Experimental procedures
Bacterial strains, media, and growth conditions.
Ruegeria pomeroyi strain DSS-3 and the R. pomeroyi phoX mutant were routinely
cultured at 30°C with shaking at 150 rpm using Marine Broth (Difco 2216). For the
experiments R. pomeroyi strain and the phoX mutant were grown in a marine basal
medium (MBM) (12.1 g of Tris HCl, 1.0 g of NH4Cl, 20 g of ASW, supplemented
with 1µM of Fe, 1ml of vitamin solution (Sebastian and Ammerman, 2009), 0.2 mM
of K2HPO4 and glucose as a carbon source to a final concentration of 10 mM.
Growth on different organic P sources
To obtain phosphorus-starved cultures, phosphorus-free MBM was inoculated with a
1% (vol/vol) overnight-grown MB culture and incubated overnight. These
phosphorus-starved cultures were used as inocula for growth experiments. We
compared growth of the wild-type and the phoX mutant on phosphate (Pi), Glucose-6P (G6P), -glycerol-phosphate (BGP), AMP, ADP, ATP and low-molecular-weight
herring sperm DNA (SIGMA). All the P sources were added at a final concentration
of 0.2 mM except DNA that was added at a final concentration of 78.3 mg L-1
following Pinchuk et al., (2008). Samples for OD were taken at different time
intervals. All growth experiments were independently repeated at least twice and
performed in triplicate.
Starvation experiments
R. pomeroyi wild-type and phoX mutant were grown in MBM supplemented with
phosphate and cells were harvested by centrifugation at the onset of stationary phase.
Half of the cells were resuspended in P-replete medium as a control (+P, 0.2 mM
K2HPO4) and the other half in medium without added Pi (-P). The experiment was
carried out by duplicate. Samples were taken at different time points for OD and
alkaline phosphatase activity assays. Additional samples were taken for kinetics and
substrate competition experiments.
Alkaline phosphatase activity measurements and kinetics
Alkaline phosphatase activity was determined in duplicate subsamples by monitoring
the rate of hydrolysis of the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl
phosphate (DifMUP, Invitrogen, Eugene, OR, USA) at a final concentration of 10
µM. The DifMU produced during the assay was detected as an increase in
fluorescence over a 10 minute incubation using a Tecan Genios 96-well fluorescence
microplate reader. A standard curve with DifMU (Sigma) was used to quantify the
hydrolysis rates (initial slope of the phosphatase assay divided by the slope of the
standard curve). Kinetic experiments were performed by adding the substrate in a
range of concentrations from 0.5 to 100 M. Kinetic curves followed MichaelisMenten and therefore a non-linear fit of this equation was used to calculate the kinetic
parameters.
Substrate competition experiments
Substrate competition experiments were carried out to investigate which of the
compounds used in the growth experiments can be more efficiently hydrolyzed by the
wild type and the mutant strains. For these experiments increasing concentrations of
Pi, G6P, AMP, ADP, and ATP were added to duplicate samples just before adding the
DifMUP substrate. The concentrations used were: 0, 5, 10, 20, 50 and 100 M.
Alkaline phosphatase assays were performed as described above.
References
Pinchuk, G.E., Ammons, C., Culley, D.E., Li, S.M.W., McLean, J.S., Romine, M.F.,
et al. (2008) Utilization of DNA as a sole source of phosphorus, carbon, and
energy by Shewanella spp.: ecological and physiological implications for
dissimilatory metal reduction. Applied and Environmental Microbiology 74:
1198.
Sebastian, M., and Ammerman, J.W. (2009) The alkaline phosphatase PhoX is more
widely distributed in marine bacteria than the classical PhoA. The ISME
Journal 3: 563-572.