Journal of Microbiological Methods 77 (2009) 109–118
Contents lists available at ScienceDirect
Journal of Microbiological Methods
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j m i c m e t h
16S-23S rDNA internal transcribed spacer regions in four Proteus species
Boyang Cao a,b,c,d,1, Min Wang a,b,c,d,1, Lei Liu a,b,c,d, Zhemin Zhou a,b,c,d, Shaoping Wen a,b,c,d,
Antoni Rozalski f, Lei Wang a,b,c,d,e,⁎
a
TEDA School of Biological Sciences and Biotechnology, Nankai University, 23# HongDa Street, TEDA, Tianjin 300457, China
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China
Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin 300457, China
d
Tianjin State Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, 23# HongDa Street, TEDA, Tianjin 300457, China
e
Tianjin Biochip Corporation, 23# HongDa Street, TEDA, Tianjin 300457, China
f
Department of Immunobiology of Bacteria, Institute of Microbiology and Immunology, University of Lodz, PL 90-237 Lodz, Banacha Str. 12/16, Poland
b
c
a r t i c l e
i n f o
Article history:
Received 21 August 2008
Received in revised form 16 January 2009
Accepted 19 January 2009
Available online 8 February 2009
Keywords:
Proteus
ITS
Internal transcribed spacer
a b s t r a c t
Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal
transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P.
mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P.
myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITSGlu (containing tRNAGlu (UUC) gene)
and ITSIle + Ala (containing tRNAIle (GAU) and tRNAAla (UGC) gene), and are in the forms of 28 variants with 25
genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and
two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2%
to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or
horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable
region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P.
penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely
related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and
laid solid foundation for the development of high-throughput technology for quick and accurate
identification of the important foodborne and nosocomial pathogens.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Proteus belongs to the Family Enterobacteriaceae within the
Proteobacteria. Proteus genus includes five species, P. vulgaris, P.
mirabilis, P. penneri, P. hauseri and P. myxofaciens, exist in manure, soil,
polluted water, and in intestines of human and wide variety of animals
(Garrity et al., 2005). P. vulgaris, P. mirabilis, P. penneri and P. hauseri
are important facultative human pathogens causing foodborne and
primary and secondary nosocomial infections under favorable conditions. P. mirabilis is the third most common cause of urinary tract
infection (UTI) after Escherichia coli and Klebsiella pneumoniae,
causing 12% of the infections; and the second most common cause,
after Providencia stuartii, of catheter-associated bacteria in long-term
catheterized patients responsible for 15% of the infections (Woese,
1987). P. vulgaris and P. penneri are also causative agents of UTI
(Penner, 1992). UTI caused by Proteus is difficult to treat and sometime
⁎ Corresponding author. TEDA School of Biological Sciences and Biotechnology,
Nankai University, 23# HongDa Street, TEDA, Tianjin 300457, China. Tel.: +86 22
66229588; fax: +86 22 66229596.
E-mail address: [email protected] (L. Wang).
1
B. C. and M. W. contributed equally to this report.
0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2009.01.024
can be fatal. These three species may also cause infections of wounds,
burns, skins, and respiratory tracts. P. myxofaciens has been found in
larvae of the gypsy moth, Porthetria dispar (Cosenza and Podgwaite,
1966).
DNA related studies have shown that members of the Proteus
genus are distantly related to other species in the Family Enterobacteriaceae; and from the standpoint of evolution, they are at the
periphery of the Family (Brenner et al., 1978). Previously, P. vulgaris
was divided into three Biogroups, Biogroup 1, 2 and 3. In 1982, P.
vulgaris Biogroup 1 was renamed as Proteus penneri (Hickman et al.,
1982) based on its negative reactions for indole production, salicin
fermentation and aesculin hydrolysis. For the remaining two
Biogroups, Biogroup 2 includes majority of the P. vulgaris strains
isolated from clinical sources and Biogroup 3 is a heterogeneous group
that includes four genomospecies (Brenner et al., 1995; Garrity et al.,
2005). O'Hara et al. showed that genomospecies 3 contained only two
strains exhibiting low relatedness to other P. vulgaris genomospecies
and had unique biochemical features (DNase, lipase and tartrate
negative). Therefore the authors separated the genomospecies 3 from
the other genomospecies as well as to propose that it should be named
Proteus hauseri, whereas the other genomospecies remained unnamed
as Proteus genomospecies 4, 5, and 6 (O'Hara et al., 2000).
