Diversity of Growth Patterns Probed in Live Cyanobacterial Cells Using a Fluorescent Analog of a Peptidoglycan Precursor

Abstract

Cyanobacteria were the first oxygenic photosynthetic organisms during evolution and were ancestors of plastids. Cyanobacterial cells exhibit an extraordinary diversity in their size and shape, and bacterial cell morphology largely depends on the synthesis and the dynamics of the peptidoglycan (PG) layer. Here, we used a fluorescence analog of the PG synthesis precursor D-Ala, 7-Hydroxycoumarin-amino-D-alanine (HADA), to probe the PG synthesis pattern in live cells of cyanobacteria with different morphology. They displayed diverse synthesis patterns, with some strains showing an intensive HADA incorporation at the septal region, whereas others gave an HADA signal distributed around the cells. Growth zones covering several cells at the tips of the filament were present in some filamentous strains such as in Arthrospira. In Anabaena PCC 7120, which is capable of differentiating heterocysts for N₂ fixation, PG synthesis followed the cell division cycle. In addition, an HADA incorporation was strongly activated from 12 to 15 h following the initiation of heterocyst development, indicating a thickening of the PG layer in heterocysts. The PG synthesis pattern is diverse in cyanobacteria and responds to developmental regulation. The use of fluorescent analogs may serve as a useful tool for understanding the mechanisms of cell growth and morphogenesis operating in these organisms.

Keywords: HADA; cell wall; cyanobacteria; growth pattern; heterocyst; peptidoglycan.

FIGURE 1

HADA labeling in three unicellular cyanobacteria: Synechocystis PCC 6803, Synechococcus elongatus PCC 7942, and Microcystis PCC 7806. (A) Pulse labeling of Synechocystis and Synechococcus elongatus with a high concentration of HADA (800 μM) for 30 min, followed by removal of HADA by washing and observation by fluorescence microscopy. (B) Continuous labeling with a low concentration of HADA in Synechocystis, Synechococcus elongatus, and Microcystis. For Synechocystis and Synechococcus elongatus, three or four sets of photos are shown, and each demonstrates a representative HADA labeling pattern. HADA signal is in blue, and photosynthetic pigment in red, and the overlay for the images of same cells acquired through the two channels. Arrows indicates the pre-septal division mode in Microcystis.

FIGURE 2

Growth pattern of the filamentous cyanobacteria Arthrospira sp. FACHB 792 and Oscillatoria animalis. (A,C) Arthrospira pulse-labeled with 800 μM of HADA for half an hour or long-term labeled with 200 μM of HADA for 24 h. (B) The HADA signal at the tips of some pulse labeled Arthrospira quantified with ImageJ. Every curve in the graph represents the fluorescence intensity along one end of a filament as exemplified above the graph. The filaments of long-term labeled Arthrospira were straightened with ImageJ. (D) Oscillatoria incubated with 200 μM of HADA for 24 h. Bright-field photos (gray background pictures in panel A), as well as images of HADA fluorescence (blue), photosynthetic pigment fluorescence (red), and their merges are shown.

FIGURE 3

HADA incorporation into Leptolyngbya sp. CB006 and Spirulina subsalsa. Leptolyngbya requires a longer incubation time with HADA. Photos of HADA fluorescence (blue), photosynthetic pigment fluorescence (red), and their merges are shown.

FIGURE 4

PG synthesis during vegetative growth and heterocyst development in Anabaena PCC 7120. Filaments of Anabaena were grown in BG11 using nitrate as a combined nitrogen source, then incubated with 200 μM of HADA in BG11 (A), or in BG11₀ (B) to induce heterocyst development. Arrows in A indicate elongated cells that just started to incorporate HADA at the division site, and in B heterocysts. Bright-field (gray) and HADA fluorescence (blue) images are shown.

FIGURE 5

Relationship between cell division and PG synthesis in Anabaena PCC 7120. (A) An Anabaena strain in which the cell division gene ftsZ was replaced by a ftsZ-yfp translational fusion was treated with HADA, and imaged under a fluorescence microscope. The top panel shows red photosynthetic pigment and the HADA signal in blue; the middle panel shows the Z-ring in yellow; and the bottom panel shows a superimposition of the same filaments with both HADA and FtsZ-YFP signals. Asterisks indicate cell–cell junctions labeled by HADA, white arrows indicate septa having both HADA signal and FtsZ-YFP signal, and red arrows indicate septa having FtsZ-YFP signal only. (B) The same recombinant strain as in A was treated with aztreonam (Az), an antibiotic targeting to FtsI, a PG synthase involved in cell division. Cells elongated as a consequence of cell division inhibition as previously reported (Sakr et al., 2006). The same filaments were pictured in red (photosynthetic pigments), blue (HADA), yellow (FtsZ-YFP) and superimposition of the HADA and YFP signals.

FIGURE 6

Determination of the timing of PG synthesis in heterocysts. Hetercoysts differentiation takes approximately 20–24 h after the deprivation of combined nitrogen from the growth medium. Heterocysts were induced in the BG11₀ medium containing 200 μM HADA. Seven hours after the induction, filaments were stained with Alcian blue which binds to heterocyst-specific polysaccharide layer, to better recognize the developing cells (arrows). (A) The filaments were examined under both bright-field and for HADA fluorescence to determine the incorporation of HADA into developing heterocysts. The same procedure was followed after 10 h, 12 h and 15 h of induction. All HADA fluorescence images were taken using the same exposure time (1 s) and were processed with the same procedures using ImageJ. (B) Quantification of HADA incorporation in developing and mature heterocysts at 8 h, 10 h, 12 h, and 15 h. At each time point, the fluorescence signal along seven representative short filaments, each of which consists of one heterocyst (Het) at the center and a few vegetative cells (Veg) on each side, were quantified with ImageJ. Every curve in the graphs represents the fluorescence intensity along one filament. The signal from developing or mature heterocysts in the graphs is indicated by the center shadow area. The bright-field microscopy image and the HADA fluorescence picture of one filament from the 15 h sample were shown at the top of the panel for illustration purpose.