated above, of the 22 families under study, 18 are pre-

sent in a single Group and four are present in more than

one Group. These latter four families encompass the

marine bacteria [35]. They are Vibrionaceae, in Groups I

and III; and the Alteromonas-related protobacteria

(Ivanova et al., 2004): Alteromon adaceae, in Groups I

and II; Pseudoalteromon adaceae, in Groups I and III;

and Shewanellaceae, in Groups I and III. Interestingly,

within a Group, these marine bacteria are found in close

proximity of one another. It reflects the varying level of

heterogeneity among Alteromonas-related protobacteria.

The grouping showed here is based on a 232 bp marker.

The biological significance of this grouping is unknown.

Clearly, however, the phylogenetic analyses of these

related marine heterotrophic bacteria is a work in pro-

gress [36].

4. CONCLUSIONS

In conclusion, the use of this 232 bp marker presents

several advantages over the use of the entire 16S rRNA

gene or the generation of extensive phenotypic and gen-

otypic data in phylogenetic analyses. First, this marker is

not allele-dependant. The 3’ end of the 16S rRNA gene

is highly conserved at the intra-strain level. We have

shown here that although the16S-23S ITS allelic se-

quences can be very heterogeneous within a strain, the

first 75 bp, however, are conserved among alleles from

same strain in γ-proteobacteria. Clearly, any allele would

generate the same results. Second, this 232 bp marker

contains 157 bp from the 3’ end of the 16S rRNA gene

and 75 bp from the 5’ end of the 16S-23S ITS. The 157

bp is highly conserved among closely related species.

Owing to its higher rate of nucleotide substitutions, the

75 bp adds discriminating power among closely related

species from same genus and closely related genera from

same family. Because of its higher percentage of nucleo-

tide sequence divergence than the 16S rRNA gene, the

232 bp marker can better discriminate among closely

related γ-proteobacteria species. Third, the method is

simple, rapid, suited to large screening programmes and

easily accessible to most laboratories. More importantly,

however, this 232 bp marker can group γ-proteobacteria

families and genera in accordance with established phy-

logenies, with the exceptions indicated above. It can also

reveal γ-proteobacteria species which may appear mis-

assigned and for which additional characterization app-

ear warranted.

REFERENCES

[1] Stackebrandt, E., Murray, R.G.E. and Trüper, H.G. (1988)

Proteobacteria classis nov., a name for the phylogenetic

taxon that includes the “purple bacteria and their rela-

tives”. International Journal of Systematic Bacteriology,

38(3), 321-325.

[2] Gupta, R.S. (2000) The phylogeny of proteobacteria:

relationships to other eubacterial phyla and eukaryotes.

FEMS Microbiology Reviews, 24(4), 367-402.

[3] Fox, G.E., Stackebrandt, E., Hespell R.B., Gibson J.,

Maniloff, J., Dyer, T.A., Wolfe, R.S., Balch, W.E., Tanner,

R.S., Magrum, L.J., Zablen, L.B., Blakemore, R., Gupta,

R., Bonen, L., Lewis, B.J., Stahl, D.A., Luehrsen, K.R.,

Chen, K.N. and Woese, C.R. (1980) The phylogeny of

prokaryotes. Science, 209(4455), 457-463.

[4] Woese, C.R., Stackebrandt, E., Macke, T.J. and Fox, G. E.

(1985a) A phylogenetic definition of the major eubacterial

taxa. Systematic and Applied Microbiology, 6, 143-151.

[5] Woese, C.R. (1987) Bacterial Evolution. FEMS Mic-

robiology Reviews, 51(2), 221-271.

[6] De Ley, J. (1992) The proteobacteria: Ribosomal RNA

cistron similarities and bacterial taxonomy. In: by Balows,

A., Trüper, H.G., Dworkin, M., Harder, W., and Schleifer,

K.H., Eds., The Prokaryotes, 2nd Edition, Springer-Verlag,

Berlin, 2111-2140.

[7] Woese, C.R., Stackebrandt, E., Weisburg, W.G., Paster, B.

J., Madigan, M.T., Fowler, V.J., Hahn, C.M., Blanz, P.,

Gupta, R., Nealson, K.H. and Fox, G.E. (1984a) The

phylogeny of purple bacteria: The alpha subdivision.