110
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
16S rDNA sequencing is a powerful tool for elucidating phylogenetic relationships among prokaryotes (Stackebrandt et al., 1997;
Woese, 1987) and for differential identification of bacterial species and
genera (Goto et al., 2000; Joung and Cote, 2002). However, the
evolutionary conservation of 16S rDNA sequences makes it difficult to
define phylogenetic relationships among closely related species
(Woese, 1987). The genetic relationships of closely related species P.
vulgaris and P. penneri cannot be clearly resolved by 16S rDNA
sequence analysis (Rogall et al., 1990). In comparison with 16S rDNA,
sequence of 16S-23S rDNA internal transcribed spacer (ITS) is
considered to be under less evolutionary pressure and therefore is
prone to more genetic variation (Barry et al., 1991; Gurtler and
Stanisich, 1996). ITS sequences have been shown to be useful in
inferring the phylogenetic relationships of closely related organisms
(Gurtler and Stanisich, 1996), and has successfully differentiated many
bacterial species and subspecies (Conrads et al., 2002; Guasp et al.,
2000; Motoyama and Ogata, 2000; Yoon et al., 1997).
Prior to this study, there is only one ITS sequence of P. mirabilis
available in GenBank. In this report, except of P. hauseri, 813 Proteus
ITS sequences from the other four species were studied, and their
conservation and variability analyzed. The phylogenetic analysis
based on the second variable region of the 16S-23S ITS sequences
were constructed and used to differentiate the closely related P.
vulgaris and P. penneri. This work provides theoretical basis of
molecular separation of individual pathogenic species within Proteus
genus.
Table 1
Length variation and intraspecies identity levels of the Proteu ITS
Species
Strain number
ITSGlu size (bp)
Intraspecies identity
ITSIle + Ala size(bp)
Intraspecies identity
P. mirabilis
3-CCUG4637a
6-CCUG19016a
9-Prk 50/57b
9-Prk 51/57b
5-Prk 20/57b
PrK 47/57b
PrK 53/57b
C1875d
C5735d
G2372c
C2299d
C3731d
C3759d
C3725d
C2297d
7-Prk 42/57b
PrK 34/57b
4-G1c
PrK 12/57b
C2381d
C4305d
PrK 28/57b
CCUG-1 4635a,B2
CMCC 834e,B2
PrK 48/57b,B2
PrK 57/57b,B2
11-CCUG 4677a,B2
PrK 9/57b,B2
PrK 17/57b,B2
TG276-1c,B3
2-Prk 5/57b,B2
PrK 39/57b,B2
CCUG4680a,B3
PrK 37/57b, B3
CCM 3672f
CCM 3674f
16c
63c
60c
52c
26c
41c
1c
19c
ATCC 19692g
G2656c
392, 501
392, 521
392, 521
392, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501, 521
392, 501
392, 501
521, 524
501, 521
501, 521
501, 521
521
528
528
528
528
528
528
528
505, 528
401, 528
401, 528
401, 505, 528
401, 505
525, 527
525, 527
525, 527
525, 527
525, 527
525, 527
527
527
527
527
398, 399
398, 399
/, /
0.997, /
0.989, 0.992,
0.994, 0.986
0.992, 0.990, 0.990
0.987, 0.990, 0.990
0.994, 0.994, 0.994
0.979, 0.909, 0.990
0.987, 0.988, 0.998
0.997, 0.992, 0.996
0.992, 0.990, 0.992
0.989, 0.990, 0.994
0.987, 0.992, 0.998
0.987, 0.994, 0.990
0.992, 0.990, 0.990
0.984, 0.986
0.992, 0.998
0.990, /
1.000, 0.996
0.990, 0.990,
0.994, 0.994,
0.996
/
1.000,
0.998
0.994
0.998
0.996,
0.998
0.988, 0.996
/, 0.996
0.990, 0.998
0.965, /, 0.996
0.965, 1.000,
/, /
1.000, 0.998
0.996, 1.000
0.998, 0.998
0.996, 0.998
0.996, 0.996
1.000
0.998
0.994
0.998
/, /
1.000, 1.000
570, 693, 699, 702
564, 570, 573, 673, 693
564, 570, 573, 673, 693
564, 573, 693, 701
564, 693
573, 693
573, 693
573, 693
573, 693
564, 693
564, 693
564, 693
564, 693
564, 693, 702
564, 693, 702
693, 702
570, 693, 699, 702
693
564, 570, 693, 702
564, 570, 693, 702
