figure’s1: transferof p.ubique … · 2013-02-19 ·...
TRANSCRIPT
Figure S1: Transfer of P. ubique batch cultures from natural seawater to AMS1. ▲: P. ubique transferred from natural seawater amended with pyruvate (50 µM), glycine (1 µM), methionine (1 µM) and vitamins to identically amended natural seawater medium. ●: P. ubique transferred from natural seawater amended with pyruvate (50 µM), glycine (1 µM), methionine (1 µM) and vitamins to identically amended AMS1. Points are the mean of triplicate cell density measurements. Error bars: ± 1.0 SD, n=3. When error bars are not visible, they are smaller than the size of the symbols.
cell
dens
ity (c
ells
ml-1
)
104
105
106
107
time (days)0 2 4 6 8 10 12
Figure S2: Flow cytometry profiles and corresponding microscopic images at different pyruvate:glycine molar ratios. All cultures were grown to stationary-‐phase in AMS1 containing methionine (10 µM) with the following glycine and pyruvate concentrations: A) 0 µM pyruvate, 50 µM glycine; B) 0.1 µM pyruvate, 50 µM glycine; C) 0.9 µM pyruvate, 50 µM glycine; D) 5 µM pyruvate, 50 µM glycine; E) 25 µM pyruvate, 50 µM glycine; F) 50 µM pyruvate, 0 µM glycine; G) 50 µM pyruvate, 0.1 µM glycine; H) 50 µM pyruvate, 0.9 µM glycine; I) 50 µM pyruvate, 5 µM glycine; J) 50 µM pyruvate, 25 µM glycine. For each treatment, the upper panel shows relative DNA fluorescence profiles of SYBR Green I-‐stained cells; the lower panel shows microscopic images of SYBR green I-‐stained cells. In (A), †: the relative DNA fluorescence of single cells; ‡: the relative DNA fluorescence of doublets
F G H I
A B C D E
J
F G H I
A B C D E
J
† ‡
Figure S3: DNA fluorescence profiles and maximum cell densities in response to alanine additions. Top panel: Relative DNA fluorescence time course of batch cultures grown in AMS1 amended with limiting pyruvate (0.5 µM) glycine (1 µM), methionine (1 µM) and different L-‐alanine concentrations, as indicated. Bottom panel: Maximum cell densities of cultures for which relative DNA fluorescence values are shown in the top panel.
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0
2
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10
alanine concentration
time
afte
r ino
culat
ion (d
ays)
2000 nM 500 nM 250 nM 125 nM 0 nM
2000 nM 500 nM 250 nM 125 nM 0 nM
cell d
ensit
y (x 1
06 cells
ml-1
)
Figure S4: Effect of vitamin additions on P. ubique growth. P. ubique grown in AMS1 without added organic carbon and different doses of freshly prepared vitamins (as listed in Table 1). Points show the maximum density achieved of single flasks (triplicates for each dose are shown).
Figure S4 Discussion: Upon exclusion of the vitamin mixture and in the absence of added organic carbon, P. ubique grew to densities of ca. 2.0 ×105cells ml-‐1. Higher densities, exceeding 1.2 ×106 cells ml-‐1, were observed, in a dose-‐dependent manner, when a freshly prepared vitamin solution was added (Figure S4). We speculate that Pelagibacter makes efficient use of organic carbon that enters the cultures as reagent contaminants (for example, in the vitamin stocks), or by diffusion from plastic culture ware and the air. Previously P. ubique has been shown to use a variety of volatile organic carbon compounds as sources of electrons for respiration (Sun, et al). Also in support of this interpretation, we note that the growth of bacteria in water purification systems is not uncommon (Penna et al, 2002). Collectively, we conclude that unintentional trace levels of nutrients entering our experiments, coupled with the low amounts of carbon required by P. ubique, is a probable explanation for the observed growth without added carbon.
0.00E+00
2.00E+05
4.00E+05
6.00E+05
8.00E+05
1.00E+06
1.20E+06
1.40E+06
1.60E+06
0 2 4 6 8 10 12
maximum
density (cells ml-‐1)
Dose of vitamin mix added (fold)