Popular molecular markers in bacteria
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МОЛЕКУЛЯРНАЯ ГЕНЕТИКА, МИКРОБИОЛОГИЯ И ВИРУСОЛОГИЯ № 3, 2012
© Коллектив авторов, 2012 УДК 579. 083. 1:577.2. 08
Weilong Liu, Lv Li, Md. Asaduzzaman Khan and Feizhou Zhu
Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan, 410 013, China
popular molecular markers in bacteria
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. Taking 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 bacteria, 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. Key words: Molecular markers, Bacteria, Classificatio, RNA fragments
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 classification 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 polymorphismic, 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 .
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 purposes of bacteria [2,3]. The 16S rRNA gene sequence is about 1. 5kb, and composed of variable and conserved regions. It has higher degree of conservation than genes encoding enzymes, since the uniqueness and importance of rRNA make it resistant to frequent mutations that may affect the essential structures . The sequence of 16S rRNA gene shows evolutionary distance and relationships between organisms, and provides statistical and valid measurements for bacterial identification, owning to its sufficient inter-species 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 species 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 phenotypic methods, allows more precise identification of poorly described, rarely isolated, or phenotypically aberrant strains . In addition, this molecular marker could also be used to identify non-cultured bacteria . Moreover, some clinical microorganisms, which can hardly be identified by phenotypic methods, have been distinguished by 16S rRNA gene sequence analysis . Recently, this technology proved the fact that on the species level they could identify the mycobacteria more accurately than phenotypic methods. The significance of 16S rRNA gene sequencing for clinical microbiology has been established [2, 9−11]. However, 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 (in-tergenic 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 most commonly used method for identifying unknown bacteria [ 14, 15].
23s rma gene and 16s-23sNA IsR
Although 16S rRNA gene is the most ubiquitous molecular marker, it is necessary for taxonomic decision to find supports offered by other molecular markers, such as 23S rRNA gene . 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 . 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 microarray-based approach by amplifying a variable region of bacterial 23S rDNA to classify bacteria quickly and precisely . 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 responsible for bacteremia .
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 . 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 specific 16s rRNA sequence to distinguish closely related species . 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 classifica-
tion of certain 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 populations , and is applicable to identify bacteria at the species level . In addition, 16S-23S rDNA ISR also can be used for Streptococcus classification  and species-specific primer design to distinguish species . 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 . Researchers examined 16S-23S rDNA ISR, and reported a result similar to the study of 23S-5S rDNA spacer region . In summary, it is appropriate for these regions to become potential candidates for the research of species-specific probe.
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 classification . 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 16S rRNA and mip (macrophage infectivity potentiator) in some cases . 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, 30, 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 . To overcome the difficulties of identification 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 .
The gyrB gene encodes the P-subunit of DNA gyrase, which is a type II DNA topoisomerase and introduces negative supercoils into closed circular DNA molecules . The gyrB gene, which can infer interspecies and intraspecies relationships, has been investigated in a number of bacterial species . 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 . As the base substitution in gyrB is more frequent than that in 16S rDNA gene, analysis based on gyrB is more discriminating than 16S rDNA in some species, such as, Pseudomonas putida . 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 . Furthermore, Kawasaki identified the sequence polymorphisms in the Campylobacter gyrB gene
and developed species-specific PCR assays and PCR-RFLP using the restriction enzymes DdeI and XspI to differentiate 12 Campylobacter species .
The 70-kDa heat shock protein (HSP70) is encoded by dnaK gene and plays an important role in protein folding and unfolding as a chaperone. It is believed to be a suitable marker for classifying bacteria, 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 . The sequence of the dnaK is more polymorphic than that of 16S rRNA gene . Furthermore, 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 . Therefore, the dnaK is suggested to be a complete molecular marker to classify some bacteria.
The dsrAB gene encodes the a and P subunits of an enzyme catalyzing the six-electron reduction of sulfite to sulfide. It is considered to be available for phylogenetic studies of sulfate-reducing bacteria (SRB) and archaea. It is also used as a molecular marker to identify and discriminate metabolic active SRB in different habitats [42, 43]. dsrAB owns a highly observed conservation in SRB and archaea, and the phylogeny of different SRB lineage is congruent with 16S rRNA gene-based phylogenetic tree . Furthermore, the denaturing gradient gel electrophoresis (DGGE) of PCR-amplified dsrB gene fragments has been described to follow population dynamics of SRB .