564, 570, 693, 702
564, 693
594, 720, 721
594, 720, 721
594, 720, 721
594, 720, 721
594, 720
594
594
571, 676
594, 720, 721
594, 721
697, 720
697, 720, 721
721, 722
721, 722
721, 722
721, 722
721
722
721, 722
721, 722
721, 722
722
591
591
1.000, 0.992, /, 0.992
/, /, /, /,/
0.996, 0.998, 0.998, 0.986, 0.992
0.994, 1.000, 0.992, /
0.994, 0.992
0.996, 1.000
0.994, 1.000
0.994, 0.994
1.000, 0.987,
0.996, 0.997,
0.994, 0.997
0.996, 0.989
0.994, 0.995,
0.996, 0.994, 0,995
0.992, 0.994, 0.994
0.995, /
/, 0.998, 1.000, 0.925
0.989
0.994, 0.991, 1.000,
0.996, 1.000, 0.992, 0.997
0.994, 0.998, 0.988, 0.994
0.994, 0.992
/, /, /
0.998, 0.997, 0.994
0.994, 0.998, 0.995
0.996, 0.997, 0.995
0.998, 0.997
0.994,
0.998,
/, /
0.998, 0.998, 0.997
0.996, 1.000
/, 0.983
1.000, 0.983, 0.979
/, /
0.993, 0.994
0.993, 0.994
0.994, 0.99
0.968
0.994
0.987, 0.994
0.995, 0.994
0.988, 0.997
0.994
/
1.000
P. vulgaris
P. penneri
P. myxofaciens
a
Culture Collection, University of Göteborg (CCUG), Göteborg, Sweden.
Czech Culture Collection of Type Culture, Institute of Hygiene, Prague (Prk), Czech Republic.
c
Department of Immunobiology of Bacteria Institute of Microbiology and Immunology, University of Lodz, Loda, Poland.
d
Clinical isolates.
e
National Centre for Medical Culture Collection (CMCC), China.
f
Czech Collection of Microorganisms (CCM), Czech Republic.
g
American Type Culture Collection (ATCC), USA.
/The ITS was used as the reference for calculation of identity levels.
B2
Biogroup 2.
B3
Biogroup 3.
b
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
111
2. Materials and methods
2.7. Nucleotide sequence accession numbers
2.1. Bacterial strains and culture conditions
A total of 813 ITS sequences analyzed in this study have been
submitted to the EMBL/DDBJ/GenBank Database under the following
accession numbers FJ517769-FJ518370 and FJ518388-FJ518598.
A total of 46 strains were used in this study (Table 1),
including 22 P. mirabilis, 12 P. vulgaris, 10 P. penneri, and 2 P.
myxofaciens. Clinical isolates were collected from six hospitals in
Tianjin, China, during the period of 2005 to 2006. All Proteus
strains were grown in LB medium at 37 °C overnight with shaking.
2.2. DNA extraction
Total DNA was extracted from pure cultures of Proteus using
TIANGEN DNA extraction kit (Tiangen Biotech Co., Ltd., China)
according to manufacturer's protocol. Recombinant plasmid DNA
was extracted from E. coli by alkaline-lysis method (Stephen et al.,
1990).
2.3. Primer design and amplification of 16S-23S ITS region
Thirty-three 16S rRNA sequences and eighteen 23S rRNA
sequences of Proteus were downloaded from RDP (http://rdp.
cme.msu.edu/) and NCBI (http://www.ncbi.nlm.nih.gov/). The
16S-23S Proteus ITS region was amplified by PCR using a pair of
universal ITS primers wl-5793 (5'-TGT ACA CAC CGC CCG TC-3')
and wl-5794 (5'-GGT ACT TAG ATG TTT CAG TTC-3') based on
the highly conserved sequence at the end of 16S rDNA and
the beginning of 23S rDNA. PCR mixture contained 100 ng DNA,
10 nM each primer, 200 µM dNTP, 2.5 mM MgCl2 and 2 units of
Taq DNA polymerase (TaKaRa Biotechnology (Dalian) Co. Ltd.,
China) in 1 x reaction buffer (KCl 50 mM, Tris–HCl 10 mM, [pH
8.3]) in a total volume of 50 µl. Amplification was performed
under the following conditions: 5 min at 95 °C, 35 cycles of 94 °C
for 30 s, 50 °C for 30 s, and 72 °C for 1 min, and a final extension
of 5 min at 72 °C with a DNA Thermal Cycler (Biometra Tpersonal
PCR System, German). Amplicons were visualized by agarose gel
electrophoresis.