Systematic and Applied Microbiology, 5, 315-326.

[8] Woese, C.R., Weisburg, W.G., Paster, B.J., Hahn, C.M.,

Tanner, R.S., Krieg, N.R., Koops, H.-P., Harms, H. and

Stackebrandt, E. (1984b) The phylogeny of purple bacte-

ria: The beta subdivision. Systematic and Applied Micro-

S. Yakoubou et al. / Natural Science 2 (2010) 535-543

543

biology, 5, 327-336.

[9] Woese, C.R., Weisburg, W.G., Hahn, C.M., Paster, B.J.,

Zablen, L.B., Lewis, B.J., Macke, T.J., Ludwig, W. and

Stackebrandt, E. (1985b) The phylogeny of purple bacte-

ria: The gamma subdivision. Systematic and Applied Mi-

crobiology, 6, 25-33.

[10] Brenner, D.J., Krieg, N.R., Staley, J.T. and Garrity, G.M.,

(Eds.) (2005) Bergey’s manual of systematic bacteriology,

2nd Edition, Vol. 2 (The P roteobacteria), part C (The Al-

pha-, Beta-, Delta-, and Epsilonproteobacteria), Springer,

New York.

[11] Kersters, K., Devos, P., Gillis, M., Swings, J., Vandamme,

P. and Stackebrandt, E. (2006) Introduction to the Proteo-

bacteria. In: Dworkin, M., Falkow, S., Rosenberg, E.

Schleifer, K.H. and Stackebrandt, E., Eds., The Pro-

karyotes: A Handbook on the Biology of Bacteria.

Springer, New York, 3-37.

[12] Belda, E., Moya, A. and Silva, F.J. (2005) Genome rear-

rangement distances and gene order phylogeny in γ-pro-

teobacteria. Molecular Biology and Evolution, 22(6), 1456-

1467.

[13] Woese, C.R., Kandler, O. and Wheelis, M.L. (1990) To-

wards a natural system of organisms: Proposal for the

domains archaea, bacteria, and eucarya. Proceedings of

the National Academy of Sciences, USA, 87(12), 4576-

4579.

[14] Ludwig, W. and Klenk, H.P. (2005) Overview: A phy-

logenetic backbone and taxonomic framework for pro-

karyotic systematics. In: Brenner, D.J. Krieg, N.R. Staley

J.T. and Garrity, G.M., Eds., Bergey’s Manual of System-

atic Bacteriology, Springer-Verlag, Berlin, 49-65.

[15] Anton, A.I., Martinez-Murcia, A.J. and Rodriguez-Val-

era, F. (1998) Sequence diversity in the 16S-23S inter-

genic spacer region (ISR) of the rRNA operons in repre-

sentatives of the Escherichia coli ECOR collection. Jou-

rnal of Molecular Evolution, 47(1), 62-72.

[16] Giannino, V., Rappazzo, G., Scuto, A., Di Marco, O.,

Privitera, A., Santagati, M. and Stefani, S. (2001) rrn op-

erons in Haemophilus parainfluenzae and mosaicism of

conserved and species-specific sequences in the 16S-23S

rDNA long spacer. Research in Microbiology, 152(5),

461-468.

[17] Goncalves, E.R. and Rosato, Y.B. (2002) Phylogenetic

analysis of Xanthomonas species based upon 16S-23S

rDNA intergenic spacer sequences. International Journal

of Systematic and Evolutionary Microbiology, 52(Pt 2),

355-361.

[18] Wang, M., Cao, B., Yu, Q., Liu, L., Gao, Q., Wang, L.

and Feng, L. (2008) Analysis of the 16S-23S rRNA gene

internal transcribed spacer region in Klebsiella species.

Journal of Clinical Microbiology, 46(11), 3555-3563.

[19] Tambong, J.T., Xu, R. and Bromfield, E.S.P. (2009) In-

tercistronic heterogeneity of the 16S-23S rRNA spacer

region among Pseudomonas strains isolated from subter-

ranean seeds of hog peanut (Amphicarpa bracteata). Mi-

crobiology, 155(Pt 8), 2630-2640.

