10
G.R. Carvalho et al.
taxonomic classification of microbial species (Doi and Igarashi 1965, Dubnau et al.
1965, Pace and Campbell 1971a, b). Over the past decades the comparative analysis of homologous gene sequences has become an indispensable approach to gain
new insights into the phylogeny and diversity of microbial organisms (Díez et al.
2001, Evans et al. 2007). The genes coding for rRNA are particularly well suited for
phylogenetic analysis, because they are universal, found in all cellular organisms;
they are of relatively large size; and they contain both highly conserved and variable
regions with no evidence for lateral gene transfer (Woese 1987). The number of ribosomal sequences is continuously growing. Currently it is in the range of ∼550,000
sequences (Pruesse et al. 2007). Several publications describe the power of ribosomal sequences to identify both prokaryotic and eukaryotic micro-organisms (Amann
et al. 1990; Simon et al. 2000; Groben et al. 2004). Direct cloning and sequencing
of the small subunit ribosomal DNA (18S rDNA) from natural samples has, for
example, permitted a broader view of the structure and composition of picoplankton
communities (Giovannoni et al. 1990; López-Garcia et al. 2001, Medlin et al. 2006).
1.2.1 Diversity and Functional Analyses of Microbial Communities
With >99% of its organisms predicted to be unculturable, the marine environment
represents the largest untapped reservoir of genomic diversity on the planet (Beja
2004). Indeed, with an average of more than a million viruses per millilitre of seawater and no means of propagation outside of their host organism, the marine viral
fraction represents perhaps the most poorly sampled biomass on Earth. To date,
PCR-based approaches (e.g. clone libraries, denaturing gradient gel electrophoresis (DGGE) (Muyzer 1999) and restriction fragment length polymorphism (RFLP)
analyses) have offered us rare glimpses of the enormous diversity of marine microorganisms (Archaea, prokaryotes, eukaryotes) derived from the analysis of specific
homologous genes (e.g. 16S or 18S ribosomal DNA) used to identify species or
phylotypes (Kemp and Aller 2004). DGGE allows the rapid analysis and comparison
of microbial communities. Compositional diversity can be visualized using DGGE
where each band in principle represents a microbial phylotype (Tzeneva et al. 2008).
Sequencing of a single band provides information on the related taxon and can be
used, for example, in phylogenetic analysis. Since viruses are obligate intracellular parasites, they lack ribosomal encoding regions such as 16S and 18S, thereby
necessitating the development of markers specific for each broad family (such as
RNA polymerase and DNA polymerase) in order to perform similar biodiversity
studies (Allen and Wilson 2008).
Whereas such studies highlight the sheer abundance of natural biodiversity,
no real functional significance can be attributed to the communities in question,
because the molecular markers used for biodiversity appraisals have conserved
genomic functions. For years, the Holy Grail of biodiversity research has been not
to merely study biodiversity but to assess functional biodiversity, that is, to determine how diversity contributes to ecosystem functioning. The study of functional
G.R. Carvalho et al.
taxonomic classification of microbial species (Doi and Igarashi 1965, Dubnau et al.
1965, Pace and Campbell 1971a, b). Over the past decades the comparative analysis of homologous gene sequences has become an indispensable approach to gain
new insights into the phylogeny and diversity of microbial organisms (Díez et al.
2001, Evans et al. 2007). The genes coding for rRNA are particularly well suited for
phylogenetic analysis, because they are universal, found in all cellular organisms;
they are of relatively large size; and they contain both highly conserved and variable
regions with no evidence for lateral gene transfer (Woese 1987). The number of ribosomal sequences is continuously growing. Currently it is in the range of ∼550,000
sequences (Pruesse et al. 2007). Several publications describe the power of ribosomal sequences to identify both prokaryotic and eukaryotic micro-organisms (Amann
et al. 1990; Simon et al. 2000; Groben et al. 2004). Direct cloning and sequencing
of the small subunit ribosomal DNA (18S rDNA) from natural samples has, for
example, permitted a broader view of the structure and composition of picoplankton
communities (Giovannoni et al. 1990; López-Garcia et al. 2001, Medlin et al. 2006).
1.2.1 Diversity and Functional Analyses of Microbial Communities
With >99% of its organisms predicted to be unculturable, the marine environment
represents the largest untapped reservoir of genomic diversity on the planet (Beja
2004). Indeed, with an average of more than a million viruses per millilitre of seawater and no means of propagation outside of their host organism, the marine viral
fraction represents perhaps the most poorly sampled biomass on Earth. To date,
PCR-based approaches (e.g. clone libraries, denaturing gradient gel electrophoresis (DGGE) (Muyzer 1999) and restriction fragment length polymorphism (RFLP)
analyses) have offered us rare glimpses of the enormous diversity of marine microorganisms (Archaea, prokaryotes, eukaryotes) derived from the analysis of specific
homologous genes (e.g. 16S or 18S ribosomal DNA) used to identify species or
phylotypes (Kemp and Aller 2004). DGGE allows the rapid analysis and comparison
of microbial communities. Compositional diversity can be visualized using DGGE
where each band in principle represents a microbial phylotype (Tzeneva et al. 2008).
Sequencing of a single band provides information on the related taxon and can be
used, for example, in phylogenetic analysis. Since viruses are obligate intracellular parasites, they lack ribosomal encoding regions such as 16S and 18S, thereby
necessitating the development of markers specific for each broad family (such as
RNA polymerase and DNA polymerase) in order to perform similar biodiversity
studies (Allen and Wilson 2008).
Whereas such studies highlight the sheer abundance of natural biodiversity,
no real functional significance can be attributed to the communities in question,
because the molecular markers used for biodiversity appraisals have conserved
genomic functions. For years, the Holy Grail of biodiversity research has been not
to merely study biodiversity but to assess functional biodiversity, that is, to determine how diversity contributes to ecosystem functioning. The study of functional