2.4. Cloning and DNA sequencing
The PCR amplicons were cloned into pGEM-T Easy vector
(Promega, MA, USA) and transformed into competent E. coli DH5α
cells. White colonies were selected randomly on an ampicillin plate
containing IPTG and X-gal. The recombinant plasmid DNA was isolated
and digested with EcoR I, and visualized in agarose gels to determine
the insert size. For each PCR amplicon, ten clones were selected for
sequencing using wl-5793 and wl-5794 primers using an ABI 3730
automated DNA sequencer.
3. Results
3.1. PCR amplicons of Proteus ITS
The primer pair wl-5793 and wl-5794 was used to amplify the last
120 bp of 16S rDNA, the first 180 bp of 23S rDNA, and the entire 16S23S ITS regions from P. mirabilis (n = 22), P. vulgaris (n = 12), P.
penneri (n = 10) and P. myxofaciens (n = 2) in between. The amplified
fragments were separated by agarose gel electrophoresis to generate
multiple bands of different intensities and sizes approximately from
700 to 1000 bp. The 10 P. penneri and 2 P. myxofaciens strains
generated two bands. The P. mirabilis strains showed different banding
patterns: two amplicons were observed in two strains and three
amplicons in twenty strains. The P. vulgaris also showed two band
patterns: two bands in four strains and three bands in nine strains
(Fig. 1).
3.2. Sequence analysis of ITS and tRNA genes
To avoid errors introduced by Taq DNA polymerase during PCR
amplification and to cover all ITS amplicons, ten clones were
sequenced for each of the PCR fragments. A total of 813 ITS were
determined, including 388 from P. mirabilis, 211 from P. vulgaris, 169
from P. penneri and 45 from P. myxofaciens. After exclusion of the
flanking ends, the length of the ITS was 392–722 bp (Table 1). The
sequences of ITS were interpreted using the program tRNAscan-SE,
version 1.21 (Lowe and Eddy, 1997) and the results revealed the
existence of two distinct ITS types, ITSGlu and ITSIle + Ala, and 28 ITS
sequence variations (based on the size and type of ITS). The two ITS
types are similar to the most common arrangement of the 16S-23S ITS
region in Gram-negative bacteria previously reported (Gurtler and
Barrie, 1995; Perez Luz et al., 1998; Rogall et al., 1990; Cosenza and
Podgwaite, 1966). The sequences of ITSGlu, ITSIle and ITSAla, in four
species of Proteus are 100% in homology, and are 73, 74 and 73 bp long,
respectively. The ITSGlu sequences contain 11 variants with sizes of
392, 501, 521 and 524 bp in P. mirabilis; 401, 505 and 528 bp in P.
vulgaris; 525 and 528 bp in P. penneri; and 398 and 399 bp in P.
myxofaciens. The ITSIle + Ala sequences include 17 variants with sizes of
564, 570, 573, 673, 693, 699, 701 and 702 bp in P. mirabilis; 571, 594,
676, 697, 720 and 721 bp in P. vulgaris; 721 and 722 bp in P. penneri;
and 591 bp in P. myxofaciens (Table 1).
2.5. Sequence analysis of ITS regions
After sequencing the PCR fragments, the portions of the 16S
and 23S rDNA regions were edited out to obtain full ITS sequence.
The 16S-23S ITS sequences determined in this study were aligned
using the ClustalX 1.83 (Jeanmougin et al., 1998). The intraspecies
identity levels for the ITS sequences were calculated using BioEdit
program version 7.0.5.
2.6. Phylogenetic analysis
The phylogenetic trees were constructed by the neighborjoining method and plotted by program MEGA 3.1 (Kumar et al.,
2004). For neighbor-joining (NJ) analysis, the distance between the
sequences was calculated using Kimura's two-parameter model.
The robustness of the trees was statistically evaluated by bootstrap
analysis with 1,000 bootstrap samples.
