333
16.5.2.2 Microarray
Microarray has gained much popularity in coral-bacterial studies. It is a nucleic acid
hybridization-based technique in which nucleic acid is hybridized to a very large set
of oligonucleotide probes immobilized on a solid support generally called gene chip
(Fig. 16.8b). It is mainly used to study gene expression and genetic variation in different samples. A typical microarray experiment involves extraction of total RNA
separately from the different samples. Then, these RNA samples are reverse transcribed into cDNAs and labeled with different fluorescent dyes. Samples are mixed
in equal proportion and hybridized with the cloned sequences on the gene chip.
Hybridization of cDNA with the DNA on gene chip results into production of a
fluorescent signal that indicates the expression level of the various genes. In coral
bacteriology, 16s rRNA gene microarray is used to assay differences in bacterial
assemblages of healthy and diseased colonies. Recent studies found increased bacterial richness in diseased samples highlighting the role of opportunistic conditions
in structuring microbial community patterns during disease. They observed that
host transcriptome under yellow-band disease (YBD) showed a reduced cellular
expression of defense- and metabolism-related processes (Closek et al. 2014; Roder
et al. 2014).
16.5.2.3 Membrane-Based Methods
Membrane-based techniques such as colony hybridization and ribotyping are also
gaining momentum in coral-bacterial studies (Fig. 16.8c):
a. Colony hybridization is a molecular approach for microbial community analysis.
The technique utilizes a membrane usually nylon or nitrocellulose on which
blotted colonies are lysed to release nucleic acids which are denatured and
hybridized with a labeled probe (Fig. 16.8 (c1)). The membrane is then visualized by UV or autoradiography. It can be used for screening clones or bacterial
isolates based on probes utilized in the experiment. The probes can be a fragment
of DNA or RNA of variable length constructed on the basis of specific genes. It’s
a versatile technique and can be used for a large number of samples. Bourne et al.
(2008) have used probes Vib-sp1 and Vib-GV to screen for Vibrionaceae- positive
clones. As it identifies a target gene, this technique can be very useful for the
identification of potential pathogens from the corals. The limitation of this
method is that there is a probability of cross-hybridization and misidentification
of closely related species (Cerdà-Cuéllar and Blanch 2002).
b. Ribotyping is another membrane-based technique which entails digestion of bacterial genomic DNA with specific restriction enzymes followed by gel electrophoresis
and then the subsequent transfer of the DNA fragments onto nylon or nitrocellulose
membranes (Fig. 16.8 (c2)). The DNA on the membrane is then hybridized with a
labeled 16S or 23S rRNA probe which can be visualized and compared with reference organisms from the available database. Basically, this method has been used
for the identification of pathogenic species (Prevost et al. 1992; Carson et al. 2001;
Gerner-Smidt 1992). Among pathogens, also there are many clones and it is difficult
16 Role of Bacteria in Coral Ecosystem
16.5.2.2 Microarray
Microarray has gained much popularity in coral-bacterial studies. It is a nucleic acid
hybridization-based technique in which nucleic acid is hybridized to a very large set
of oligonucleotide probes immobilized on a solid support generally called gene chip
(Fig. 16.8b). It is mainly used to study gene expression and genetic variation in different samples. A typical microarray experiment involves extraction of total RNA
separately from the different samples. Then, these RNA samples are reverse transcribed into cDNAs and labeled with different fluorescent dyes. Samples are mixed
in equal proportion and hybridized with the cloned sequences on the gene chip.
Hybridization of cDNA with the DNA on gene chip results into production of a
fluorescent signal that indicates the expression level of the various genes. In coral
bacteriology, 16s rRNA gene microarray is used to assay differences in bacterial
assemblages of healthy and diseased colonies. Recent studies found increased bacterial richness in diseased samples highlighting the role of opportunistic conditions
in structuring microbial community patterns during disease. They observed that
host transcriptome under yellow-band disease (YBD) showed a reduced cellular
expression of defense- and metabolism-related processes (Closek et al. 2014; Roder
et al. 2014).
16.5.2.3 Membrane-Based Methods
Membrane-based techniques such as colony hybridization and ribotyping are also
gaining momentum in coral-bacterial studies (Fig. 16.8c):
a. Colony hybridization is a molecular approach for microbial community analysis.
The technique utilizes a membrane usually nylon or nitrocellulose on which
blotted colonies are lysed to release nucleic acids which are denatured and
hybridized with a labeled probe (Fig. 16.8 (c1)). The membrane is then visualized by UV or autoradiography. It can be used for screening clones or bacterial
isolates based on probes utilized in the experiment. The probes can be a fragment
of DNA or RNA of variable length constructed on the basis of specific genes. It’s
a versatile technique and can be used for a large number of samples. Bourne et al.
(2008) have used probes Vib-sp1 and Vib-GV to screen for Vibrionaceae- positive
clones. As it identifies a target gene, this technique can be very useful for the
identification of potential pathogens from the corals. The limitation of this
method is that there is a probability of cross-hybridization and misidentification
of closely related species (Cerdà-Cuéllar and Blanch 2002).
b. Ribotyping is another membrane-based technique which entails digestion of bacterial genomic DNA with specific restriction enzymes followed by gel electrophoresis
and then the subsequent transfer of the DNA fragments onto nylon or nitrocellulose
membranes (Fig. 16.8 (c2)). The DNA on the membrane is then hybridized with a
labeled 16S or 23S rRNA probe which can be visualized and compared with reference organisms from the available database. Basically, this method has been used
for the identification of pathogenic species (Prevost et al. 1992; Carson et al. 2001;
Gerner-Smidt 1992). Among pathogens, also there are many clones and it is difficult
16 Role of Bacteria in Coral Ecosystem