The recA gene, which plays a central role in DNA recombination in many processes relevant to DNA metabolism, has recently been accepted as a molecular marker
. 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)
. The recA gene is reported to identify entire Burkholderia genus with BuR1 and BuR2, but BuR3 and BuR4 are more acurate and suitable to identifyBur-kholderia genus than BuR1 and BuR2. Besides, recA gene can also be used as a phylogenetic marker in the classification of Aeromonas strains and dairy propi-onibacteria [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 .
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,
МОЛЕКУЛЯРНАЯ ГЕНЕТИКА, МИКРОБИОЛОГИЯ И ВИРУСОЛОГИЯ № 3, 2012
AmoB, and AmoC . 16S rRNA is only suitable for certain phylogenetic groups belonging to the Proteobac-teria, but not for AOB subgroups of Proteobacteria. However, the gene amoA is present in all of AOB subgroups of Proteobacteria and can be used to classify them [51, 52]. The gene amoA is less resolutive than 16S rDNA in phy-logeny inference . The amoB with its suitable size and essential role is suggested to be an appropriate molecular marker for the classification of AOB . 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 .
mip gene: The Mip protein, which is a surface-exposed protein on the Legionella species, plays an important role in virulence . The comparison between 16S rRNA and mip gene of Legionella species shows that the variation in mip gene is much more than that in the 16S rRNA gene . In spite of greater mutational variation, the mip gene has genetic stability. 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, incongru-ence studies between the phylogeny deduction from the 16S rRNA gene and mip gene suggest that neither of them is able to completely distinguish Legionella species .
horA and hitA genes: The horA gene, which encodes an ATP-binding cassette (ABC) transporter, and acts as cell-membrane ionophores related to hop tolerance, has been proved to export ionophoric a-acids of hop out of bacteria . The hitA gene of L. Brevis that is strongly similar to other divalent-cation transporter genes in sequence is reported to function in hop tolerance . These two genes have been applied altogether in identifying beer-spoilage Lactobacillus strains of lactic acid bacteria and explaining the mechanism of hop tolerance. A recent study has used the RAPD-PCR for cloning these two genes and identified Pediococcus damnosus, Lactobacillus collinoides, L. coryniformis and L. Brevis of the beer-spoilage strains successfully, which once brought confusions to identification .
ica gene and is256
ica gene product has large influence on formation of biofilm, encodes polysaccharide intercellular adhesion (PIA) protein, which plays an important role in immune evasion, biomembranous formation and virulence in bio-film-associated infection [60, 61]. IS256, the common insertion sequence in gram-positive cocci, is previously reported as the flanking region of aminoglycoside resistance-mediating trasposon Tn4001 . Both of ica gene and IS256 can differentiate invasive strains and commensal strains of Staphylococcus epidermidis, which are the most important causes of nosocomial infections . 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 powerful tool in the discrimination of bacteria [63, 64].
The DNA molecular markers have been increasingly applied to identify and classify numerous microorganisms. Each of them has their advantages and disadvantages. 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 . With the discovery of more and more new molecular markers, the technique of DNA molecular markers will be applied in the research of bacterial identification and classification more frequently in the future.
This work has been supported by Hunan Science and Technology Project (GrantNo. 2009SK3192).
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Поступила 23. 01. 12
POPULAR MOLECULAR MARKERS IN BACTERIA
Weilong Liu, Lv Li, Md. Asaduzzaman Khan, and Feizhou Zhu
Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan, China.
Molecular markers (also called genetic markers) are defined as the fragments of the DNA sequence associated with a genome, which are used to identify a particular DNA sequence. Presently, with the explosive growth of genetic research and bacterial classification, molecular marker is an important tool to identify bacterial species. Taking into account its significant roles in clinic, medicine and food industry, the conventional research and new development about molecular markers in bacteria, 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 are reviewed.
Key words: molecular markers, bacteria, classification