Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

of carbohydrate metabolism under cold stress that resulted in a rapidly generated
energy to overcome the stress. Cold stress resulted in a decrease in the polyb-hydroxybutyrate
in a psychrotolerant Rhizobium, owing to PHB inhibition rather
than an increase in its breakdown at low temperature. P. fluorescens accumulated
2-ketogluconate in medium (consisting of 0.3% NH4H2PO4; 0.2% K2HPO4; 0.05%
MgSO4 7H2O; 0.5 mgml 1 FeSO4 7H2O; and 0.2% filter-sterilized glucose as carbon
source) as the major oxidation product of glucose [23].
10.2.3
Expression of Antifreeze Proteins
10.2 Cold Adaptation of PGPR Strainsj199
There are a number of substances described in the published literature that inhibit
ice nucleation. Certain bacterial strains, mostly found in the nonfluorescent pseudomonad
species, release materials into the growth medium that reduce the nucleation
temperature of water droplets to below that of distilled water [24]. These
substances include sucrose, unsaturated fatty acids and phospholipids; however,
in psychrophilic bacteria and some other psychrophilic organisms, specific proteins
are produced that reduce freezing temperature and protect them from freeze injury.
These are known as antifreeze proteins and help bacteria to survive the freezing
conditions.
Antifreeze proteins (AFPs) are structurally a diverse group of proteins with the
ability to modify ice crystal structure [25] and inhibit recrystallization of ice by
adsorbing onto the surface of ice crystals via van der Waals interactions and/or
hydrogen bonds [26–28]. During cold acclimation, many freeze-tolerant organisms
accumulate antifreeze proteins [29–31].
A novel AFP assay, designed for high-throughput analysis in Antarctica, demonstrated
putative activity in 187 of the cultures tested. Subsequent analysis of the
putative positive isolates showed 19 isolates with significant recrystallization inhibition
(RI) activity. The 19 RI-active isolates were characterized using ARDRA
(amplified rDNA restriction analysis) and 16S rDNA sequencing. They belong to
genera from the a-proteobacteria, with genera from the g subdivision being predominant.
The 19 AFP-active isolates were isolated from four physicochemically
diverse lakes [32].
The structural and functional features of AFPs enable them to protect living
organisms by suppressing the effect of freezing temperatures and modifying or
suppressing ice crystal growth. The plant growth promoting rhizobacterium Pseudomonas
putida GR12-2 was isolated from the rhizosphere of plants growing in the
Canadian High Arctic. This bacterium was able to grow and promote root elongation
of spring and winter canola at 5 C, a temperature at which only a relatively small
number of bacteria were able to proliferate and function. In addition, the bacterium
survived exposure to freezing temperatures, ranging from 20 to 50 C; and it was
discovered that at 5 C, P. putida GR12-2 synthesized and secreted an antifreeze
protein into the growth medium [33,34].
Katiyar and Goel [35] reported the presence of antifreeze proteins in a coldtolerant
mutant of P. fluorescens. It was observed that AFP was capable of protecting