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[Identification of pathogenic microorganism by sequencing 16S rRNA gene]

Authors:

Abstract

Objective: To identify 14 bacteria by sequencing the 16S rRNA gene and establish the basis for clinical application in the future. Methods: DNA samples of the 14 bacteria were extracted. The 16S rRNA genes were amplified by PCR and sequenced with common primers. The sequences of the 16S rRNA genes were aligned by online software Blastn in nucleotide database. The bacteria were identified according to the homology of their 16S rRNA genes. Results: Twelve bacteria were classified to species, the other 2 bacteria were classified to genus. Conclusion: 16S rRNA gene sequence analysis is useful in identifying pathogenic bacteria.
ISSN 08914168, Molecular Genetics, Microbiology and Virology, 2012, Vol. 27, No. 3, pp. 103–107. © Allerton Press, Inc., 2012.
103
1
INTRODUCTION
Molecular markers, which are closely linked to
allelic variations and its target traits, can be segregated
with those traits together, and play an essential role in
genetic research and bacterial classification. With the
explosive growth of genetic research, bacterial classifi
cation has gone from morphological, cytological and
biochemical phases into molecular marker phase.
Molecular markers of bacteria, widely located in both
coding gene and noncoding regions of DNA, should
have several characteristics: firstly, most of them are
housekeeping genes present in all bacterial species;
secondly, molecular markers are highly polymorphis
mic, which make them distinguishable in different
bacterial species; thirdly, molecular markers are highly
conserved in some regions, which are easy to design
appropriate primers to amplify by PCR [1].
The development of molecular markers is based on
the research of DNA polymorphism. According to its
significant roles in clinical medicine, food industry
and microbiology, it is essential for us to have a review
on the achievements in molecular markers.
16S rRNA Gene
It is wellknown that the genes coding for 5S, 16S
and 23S rRNA, and the spaces between these genes are
significant for bacterial identification. Among these
stable genetic fragments, the 16S rRNA gene is the
most commonly used marker for the taxonomic pur
poses of bacteria [2, 3]. The 16S rRNA gene sequence
is about 1.5 kb, and composed of variable and con
served regions. It has higher degree of conservation
1
The article is published in the original.
than genes encoding enzymes, since the uniqueness
and importance of rRNA make it resistant to frequent
mutations that may affect the essential structures [4].
The sequence of 16S rRNA gene shows evolutionary
distance and relationships between organisms, and
provides statistical and valid measurements for bacte
rial identification, owning to its sufficient interspe
cies polymorphisms [5, 6]. Furthermore, the 16S
rRNA gene is so universal in bacteria that it has the
ability to measure all bacteria from phyla level to spe
cies level [7, 8]. Because of the unique characteristics
of 16S rRNA, it has applications in several fields. The
discrimination of 16S rRNA gene sequence analysis
among strains of bacteria, which is better than pheno
typic methods, allows more precise identification of
poorly described, rarely isolated, or phenotypically
aberrant strains [4]. In addition, this molecular marker
could also be used to identify noncultured bacteria
[4]. Moreover, some clinical microorganisms, which
can hardly be identified by phenotypic methods, have
been distinguished by 16S rRNA gene sequence anal
ysis [4]. Recently, this technology proved the fact that
on the species level they could identify the mycobacte
ria more accurately than phenotypic methods. The
significance of 16S rRNA gene sequencing for clinical
microbiology has been established [2, 9–11]. How
ever, some researchers have found that 16S rRNA gene
is indistinguishable for a few species, which are able to
be identified by 23S rRNA gene or 16S23S rDNA
ISR (intergenic spacer region) more precisely [12, 13].
It is true that the usage of 16S rRNA gene is limited,
for the closely related species have high percentage of
sequence similarity and lack enough variation. In spite
of these facts, 16S rRNA gene sequencing is still the
Popular Molecular Markers in Bacteria
1
Weilong Liu, Lv Li, Md. Asaduzzaman Khan, and Feizhou Zhu
Department of Biochemistry, School of Biological Science and Technology, Central South University,
Changsha, Hunan, 410013 China
Received January 23, 2012
Abstract
—Molecular markers are defined as the fragments of DNA sequence associated with a genome,
which areused to identify a particular DNA sequence. Nowadays, with the explosive growth of genetic
research and bacterial classification, molecular marker is an important tool to identify bacterial species. Tak
ing account to its significant roles in clinic, medicine and food industry, in this review article, we summarize
the traditional research and new development about molecular markers (also called genetic markers) in bac
teria, including genes of 16S rRNA, 23S rRNA,
rpoB
,
gyrB
,
dnaK
,
dsrAB
,
amoA
,
amoB
,
mip
,
horA
,
hitA
,
recA
,
ica
,
frc
,
oxc
, 16S23S rDNA ISR and IS256.
