Environmental Microbiology of Aquatic and Waste Systems

44 3 Aspects of the Molecular Biology of Microorganisms of Relevance to the Aquatic Environment

is a short sequence of nucleotides upstream of

the translational start site that binds to ribo-

somal RNA and thereby brings the ribosome to

the initiation codon on the mRNA. The com-

puter program searches for a Shine–Dalgardo

sequence and finding it helps to indicate not

only which start codon is used, but also that the

ORF is likely to be functional.

(e) If the ORF is preceded by a typical promoter (if

consensus promoter sequences for the given

organism are known, check for the presence of

a similar upstream region).

(f) If the ORF has a typical GC content, codon

frequency, or oligonucleotide composition of

known protein-coding genes from the same

organism, then it is likely to be a functional

ORF.

2. Comparison with Existing Genes
   Sometimes, it may be possible to deduce not only
   the functionality or not of a gene (i.e., a functional
   ORF), but also the function of a gene. This can be
   done by comparing an unknown sequence with the
   sequence of a known gene available in databases
   such as The Institute for Genomic Research
   (TIGR) in Maryland (Rogic et al. 2001 ).

3.6 Metagenomics

Metagenomics is the genomic analysis of the collective
genome of an assemblage of organisms or “metage-
nome.” Metagenomics describes the functional and
sequence-based analysis of the collective microbial
genomes contained in an environmental sample
(Fig. 3.9). Other terms have been used to describe the
same method, including environmental DNA libraries,
zoolibraries, soil DNA libraries, eDNA libraries,
recombinant environmental libraries, whole genome
treasures, community genome, and whole genome
shotgun sequencing. The definition applied here
excludes studies that use PCR to amplify gene cas-
settes or random PCR primers to access genes of inter-
est since these methods do not provide genomic
information beyond the genes that are amplified.
Many environments have been the focus of metag-
enomics, including soil, the oral cavity, feces, and
aquatic habitats, as well as the “hospital metagenome”
a term intended to encompass the genetic potential of

organisms in hospitals that contribute to public health

concerns such as antibiotic resistance and nosocomial

infections.

Uncultured microorganisms comprise the majority

of the planet’s biological diversity. In many environ-

ments, as many as 99% of the microorganisms cannot

be cultured by standard techniques, and the uncultured

fraction includes diverse organisms that are only dis-

tantly related to the cultured ones. Therefore, culture-

independent methods are essential to understand the

genetic diversity, population structure, and ecological

roles of the majority of microorganisms in a given

environmental situation. Metagenomics, or the culture-

independent genomic analysis of an assemblage of

microorganisms, has the potential to answer funda-

mental questions in microbial ecology. Several mark-

ers have been used in metagenomics, including 16S

mRNA, and the genes encoding DNA polymerases,

because these are highly conserved (i.e., because they

remain relatively unchanged in many groups). The

marker most commonly used, however, is the sequence

of 16S mRNA. The procedure in metagenomics is

described in Fig. 3.9. Its potential application in bio-

technology and environmental microbiology is that it

can facilitate the identification of uncultured organ-

isms whose role in a multi-organism environment such

as sewage or the degradation of a recalcitrant chemical

soil may be hampered because of the inability to

culture the organism.

Environmental sample

Metagenomic library construction

Metagenomic library

Metagenomic

analysis

screen for particular

sequences by PCR or

hybridization

random

sequencing

screen for

expression of

particular phenotypes

Extract

DNA Clone

Transform into a

host bacterium

(e.g.E. coli)

Fig. 3.9 Schematic procedure for metagenomic analysis

(Modified from Riesenfeld et al. 2004 )