Fig. 1. ITS amplicons of Proteus species using primers wl-5793 and wl-5794. M,
molecular weight standards (DL2000 Marker); Lane 1, P. myxofaciens; Lane 2, P. penneri;
Lanes 3–4, P. vulgaris Biogroup 3; Lanes 5–6, P. vulgaris Biogroup 2; Lanes 7–8,
P. mirabilis.
112
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
Fig. 2. Sequence alignments of representative 16S-23S ITS blocks of Proteus species: i to vii (A), viii to partial xiii (B), partial xiii to xv (C). ITS types and variants are shown on the left. The dots (…) indicate identical nucleotides, the dashes (—)
indicate a gap or absence of respective nucleotides in the consensus sequence, and gray boxes highlight sequence of tRNA and antiterminator box A.
113
Fig. 2 (continued).
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
114
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
Fig. 2 (continued).
3.3. ITS sequence alignments
The ITS identity is in the range of 96.2–100% (Table 1) for the
strains of the same species. The individual sequences of the 28 ITS
variants are listed in Fig. 2 and their organization structures are
presented in Fig. 3. The aligned ITS sequences without tRNA genes
were further grouped into 15 different blocks (i to xv) after ClustalX
1.83 analysis with manual revision (Fig. 4). Each block, its length
ranges from 8 to 125 bp, was subdivided into different groups
(represented by Arabic numeral) based on sequence similarity. The
sequence analysis indicates that these strains carry 28 ITS variants and
come from 25 genomic origins (Fig. 4). It has been found that a
number of the block arrangements shared by more than one ITS
variants, for example, block ii is shared by all the 28 ITS variants, and
block iv-5, by 9 ITS variants.
3.4. ITS conservative and variable regions of Proteus
Three conservative regions (c1: block i and ii; c2: block x and xi;
c3: block xv) were identified. The c1 and c3, located at the 5' and 3'
ends of all types of ITS, are 55 bp and 12 bp in length. The c2 in the
noncoding regions downstream the tRNA genes of ITS is 70 bp in
length. The antiterminator box A, which is a conservative element in
most of bacteria (Brenner et al., 1978), was found at the start of block x
in c2. Two variable regions (v1: block iv–vii; v2: block xii–xiv) were
located, with the first variable region (v1) flanks tRNA genes, and the
second variable region (v2) exists between the two conservative
regions (c2 and c3) in all types of ITS. The conserved regions and
variable regions are scattered arrangement to make the ITS region a
mosaic-like structure (Fig. 3).
3.5. Species-specific characteristics at v2 regions
The different arrangements of sequence block at v2 regions tag
Proteus strain with species-specific characteristics. There are three
classes of sequence patterns: the first class is xii-1_xiii-1_xiv-1 and
xii-1_xiii-2_xiv-2 for P. mirabilis, xii-1_xiii-5_xiv-3 for P. vulgaris, and
xii-1_ xiii-6_ xiv-7 for P. penneri, characterized by the presence of the
same xii-1 block; the second class is xii-5_xiii-3_xiv-5 for P. mirabilis,
and xii-5_xiii-4_xiv-6 for P. vulgaris, characterized by the presence of
the same xii-5 block; the third class is xii-4_xiv-1 for P. mirabilis, xii3_xiv-2 and xii-2_xiv-2 for P. vulgaris, and xii-2_xiv-2 for P. myxofaciens, characterized by the absence of xiii block (Fig. 4).
3.6. Phylogenetic analysis
The dendrogram based on 16S rDNA sequence of Proteus was
plotted using E. coli K12 as the outgroup strain. The tree is made up of
three clusters, and bootstrap values are higher than 50% (Fig. 5a).
Cluster I comprises P. vulgaris and P. penneri; cluster II, P. mirabilis;
and cluster III, P. myxofaciens. P. penneri is closer to P. vulgaris than
other two species, which is consistent with the fact that P. penneri
was originally separated from P. vulgaris based on biochemical
assays. We have also constructed the dendrogram, for the three
facultative human pathogenic species P. mirabilis, P. vulgaris and P.
penneri using the sequences of the first class of sequence pattern at
the second variable region (v2) (Fig. 5B). The tree carries three
clusters. Cluster I comprises P. vulgaris; cluster II, P. penneri; and
cluster III, P. mirabilis. It is clear that the ITS based tree has much
better resolving power than the 16S rRNA based tree in separating P.
vulgaris from P. penneri.
