Horizontal Gene Transfer in Planktonic and Biofilm Modes - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 83

environments (Sørensen et al. 2005 ; Reisner et al. 2012 ). This methodology
involves integration of genes encoding reporter proteins such as GFP in the
conjugative plasmid of interest. In this way, the fate of plasmids in a bacterial
community can be monitored in situ nondestructively. By this approach, spread of
different IncP-1 and IncP-9 plasmids was monitored in a variety of environments
including agar surface-grown colonies (Christensen et al. 1998 ; Krone et al. 2007 ;
Fox et al. 2008 ), biofilm model systems (Christensen et al. 1998 ; Hausner and
Wuertz 1999 ;Kr´ l et al. 2011 ; Seoane et al. 2011 ), freshwater microcosms
(Dahlberg et al. 1998 ), or plant leaves (Normander et al. 1998 ).
Nancharaiah and coworkers were the first to use a dual-labeling technique
involving GFP and the red fluorescent protein (DsRed) for in situ monitoring of
HGT via conjugation (Nancharaiah et al. 2003 ). A GFPmut3b-tagged derivative of
narrow-host-range TOL plasmid (pWWO) was delivered to P. putida KT2442,
which was chromosomally labeled with dsRed by transposon insertion via biparen-
tal mating. GFP and DsRed were coexpressed in donor P. putida cells (Nancharaiah
et al. 2003 ). Donors and transconjugants in mixed culture or sludge samples were
discriminated on the basis of their fluorescence through confocal laser scanning
microscopy. Conjugative transfer frequencies on agar surfaces and in sludge micro-
cosms were determined microscopically without cultivation. The new method
worked well for in situ monitoring of HGT in addition to tracking the fate of
microorganisms released into a laboratory sequencing batch biofilm reactor treating
synthetic wastewater (Nancharaiah et al. 2003 ).
Interestingly, spatial analysis of green fluorescence in various studies conducted
by different laboratories revealed that invasive spread of IncP plasmids was neither
detectable in recipient colonies on agar surfaces nor in recipient microcolonies in
flow-chamber biofilms suggesting that local factors limit plasmid transfer
(Christensen et al. 1998 ; Fox et al. 2008 ). Reisner et al. ( 2012 ) aimed to reveal
the local distribution of IncF plasmid transfer in agar surface-grown colonies. To
this goal, they developed a dual-color labeling strategy: E. coli donor cells
expressing a chromosomally encoded LacI repressor and carrying P A1/04/03 - cfp *-
tagged conjugative plasmids were combined with recipient cells lacking a func-
tional LacI protein. Since cfp *expression from P A1/04/03 is under tight control of the
LacI repressor in the donor cells, cyan fluorescence can only emerge after transfer
of the tagged conjugative plasmid to a recipient cell. To differentiate donor from
recipient cells a P A1/04/03 - yfp *-tagged E. coli CSH26 strain was utilized as recipient
strain. Transconjugant cells are therefore distinguishable by expressing both cyan
and yellow fluorescence (Reisner et al. 2012 ). Reisner and coworkers investigated
two different plasmids, R1 and R1 drd 19. High-resolution in situ analysis through
epifluorescence and confocal microscopy revealed that plasmid invasion did not
reach beyond the first five recipient cell layers at the donor/recipient interface for
both plasmids (Reisner et al. 2012 ). Extension of in situ analysis to other prototyp-
ical plasmids of the IncF, IncI, and IncW families revealed similarly limited levels
of recipient colony invasion. The results were in agreement with previous studies
monitoring IncP plasmid invasion in agar colonies and biofilm setups (Christensen
et al. 1998 ; Fox et al. 2008 ). Fox and coworkers found that replenishment of