References

Marker Genes in Soil Microbiology 369
7
Expression of Intrinsic Marker Genes and Detection
of Gene Transfer Potentials
Marker genes themselves cannot directly indicate activity, but rather genetic
potentials. For many ecological studies, however, it is more meaningful
to analyze general or specific activities within a microbial community.
A certain indicator for activity is the cell concentration of ribosomes and
consequently SSU rRNA, which can specifically be detected by FISH, and
studieshavetriedtocompareSSUrRNAdiversitytothatofSSUrRNA
genes in order to identify active members of a microbial community. To
detect a specific activity of an intrinsic gene it would be ideal to quantify
the transcripts of the gene, the mRNA, instead of the gene. For eukaryotic
mRNA, e.g., fungal genes in soil, this approach is feasible (Bogan et al.
1996), but for bacterial mRNA it is problematic, as the latter molecules only
have an average half-life value of some minutes (Wellington et al. 2003).
Nevertheless, with an improvement of RNA extraction methods, there is
recent evidence in the literature that in selected soil environments transcripts
of bacterial genes can be isolated and characterized. Alfreider and
coworkers (2003) demonstrated the presence of mRNA of two catabolic
chlorocatechol dioxygenases genes which indicated active bioremediation
in an aquifer.
The detection of intrinsic marker genes with less conservation than the
SSU rRNA genes will often be a trade-off between choosing primer specificities
that are high enough to avoid the amplification of false-positives
(DNA fragments that have nothing to do with the functional gene that
was targeted), and still low enough to hybridize to conserved regions that
may be somewhat different from the predicted sequence and have the risk
of being false-negative if primer hybridizations should fail. An interesting
exception, however, are genes that are frequently transferred between
different phylogenetic lineages, such as genes encoding for replication and
transfer functions of broad-host range plasmids (mobile genetic elements).
On such plasmids, these genes build the backbone structure which is often
complemented with different functional genes, e.g., encoding for antibiotic
or heavy metal resistances, toxin production or biodegradative pathways.
Plasmids can be differentiated according to the compatibility of their replication
genes, either allowing coexistence of plasmids or incompatibilities.
Using primers specific for different incompatibility groups, it was possible
to study the diversity and prevalence of plasmids in a variety of environmental
substrates after direct extraction of DNA, PCR and DNA–DNA
hybridization (Smalla et al. 2000).