Roof-Dwelling Desiccation-Tolerant Cyanobacteria (Extremophiles)

Introduction Figure 1A shows a domicile on the Virginia Tech campus with a roof shingle biofilm community.   Such a community is black in color and usually observed on more north-facing roofs (away from full sun).  Isolation of the biofilm can be accomplished with the aid of a toothbrush and a small amount of water (Fig. 1B). The slurry is collected in sterile bags and includes some debris and roof shingle mineral (Fig. 1C).  The sample is returned to the lab and incubated in conditions selective for cyanobacterial growth.   Figure 2 shows a confocal laser scanning microscopic image of an unstained cyanobacterial biofilm roof isolate. The cyanobacterial cells autofluoresce due to the presence of light-harvesting proteins (phycobiliproteins and chlorophylls). The cells are encased in a protective extracellular matrix comprised predominantly of polysaccharides (EPS or bulk glycan). This matrix protects the cells from the environment. Based upon analyses that evaluate the genomic sequences found in specific regions of the genome (phylogenetic analysis, performed as described for the cyanobacterium Nostoc commune (4)), this organism is tentatively identified as Gloeocapsa sanguinea.   Figure 3 shows a cyanobacterial biofilm roof isolate when grown in culture using the cyanobacterial-selective medium BG11 (2). As the growth medium contains nitrates, the cyanobacteria invest less in EPS production (1) and thus appear as individual cells rather than several cells encased in an EPS. The colors in the confocal images are the result of specific excitation (Ex) and emission (Em) wavelength settings on the microscope:  blue (Ex = 364 nm, Em = 385-470 nm), green (Ex = 488 nm, Em = 505-530 nm), red (Ex = 543 nm, Em = 560-615 nm), purple (Ex = 633 nm, Ex = 655-719 nm).  The blue and green emissions are the result of the EPS interacting with the SYTOX green stain, while the red and purple emissions are from phycobiliprotein autofluorescence. The image at the lower right is an overlay (or merge) of the blue, green, red, and purple emissions images. Methods Figures 1A, B, and C were taken with a standard commercially available camera (Minolta Maxxum 5 SLR) with a Sigma lens (28-300 mm) and a focal length of 28 mm.  For film, FUJIFILM Superia X-TRA 400 was used and the images were digitized from a negative with Nikon SUPER COOLSCAN 5000 ED.  Figures 2 and 3 were obtained with a confocal laser scanning microscope (Zeiss 510 META; Carl Zeiss, Jena, Germany) and visualized with a 40x/1.2 W (C-Apochromat) water immersion objective lens.  Laser intensities were 543 nm (100%), 633 nm (20%), 364 nm (5.0%), and 488 nm (5.0%). The cells shown in Fig. 2 were from a roof biofilm isolate and imaged to view the autofluorescence (i.e., no staining). The cells in Fig. 3 were obtained by culturing in BG11 (2). An aliquot from a 4-week-old cell culture (1 ml) was pelleted and the supernatant was discarded.  SYTOX green (Invitrogen) was then added (500 ml of a 1:100 stock dilution) to resuspend the pellet, which was then placed in the dark for 10 minutes, pelleted again, and resuspended in 500 ml of deionized water. A sample volume of 20 ml was used for imaging. References 1.    Otero, A., and M. Vincenzini. 2004. Nostoc (Cyanophyceae) goes nude: extracellular polysaccharides serve as a sink for reducing power under unbalanced C/N metabolism. J. Phycol. 40 :74–81. 2.    Rippka , R., J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria, J. Gen. Microbiol. 111:1–61. 3.    Wright, D. J., S. C. Smith, V. Joardar, S. Scherer, J. Jervis, A. Warren, R. F. Helm, and M. Potts.  2005. UV irradiation and desiccation modulate the three-dimensional extracellular matrix of Nostoc commune (cyanobacteria). J. Biol. Chem. 280:40271–40281. 4.    Wright, D., T. Prickett, R. F. Helm, and M. Potts.  2001. Form species Nostoc commune (cyanobacteria). Int. J. Syst. Evol. Microbiol. 51:1839–1852.