plant surface microbiology.pdf

selection using GFP-expressing bacteria, this can be easily overcome by the
addition of 0.45 mM FeSO 4 ◊H 2 O. Since most GFP gene sequences are known,
gfp-tagged cells can also be detected by molecular methods such as gene
probing, DNA hybridisation, or PCR.
4.2 Rhizosphere-Stable Plasmids
To understand the biological significance of genes and mutations, they need
to be studied or expressed in the context in which they are assumed to function.
Also, the complementation of rhizosphere-expressed mutations and
expression of reporter genes need to be performed in situ. One consideration
when studying processes in complex living systems, such as under soil or rhizosphere
conditions, is that antibiotic selection often cannot be applied. In
addition, bacteria in the rhizosphere are assumed to be covered by a mucigel
layer or form biofilms which are known to have increased resistance to antibiotics.
Therefore, field and rhizosphere studies often require the use of rhizosphere-stable
plasmids, e.g. for complementation of mutations or for tracking
bacteria. While naturally occurring plasmids are often stably maintained
within a bacterial population in the absence of selection pressure, many
cloning vectors disappear without the appropriate selection. Plasmids with
genes for complementation or reporter studies should therefore be stably
maintained in strains without antibiotic pressure or be integrated into the
chromosome.
The Pseudomonas replicon pVS1 is stably maintained in many genera
including Pseudomonas, Agrobacterium, Rhizobium, Burkholderia, Aeromonas,
and Comamonas. Cloning vectors harbouring a 3.8-kb region of pVS1
with functions for replication (rep) and stability (sta) also appear to be stably
maintained. pVS1 derivatives pVSP41, pWTT2081, pME6010, pME6030,
pME6040, and derivatives have been shown to be completely stable in various
rhizosphere bacteria in the rhizospheres of various crop plants. Although the
incompatibility group of pVS1 has not been determined, the replicon appears
to be compatible with IncP-1, IncP-4, IncP-8, IncP-10, and IncP-11 plasmids in
P. aeruginosa.
4.3 Genetic and Metabolic Burdens
2 Root Colonisation Following Seed Inoculation 21
Another consideration when introducing foreign or additional DNA on plasmids
into bacterial strains is a plasmid or metabolic burden. The presence of
a plasmid may confer a metabolic burden on the cells because of the presence
of additional DNA and/or the expression of the reporter gene. Although the
effects are often not visible under laboratory conditions, the presence of a
plasmid may very well cause a genetic or metabolic burden in the rhizosphere