4. Discussion
Based on highly conserved regions of the 16S and 23S rDNA genes,
the primers wl-5793 and wl-5794 have been designed and used
successfully to amplify the ITS regions of the four Proteus species. The
sequence analysis suggests 25 genomic origins for the 46 Proteus
strains (Table 1 and Fig. 4). The minimal number of rrn operons for
each strain can be deduced. For example, at least six rrn copies of rrnA,
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
Fig. 3. Mosaic-like structures of ITS based on the sequence alignments in Fig. 2. Different patterns represent discrete sequence blocks,
names of the species and the lengths of the ITS sequences are shown on the left.
115
indicates block and
indicates box A. The
116
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
Fig. 4. Genetic properties of 28 Proteus ITS variants corresponding to 25 genomic origins (A to Y) and 15 ITS blocks (i to xv). The aligned ITS sequences minus tRNA sequences are
divided into 15 different blocks. The sequences of the 15 blocks, which ranged from 8 to 125 bp, are further subdivided into groups.
rrnB, rrnR, rrnT, rrnV and rrnX exist in P. mirabilis 9-Prk51/57.
Nevertheless, it is difficult to determine the exact numbers of rrn
operons, for the possible presence of additional ITS loci. The number of
rRNA operons in bacteria varies from one to fifteen (Osorio et al.,
2005; Klappenbach et al., 2001), and is not always conserved in
different species of a genus and even in different strains of the same
species. For example, it is reported that in genus Streptococcus, there
are six rrn operons in Streptococcus agalactiae, five in Streptococcus
uberis, five to six in Streptococcus dysgalactiae, and six in Streptococcu
parauberis (Bentley and Leigh, 1995). Similarly, the rrn copy numbers
varies from seven to nine in different Vibrio cholerae strains (Lan and
Reeves, 1998).
Sadeghifard and Gürtler indicated that the high level of identity
of one ITS type in all strains of Photobacterium damselae was due to
genetic rearrangement, not mutation accumulation (Sadeghifard
et al., 2006). In our study, when the same ITS type was found in
different intraspecies strains, the identity at the nucleotide
sequences was very high, higher than 96.2%. Therefore, the main
divergence in these Proteus ITS sequences likely also resulted from
intraspecies rearrangement between the two main variable regions
via recombination or horizontal transfer. This study reveals the
exitance of mosaic-like ITS structure in all Proteus strains examined
(Figs. 2 and 3). The mosaic-like ITS organization of sequence blocks
has been described in Staphylococcus aureus (Gurtler and Barrie,
1995), Salmonella enterica (Perez Luz et al., 1998), Vibrio cholerae
(Lan and Reeves, 1998), Haemophilus parainfluenzae (Privitera et
al., 1998), Vibrio parahaemolyticus (Maeda et al., 2000), Photobacterium damselae (Osorio et al., 2005) and Clostridium difficile
(Sadeghifard et al., 2006), and attribute to horizontal gene transfer
as well.
The different arrangement patterns at v2 region are specific for
each species, with a few exceptions. The ITSIle + Ala of 571 bp found in
two strains of P. vulgaris Biogroup 3 (Genomic origin P) carries the
arrangement pattern of xii-2_xiv-2, which is identical to that in the
ITSGlu of 398 and 399 bp (Genomic origin J) and the ITSIle + Ala of
591 bp (Genomic origin K) in P. myxofaciens (Fig. 4). The same pattern
of arrangement at v2 region presents in both ITSGlu and ITSIle + Ala
suggests that the v2 region and tRNA gene may generate from
independent genetic event.
In this study, the second variable region (v2) was chosen for
phylogenetic study of the three facultative human pathogenic species
P. mirabilis, P. vulgaris and P. penneri. We found the two dendrograms
based on 16S rDNA and v2 sequence were similar topologically.
However, phylogenetic analysis based on v2 of ITS sequences was able
to clearly differentiate closely related P. vulgaris and P. penneri as
distinct species, demonstrating ITS sequence is a useful supplement to
16S rDNA in elucidating phylogenetic relationships between closely
related species.
This study is the first comprehensive study of ITS in the four Proteus species. Our investigation revealed 2 ITS types exist in Proteus:
ITSGlu (containing tRNAGlu gene) and ITSIle + Ala (containing tRNAIle
and tRNAAla gene), this is similar with E. coli, Salmonella, Shigella,
Enterobacter sakazakii, Serratia, Yersinia, and Cirtobacter koseri in
Enterobacteriaceae family Specific regions for each of the four species
were identified and can be used for species classification, especially
for the closely related species P. vulgaris and P. penneri. This study also
laid down a solid foundation for development of time-saving, accurate
and high-throughput diagnostic tools for identification of these
important human pathogens.