Keyword
s: molecular markers, bacteria, classificatio, RNA fragments.
DOI:
10.3103/S0891416812030056
REVIEWS
104
MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 27 No. 3 2012
LIU et al.
most commonly used method for identifying
unknown bacteria [14, 15].
23S rRNA Gene and 16S23S rDNA ISR
Although 16S rRNA gene is the most ubiquitous
molecular marker, it is necessary for taxonomic deci
sion to find supports offered by other molecular mark
ers, such as 23S rRNA gene [16]. 23S rRNA encoded
by 23S rRNA gene is a component of the large
prokaryotic ribosomal subunit (50S) which contains
the ribosomal peptidyl transferase activity [17]. 23S
rRNA gene is outstanding for its numerous variations
between bacterial species of medical importance,
which is more abundant than 16S rRNA gene [18, 19].
23S rRNA gene has been applied to a DNA microar
raybased approach by amplifying a variable region of
bacterial 23S rRNA to classify bacteria quickly and
precisely [20]. Besides, 23S rRNA is also assessed for
the diagnosis of bacteremia, for it can identify almost
all bacteria commonly causing bacteremia in China
[21, 22]. An oligonucleotide suspension array based on
15 beadbound probes, which can be hybridized to
PCR amplicons of the bacterial 23S rRNA gene, has
been used for identifying 15 bacterial species responsi
ble for bacteremia [23].
16S23S rDNA ISR, as a common molecular
marker for bacterial identification, is the intergenic
spacer region between the 16S rDNA and 23S rDNA
loci in the rDNA operon [24]. Generally, the studies
on bacterial identification are mainly focused on 16S
rDNA. Nevertheless, the number of polymorphic sites
in the 16S rDNA of some bacteria species are
extremely low, which makes it difficult to define spe
cific 16S rRNA sequence to distinguish closely related
species [25]. On the contrary, the structure of 16S23S
rDNA ISR has been shown to express considerably
variable size and sequence among different organisms,
which contributes significantly in classification of cer
tain bacteria [18, 25]. It is proved that the RFLP
(restriction fragment length polymorphism) of the
PCRamplified 16S23S rDNA ISR is a rapid way to
characterize acetic acid bacterial isolates and popula
tions [26], and is applicable to identify bacteria at the
species level [26]. In addition, 16S23S rDNA ISR
also can be used for
Streptococcus
classification [27]
and speciesspecific primer design to distinguish spe
cies [24]. It has also been reported that 23S5S rDNA
ISR of
Lactobacillus
has both highly conserved
sequences and divergent regions, which make 23S5S
rDNA spacer region available for a molecular marker
[28]. Researchers examined 16S23S rDNA ISR, and
reported a result similar to the study of 23S5S rDNA
spacer region [28]. In summary, it is appropriate for
these regions to become potential candidates for the
research of speciesspecific probe.
rpoB Gene
The
rpoB
gene encodes the subunit of DNA
dependent RNA polymerase and is relevant to
rifampin resistance. It possesses a particularly highly
conserved region that may be used for bacterial classi
fication [29]. The
rpoB
gene can be used to identify
enteric bacteria,
Mycobacterium
, spirochetes and
especially the
Legionella
species, some of which cause
Legionnaires disease [29, 30]. For example, in the case
of identification of
Legionella
species, the nucleotide
variation of
rpoB
endows its ability to differentiate
these species more exactly than I6S rRNA and mip
(macrophage infectivity potentiator) in some cases
[30]. Besides, it is reported that partial
rpoB
sequence
(300 bp) is able to ensure the genotypic classification
of
Legionella pneumophila
species and bluewhite
autofluorescent species [29–31]. The partial sequence
is outstanding for its high conserved character. It can
differentiate species mentioned above clearly, which
share high similarities in 16S rRNA gene sequence and
even cannot be analyzed successfully by
mip
[31, 32].
