Table 1.
List of PCR and sequencing primers.
Figure 1.
Morphology of Gloeobacter violaceus and Aphanothece caldariorum-like samples.
(A) and (B) A. caldariorum-like samples from the botanical gardens in Liberec and Teplice, respectively, showing typical rod-shaped cells with polar granules and layered mucilaginous envelopes; (C) cell morhology in the perfectly preserved herbarium type specimen of A. caldariorum var. cavernarum; (D) “nanocytes“ in the environmental sample from Liberec dominated by A. caldariorum-like morphotype; (E) and (F) batch cultures ordered by increasing age (from the left) of A. caldariorum-like strain CCALA 981 and G. violaceus PCC 7421, respectively, showing a gradual color shift from grey-violet to yellow-orange; (G–J) and (K–N) change in cell morphology from subspherical nanocyte-like cells to rod-shaped cells with occasional mucilaginous envelopes in the batch cultures of CCALA 981 and PCC 7421, respectively (the batches are the same as in panels E and F). Scale bars, 10 µm.
Figure 2.
Morphometric analysis of cells in Aphanothece caldariorum-like and Gloeobacter violaceus cultures.
(A) and (B) increasing cell length corresponding to batch cultures of increasing age of A. caldariorum-like strain CCALA 981 and G. violaceus PCC 7421 (Kruskal-Wallis test, p<0.001), difference between individual pairs of batches was also significant (two-tailed t-test, p<0.02 except for a pair of batches 41 and 50 days old in PCC 7421, for which the difference was at the edge of statistical significance, p = 0.045); (C) and (D) increasing cell length:width ratio in the same batch cultures as previously (Kruskal-Wallis test, p<0.001), difference between individual pairs of batches was also significant (two-tailed t-tests, p<0.001) except for a pair of batches 41 and 50 days old in PCC 7421. One hundred cells were measured in each sample.
Figure 3.
Light spectroscopy analysis of photosynthetic pigments in Aphanothece caldariorum-like strain and Gloeobacter violaceus.
(A) fluorescence emission spectra of A. caldariorum-like strain CCALA 981 in comparison with G. violaceus PCC 7421 for excitation to phycobilisomes. Higher content of phycoerythrin in CCALA 981 is documented by a relative increase in its fluorescence emission at 574nm. (B) and (C) whole cell absorption spectra of CCALA 981 and PCC 7421 normalized to chlorophyll a content. Clear accumulation of carotenoids (wide absorbance peak between 450–500 nm) at the stationary phase of growth (third and fourth batch) is obvious in both strains. The absorbance of 41-day old strain PCC 7421 is raised due to the beginning of accumulation of carotenoids in this batch, but phycobilin peaks are still recognizable. Individual absorbance peaks are as noted; CAR, carotenoids; PE, phycoerythrin; PC+APC, phycocyanin and allophycocyanin; Chl a, chlorophyll a.
Table 2.
Relative concentration of particular phycoerythrobilins in phycobilisomes of Aphanothece caldariorum-like and Gloeobacter violaceus strains calculated from absorption spectra.
Table 3.
Number of phycoerythrin and phycocyanin trimers in a single phycobilisome rod of Aphanothece caldariorum-like and Gloeobacter violaceus strains.
Figure 4.
Comparison of cell ultrastructure in Aphanothece caldariorum-like strains and Gloeobacter violaceus.
Typical cell ultrastructure of G. violaceus PCC 9601(A), G. violaceus CCALA 979 (B), A. caldariorum-like strain CCALA 981(C) and A. caldariorum-like strain CCALA 980 (D), respectively. Cells did not contain any thylakoid, the photosynthetic pigments accumulated in an electron-dense layer near the multi-layered cell wall. Cells typically contained two large polyphosphate granules in polar positions. Observed ultrastructure was identical to the reference strain G. violaceus PCC 7421 [6]. Scale bars, 500 nm.
Figure 5.
Phylogenetic position of Gloeobacter violaceus and Aphanothece caldariorum.
(A) Phylogenetic tree based on a SSU rRNA gene alignment. (B) Phylogenetic tree based on a concatenated SSU rRNA gene+rpoC1 alignment. Sequences generated in this study are printed in bold font. Branch support values (%) are given at nodes in this format: Bayesian inference/maximum likelihood/maximum parsimony. A well supported basal clade of cyanobacteria consisting of G. violaceus and A. caldariorum is highlighted by blue colour.