Figure 1.
Different fates of aged cultures of the cyanobacterium S. elongatus.
(A) Cultures maintained at stationary phase were characterized by dark blue-green pigmentation up to about 3 months (culture No. 1). Older cultures either rapidly collapsed (culture No. 2), or gradually acquired a yellowish color (culture No. 3) and further survived. Cultures No. 1 and 2 are about 3 months old, whereas culture No. 3 is 6 months old. (B) Conditioned medium (CM) of a collapsing culture induced rapid cell death of exponentially growing cells of S. elongatus in contrast to medium from non-collapsing cultures, which did not affect viability. Cells were inoculated into fresh medium (FM) as a control. 5 µl of undiluted cultures or cultures diluted 1∶100 or 1∶1000 were 'spotted' on solid growth medium, following exposure to the different media (see Methods).
Figure 2.
Light-dependent toxic effect of conditioned medium from collapsing culture.
Viability assessment using Sytox green (A), and absorbance spectra (B) of exponentially growing cells 24 h after inoculation into fresh growth medium (FM), or into conditioned medium of a collapsing culture (CM). Exposure to CM under light resulted in bleaching of cell pigments including chlorophyll (Chl) and phycocyanin (PC).
Figure 3.
CM is harmful to photosynthetic microorganisms but not to heterotrophic bacteria.
(A) XAD-extract of CM was applied to variety of cyanobacteria and algae including S. elongatus, Anabaena PCC 7120, Calothrix PCC 7601, Chlamydomonas reinhardtii, Dunaliella salina and Chlorella vulgaris. Sensitivity was examined spectroscopically to detect the effect on pigmentation. Additional phytoplankton species were examined, as well (see Table S1). Substances extracted with XAD were dissolved in ethanol; this organic solvent was added to fresh medium (FM) in the control samples. (B) Escherichia coli, Staphylococcus aureus, Streptococcus faecalis and Bacillus cereus were inoculated into FM or CM. The number of colony forming units (CFU) following 12 h illumination in CM was normalized to CFU obtained following exposure to FM.
Figure 4.
The TD34-mutant of Synechocystis, which lacks a functional photosystem II reaction center, remains sensitive to conditioned medium (CM).
Shown are wild type Synechocystis PCC6803 (6803) and the mutant in which the three copies of psbA, each encoding the D1 protein of photosystem II, were inactivated (TD34). Cells were inoculated into fresh medium (FM) or CM.
Figure 5.
Cells respond to CM in a density dependent manner.
(A) Exponentially growing cells of S. elongatus were inoculated into conditioned medium (CM) at different initial cell densities, as indicated by the optical density at 750 nm (OD750). (B) Chloramphenicol, an inhibitor of protein synthesis, impaired the ability of high density cultures to cope with the deleterious effect of CM. (C) Cells coped better with CM when it was not supplemented with nutrients. (D and E) Inactivation of the nblR gene, encoding a response regulator essential for nutrient starvation responses, results in extreme sensitivity to CM. Data shown in B-D represent cells exposed to CM at OD750 = 0.04. Note the different y axis scale in (C) versus (B) and (D).
Figure 6.
Antioxidants mitigate the toxic effect of CM.
(A) The antioxidants glutathione (glut) and N-acetyl cysteine (NAC) alleviated the harmful effect of CM. Inset depicts assessment of viability by 'spotting' 5 µl of undiluted cultures onto fresh solid growth medium. (B) The catalase mutant strain, KatΩ, was highly sensitive to CM.
Figure 7.
Conditioned medium causes bleaching of pigments in a cell extract.
Absorbance maxima of chlorophyll (Chl) and phycocyanin (PC) are indicated.