[20] Kunisawa, T. (2001) Gene arrangements and phylogeny

in the class Proteobacteria. Journal of Theoretical Biol-

ogy, 213(1), 9-19.

[21] Lerat, E., Daubin, V. and Moran, N.A. (2003) From gene

trees to organismal phylogeny in prokaryotes: The case

of the γ-proteobacteria. Public Library of Science Biol-

ogy, 1(1), e19.

[22] Brown, J.R. and Volker, C. (2004) Phylogeny of γ –prot-

eobacteria: Resolution of one branch of the universal tree?

Bioessays, 26(5), 463-468.

[23] Ciccarelli, F.D., Doerks, T., von Mering, C., Creevey, C.

J., Snel, B. and Bork, P. (2006) Toward automatic recon-

struction of a highly resolved tree of life. Science, 311

(5765), 1283-1287.

[24] Mrazek, J., Spormann, A.M. and Karlin, S. (2006) Ge-

nomic comparisons among γ-proteobacteria. Environm-

ental Microbiology, 8(2), 273-288.

[25] Lee, H.Y. and Côté, J.-C. (2006) Phylogenetic analysis of

γ-proteobacteria inferred from nucleotide sequence com-

parisons of the house-keeping genes adk, aroE and gdh:

comparisons with phylogeny inferred from 16S rRNA

gene sequences. Journal of General and Applied Micro-

biology, 52(3), 147-158.

[26] Gao, B., Mohan, R. and Gupta, R.S. (2009) Phyloge-

nomics and protein signatures elucidating the evolution-

ary relationships among the Gammaproteobacteria. In-

ternational Journal of Systematic and Evolutionary Mi-

crobiology, 59(2), 234-247.

[27] Xu, D. and Côté, J.-C. (2003) Phylogenetic relationships

between Bacillus species and related genera inferred from

comparison of 3’ end 16S rDNA and 5’ end 16S-23S ITS

nucleotide sequences. International Journal of Systematic

and Evolutionary Microbiology, 53(Pt 3), 695-704.

[28] Yakoubou, S., Xu, D. and Côté, J.-C. Phylogeny of Baci-

llales inferred from 3’ 16S rDNA and 5’ 16S-23S ITS

nucleotide sequences. Nature Science, in Press.

[29] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994)

Clustal W: Improving the sensitivity of progressive mul-

tiple sequence alignment through sequence weighting,

position-specific gap penalties and weight matrix choice.

Nucleic Acids Research, 22(22), 4673-4680.

[30] Saitou, N. and Nei, M. (1987) The neighbor-joining me-

thod: A new method for reconstructing phylogenetic trees.

Molecular Biology and Evolution, 4(4), 406-425.

[31] Kimura, M. (1983) The neutral theory of molecular evo-

lution. Cambridge University Press, Cambridge.

[32] Page, R.D.M. (1996) TreeView: An application to display

phylogenetic trees on personal computers. Computer Ap-

plication in the Biosciences, 12(4), 357-358.

[33] Page, R.D.M. (2000) TreeView—tree drawing software

for apple macintosh and windows. http://taxonomy.zoo-

logy.gla.ac.uk/rod/treeview.html

[34] Gürtler, V. and Stanisich V.A. (1996) New approaches to

typing and identification of bacteria using the 16S-23S

rDNA spacer region. Microbiology, 142(Pt 1), 3-16.

[35] Suen, G., Goldman, B.S. and Welch, R.D. (2007) Pred-

icting prokaryotic ecological niches using genome se-

quence analysis. Public Library of Science Biology ONE,

2(8), e743.

[36] Ivanova, E.P., Flavier, S. and Christen, R. (2004) Phylo-

genetic relationships among marine Alteromonas-like pr-

oteobacteria: Emended description of the family Altero-

monadaceae and proposal of Pseudoalteromonadaceae

fam. nov., Colwelliaceae fam. nov., Shewanellaceae fam.

nov., Moritellaceae fam. nov., Ferrimonadaceae fam.

nov., Idiomarinaceae fam. nov. and Psychromona-daceae

fam. nov. International Journal of Systematic and Evolu-

tionary Microbiology, 54(Pt 5), 1773-1788.