Acknowledgements
This study is funded by the National High Technology Research and
Development Program of China (863 Program) (2006BAK02A14,
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
117
Fig. 5. A. Dendrogram of Proteus species constructed using the NJ method based on 16S rRNA sequence data. Sequences were obtained from GenBank. The numbers at the nodes
indicate bootstrap values (in percentage) retrieved from 1000 replicates of the NJ analyses. E coli strain ATCC 11775T was used as the outgroup reference. B. Dendrogram of Proteus ITS
for P. mirabilis, P. vulgaris and P. penneri constructed using the NJ method based on the second variable region (v2). The numbers at the nodes indicate bootstrap values (in
percentage) retrieved from 1000 replicates of the NJ analyses.
2006AA06Z409 and 2006AA020703), and Tianjin Municipal Science
and Technology Committee (07JCYBJC08500).
References
Barry, T., Colleran, G., Glennon, M., Dunican, L.K., Gannon, F., 1991. The 16s/23s ribosomal
spacer region as a target for DNA probes to identify eubacteria. PCR Methods Appl. 1,
51–56.
Bentley, R.W., Leigh, J.A., 1995. Determination of 16S ribosomal RNA gene copy number
in Streptococcus uberis, S. agalactiae, S. dysgalactiae and S. parauberis. FEMS
Immunol. Med. Microbiol. 12, 1–7.
Brenner, D.J., Farmer III, J.J., Fanning, G.R., Steigerwalt, A.G., Klykken, P., Wathen, H.G.,
Hidkman, F.W., Ewing, W.H., 1978. Deoxyribonucleic acie relatedness in species of
Proteus and Providencia. Int. J. Syst. Bacteriol. 28, 269–282.
Brenner, D.J., Hickman-Brenner, F.W., Holmes, B., Hawkey, P.M., Penner, J.L., Grimont, P.
A., O'Hara, C.M., 1995. Replacement of NCTC 4175, the current type strain of
Proteus vulgaris, with ATCC 29905. Request for an opinion. Int. J. Syst. Bacteriol. 45,
870–871.
Conrads, G., Claros, M.C., Citron, D.M., Tyrrell, K.L., Merriam, V., Goldstein, E.J., 2002. 16S23S rDNA internal transcribed spacer sequences for analysis of the phylogenetic
relationships among species of the genus Fusobacterium. Int. J. Syst. Evol. Microbiol.
52, 493–499.
Cosenza, B.J., Podgwaite, J.D., 1966. A new species of Proteus isolated from larvae of the
gypsy moth Porthetria dispar (L.). Antonie Van Leeuwenhoek 32, 187–191.
Garrity, G.M., Bell, J.A., Lilburn, T., 2005. Phylum XIV. Proteobacteria phyl. nov, 2nd ed.
Bergey's manual of systematic bacteriology, vol. 2. Springer, New York, pp. 745–753.
Goto, K., Omura, T., Hara, Y., Sadaie, Y., 2000. Application of the partial 16S rDNA
sequence as an index for rapid identification of species in the genus Bacillus. J. Gen.
Appl. Microbiol. 46, 1–8.
Guasp, C., Moore, E.R., Lalucat, J., Bennasar, A., 2000. Utility of internally transcribed
16S-23S rDNA spacer regions for the definition of Pseudomonas stutzeri genomovars
and other Pseudomonas species. Int. J. Syst. Evol. Microbiol. 50, 1629–1639.
Gurtler, V., Barrie, H.D., 1995. Typing of Staphylococcus aureus strains by PCRamplification of variable-length 16S-23S rDNA spacer regions: characterization of
spacer sequences. Microbiology 141, 1255–1265.
Gurtler, V., Stanisich, V.A., 1996. New approaches to typing and identification of bacteria
using the 16S-23S rDNA spacer region. Microbiology 142, 3–16.
Hickman, F.W., Steigerwalt, A.G., Farmer, J.J., Brenner, D.J., 1982. Identification of Proteus
penneri sp. nov., formerly known as Proteus vulgaris indole negative or as Proteus
vulgaris biogroup 1. J. Clin. Microbiol. 15, 1097–1102.
Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G., Gibson, T.J., 1998. Multiple
sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405.
Joung, K.B., Cote, J.C., 2002. A single phylogenetic analysis of Bacillus thuringiensis
strains and bacilli species inferred from 16S rRNA gene restriction fragment length
polymorphism is congruent with two independent phylogenetic analyses. J. Appl.
Microbiol. 93, 1075–1082.
Klappenbach, J.A., Saxman, P.R., Cole, J.R., Schmidt, T.M., 2001. rrndb: the Ribosomal RNA
Operon Copy Number Database. Nucleic Acids Res. 29, 181–184.
Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular
evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163.
Lan, R., Reeves, P.R., 1998. Recombination between rRNA operons created most of the
ribotype variation observed in the seventh pandemic clone of Vibrio cholerae.
Microbiology 144, 1213–1221.
Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer
RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964.
Maeda, T., Takada, N., Furushita, M., Shiba, T., 2000. Structural variation in the 16S-23S
rRNA intergenic spacers of Vibrio parahaemolyticus. FEMS Microbiol. Lett. 192,
73–77.
Motoyama, Y., Ogata, T., 2000. 16S-23S rDNA spacer of Pectinatus, Selenomonas and
Zymophilus reveal new phylogenetic relationships between these genera. Int. J.
Syst. Evol. Microbiol. 50, 883–886.
O'Hara, C.M., Brenner, F.W., Steigerwalt, A.G., Hill, B.C., Holmes, B., Grimont, P.A.D., Hawkey,
P.M., Penner, J.L., Miller, J.M., Brenner, D.J., 2000. Classification of Proteus vulgaris
biogroup 3 with recognition of Proteus hauseri sp. nov. nom. rev. and unnamed Proteus
genomospecies 4, 5 and 6. Int. J. Syst. Evol. Microbiol. 50, 1869–1875.
118
B. Cao et al. / Journal of Microbiological Methods 77 (2009) 109–118
Osorio, C.R., Collins, M.D., Romalde, J.L., Toranzo, A.E., 2005. Variation in 16S-23S rRNA
intergenic spacer regions in Photobacterium damselae: a mosaic-like structure.
Appl. Environ. Microbiol. 71, 636–645.
Penner, J.L., 1992. The genera Proteus, Providencia and Morganella. The prokaryotes, vol. III.
Springer-Verlag KG, Berlin, Germany, pp. 2849–2853.
Perez Luz, S., Rodriguez-Valera, F., Lan, R., Reeves, P.R., 1998. Variation of the ribosomal
operon 16S-23S gene spacer region in representatives of Salmonella enterica
subspecies. J. Bacteriol. 180, 2144–2151.
Privitera, A., Rappazzo, G., Sangari, P., Giannino, V., Licciardello, L., Stefani, S., 1998.
Cloning and sequencing of a 16S/23S ribosomal spacer from Haemophilus
parainfluenzae reveals an invariant, mosaic-like organisation of sequence blocks.
FEMS Microbiol. Lett. 164, 289–294.
Rogall, T., Wolters, J., Flohr, T., Bottger, E.C., 1990. Towards a phylogeny and definition of
species at the molecular level within the genus Mycobacterium. Int. J. Syst. Bacteriol.
40, 323–330.
Sadeghifard, N., Gurtler, V., Beer, M., Seviour, R.J., 2006. The mosaic nature of intergenic
16S-23S rRNA spacer regions suggests rRNA operon copy number variation in
Clostridium difficile strains. Appl. Environ. Microbiol. 72, 7311–7323.
Stackebrandt, E., Rainey, F.A., Ward-Rainey, N.L., 1997. Proposal for a new hierarchic
classification system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47, 479–491.
Stephen, D., Jones, C., Schofield, J.P., 1990. A rapid method for isolating high quality
plasmid DNA suitable for DNA sequencing. Nucleic Acids Res. 18, 7463–7464.
Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51, 221–271.
Yoon, J.H., Lee, S.T., Kim, S.B., Goodfellow, M., Park, Y.H., 1997. Inter- and intraspecific
genetic analysis of the genus Saccharomonospora with 16S to 23S ribosomal DNA
(rDNA) and 23S to 5S rDNA internally transcribed spacer sequences. Int. J. Syst.
Bacteriol. 47, 661–669.