However, a lower degree of similarities in sequence of
rpoB
than other genes is not enough to differentiate
species [30]. To overcome the difficulties of identifica
tion of
Legionella
species, it is suggested that the usage
of several marker, such as combining
rpoB
with 16S
rRNA gene or
mip
gene can develop the identification
more correctly [30].
gyrB Gene
The
gyrB
gene encodes the
β
subunit of DNA
gyrase, which is a type II DNA topoisomerase and
introduces negative supercoils into closed circular
DNA molecules [33]. The
gyrB
gene, which can infer
interspecies and intraspecies relationships, has been
investigated in a number of bacterial species [34]. The
reason for selecting
gyrB
gene for phylogenetic studies
is that the HGT (horizontal gene transfer) occurs
infrequently in informational genes which are
involved in transcription and translation [35]. As the
base substitution in
gyrB
is more frequent than that in
16S rRNA gene, analysis based on
gyrB
is more dis
criminating than 16S rDNA in some species, such as,
Pseudomonas putida
[36]. The
gyrB
provides higher
resolution for some species with lower interspecies
sequence similarities (ranging from 58.3 to 89.2%)
than those reported for the 16S rRNA gene (ranging
from 89 to 99%), such as
Campylobacter
species [37].
Furthermore, Kawasaki identified the sequence poly
morphisms in the
Campylobacter gyrB
gene and devel
oped speciesspecific PCR assays and PCRRFLP
using the restriction enzymes DdeI and XspI to differ
entiate 12
Campylobacter
species [38].
dnaK Gene
The 70kDa heat shock protein (HSP70) is
encoded by
dnaK
gene and plays an important role in
MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 27 No. 3 2012
POPULAR MOLECULAR MARKERS IN BACTERIA 105
protein folding and unfolding as a chaperone. It is
believed to be a suitable marker for classifying bacte
ria, considering its highest conserved character and its
ubiquitous in all biota [39, 40].
dnaK
gene can be used
as a molecular marker in the classification of some
species. For example, the analysis between partial
dnaK
gene sequence and 16S rRNA gene of
L. casei
shows that the former is superior to latter in several
aspects [41]. The sequence of the
dnaK
is more poly
morphic than that of 16S rRNA gene [41], Further
more, the bootstrap values of nucleotide sequences at
all nodes of the
dnaK
phylogenetic tree are higher than
those of 16S rRNA tree, and the topology of the
former reveals more clearly separated groups [41].
Therefore, the
dnaK
is suggested to be a complete
molecular marker to classify some bacteria.
dsrAB Gene
The
dsrAB
gene encodes the
α
and
β
subunits of an
enzyme catalyzing the sixelectron reduction of sulfite
to sulfide. It is considered to be available for phyloge
netic studies of sulfatereducing bacteria (SRB) and
archaea. It is also used as a molecular marker to iden
tify and discriminate metabolic active SRB in different
habitats [42, 43].
dsrAB
owns a highly observed con
servation in SRB and archaea, and the phylogeny of
different SRB lineage is congruent with 16S rRNA
genebased phylogenetic tree [44]. Furthermore, the
denaturing gradient gel electrophoresis (DGGE) of
PCRamplified
dsrB
gene fragments has been
described to follow population dynamics of SRB [45].
recA Gene
The
recA
gene, which plays a central role in DNA
recombination in many processes relevant to DNA
metabolism, has recently been accepted as a molecu
lar marker [46]. There are two types of recA gene
libraries constructed, one with broadspecificity recA
primers (BUR1 and BUR2) and the other from the
products of nested PCRs using Burkholderiaspecific
primers (BUR3 and BUR4) [47].
The
recA
gene is
reported to identify entire Burkholderia genus with
BUR1 and BUR2, but BUR3 and BUR4 are more acu
rate and suitable to identifyBurkholderia genus than
BLJR1 and BUR2 [47].
Besides,
recA
gene can also be
used as a phylogenetic marker in the classification of
Aeromonas
strains and dairy propionibacteria [48, 49].
It has been reported that researchers used colony
hybridization and PCR with 16S rRNA and
recA
gene
derived probes to identify the cultivable
B. cepacia
complex species in maizeassociated soil samples [50].
Genes encoding bacterial surface proteins
amoA
and
amoB
genes:
AMO (ammonia
monooxygenase) is a bacterial membrane surface
protein, which is an essential enzyme catalyzing the
step of transferring energy and reducing power from
oxidation of ammonia to AOB (ammoniaoxidizing
bacteria). AMO is obligate in chemolithotroph, and
consists of three subunits: AmoA, AmoB, and AmoC
[51]. 16S rRNA is only suitable for certain phyloge
netic groups belonging to the Proteobacteria, but not
for AOB subgroups of Proteobacteria. However, the
gene
amoA
is present in all of AOB subgroups of Pro
teobacteria and can be used to classify them [51, 52].
The gene
amoA
is less resolutive than 16S rDNA in
phylogeny inference [53]. The
amoB
with its suitable
size and essential role is suggested to be an appropriate
molecular marker for the classification of AOB [54].
Therefore, both of
amoA
and
amoB
genes can be
applied as molecular markers in AOB. Furthermore,
combining 16S rRNA,
amoA
and
amoB
sequences, the
data provided more information than any of these
three markers alone, making the classification and
identification more accurate [53].
mip
gene:
The Mip protein, which is a surface
exposed protein on the
Legionella
species, plays an
important role in virulence [55]. The comparison
between 16S rRNA and
mip
gene of
Legionella
species
shows that the variation in
mip
gene i s much more t han
that in the 16S rRNA gene [56]. In spite of greater
mutational variation, the
mip
gene has genetic stabil
ity. With no evidence of homologous recombination
and behavior similarity with housekeeping genes, the
mip
gene is considered to be more stable than other
gene classes. However, incongruence studies between
the phylogeny deduction from the 16S rRNA gene and
mip
gene suggest that neither of them is able to com
pletely distinguish
Legionella
species [56].
horA
and
hitA
genes:
The
horA
gene, which encodes
an ATPbinding cassette (ABC) transporter, and acts
as cellmembrane ionophores related to hop toler
ance, has been proved to export ionophoric
α
acids of
hop out of bacteria [57]. The
hitA
gene of
L. brevis
that
is strongly similar to other divalentcation transporter
genes in sequence is reported to function in hop toler
ance [58]. These two genes have been applied alto
gether in identifying beerspoilage
Lactobacillus
strains of lactic acid bacteria and explaining the mecha
nism of hop tolerance. A recent study has used the
RAPDPCR for cloning these two genes and identi
fied
Pediococcus damnosus
,
Lactobacillus
collinoides
,
L. coryniformis
and
L. brevis
of the beerspoilage
strains successfully, which once brought confusions to
identification [59].
ica Gene and IS256
ica
gene product has large influence on formation
of biofilm, encodes polysaccharide intercellular adhe
sion (PIA) protein, which plays an important role in
immune evasion, biomembranous formation and vir
ulence in biofilmassociated infection [60, 61].
IS256, the common insertion sequence in gramposi
tive cocci, is previously reported as the flanking region
of aminoglycoside resistancemediating trasposon
Tn
4001
[62]. Both of
ica
gene and IS256 can differen
106
MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 27 No. 3 2012
LIU et al.
tiate invasive strains and commensal strains of
Staphy
lococcus epidermidis
, which are the most important
causes of nosocomial infections [60–62].
ica
gene
used to be suggested as the best molecular marker for
invasiveness of
S. epidermidis
, because it exists more
frequently in clinical strains than healthy individuals.
However, the expression of
ica
gene is regulated by
other genes, and its functions are strongly affected by
environmental factors [60, 63, 64]. All of these have
limited the
ica
gene as a molecular marker. As the
IS256 is superior to
ica
gene in the sensitivity and
specificity, the combination of these two with 16s
rDNA as molecular markers would be a more power
ful tool in the discrimination of bacteria [63, 64].
CONCLUSION
The DNA molecular markers have been increas
ingly applied to identify and classify numerous micro
organisms. Each of them has their advantages and dis
advantages. Therefore, different kinds of bacteria need
different suitable ways to be identified. It has been
reported that combining two or more genetic markers
to identify or classify some species of bacteria are more
reliable. Very few molecular markers can only be used
in some particular genus of bacteria. For example, the
frc
gene and
oxc
genes are used as molecular marker to
divide
Oxalobacter formigenes
in two groups [65]. With
the discovery of more and more new molecular mark
ers, the technique of DNA molecular markers will be
applied in the research of bacterial identification and
classification more frequently in the future.
ACKNOWLEDGMENTS
This work has been supported by Hunan Science
and Technology Project (grant no. 2009SK3192).
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