Localization of acidic phospholipid cardiolipin and DnaA in mycobacteria

Abstract

Acidic phospholipids such as cardiolipin (CL) have been shown to modulate Mycobacterium tuberculosis (Mtb) DnaA interactions with ATP. In the present study, using nonyl acridine orange fluorescent dye we localized CL-enriched regions to midcell septa and poles of actively dividing cells. We also found that CL-enriched regions were not visualized in cells defective for septa formation as a consequence of altered FtsZ levels. Using Mtb cultures synchronized for DNA replication we show that CL localization could be used as a marker for cell division and cell cycle progression. Finally, we show that the localization pattern of the DnaA-green fluorescent fusion protein is similar to CL. Our results suggest that DnaA colocalizes with CL during cell cycle progression.

Fig. 1

NAO staining of M. smegmatis cells. M. smegmatis cells were grown in liquid culture from a starting optical density (600 nm) of 0.1 and 200 nM NAO was added 2 hrs prior to observation by microscopy. Exponential phase cultures (top panels) were harvested after 3 hrs and stationary phase cultures (bottom panels) after 20 hrs of growth. The NAO stain binds monoacidic phospholipids in monomer form, but forms a dimer when bound to cardiolipin, which causes a fluorescence shift from green to red. Grey panels – brightfield images, green images - NAO staining of monoacidic phospholipids and red images - cardiolipin staining. Bar = 3 μm.

Fig. 2

NAO staining of the M. smegmatis ftsZ strains. Actively growing cultures of M. smegmatis FtsZ-72 (A) or M. smegmatis ΔftsZ, FZ3-78 (B) were grown for 6 hrs (active) or 18 hrs (stationary) with acetamide to induce FtsZ overproduction (A) or without acetamide to induce FtsZ depletion (B). NAO (200 nM) was added in the last 2 hrs of incubation and live cells observed by fluorescence microscopy. Following growth, the culture media was removed by centrifugation and cells suspended in fresh media either without acetamide to reduce overproduction or with acetamide to induce FtsZ production in the depleted strain. Incubation was continued again for 8 hrs with NAO being added for the last 2 hrs. Bar = 3 μm; BF are brightfield images and fluorescence signals in NAO images show regions where cardiolipin is present. Panels i, iii, v and vii of A and B are brightfield images whereas ii, iv, vi and viii are the corresponding fluorescent images.

Fig. 3

(A) NAO staining of Mtb dnaAcos115 strain. The NAO dye was added to cultures during a temperature shift experiment and cells were fixed in paraformaldehyde following incubation at either 30ºC (non-permissive for growth) or 37ºC (permissive temperature). Cells were then observed by fluorescence microscopy. Bar = 3 μm. Fluorescence images (CL) show NAO staining of regions where CL is present. (B) NAO staining patterns observed in Mtb wild-type and the Mtb dnaAcos115 strain. Fluorescence images of NAO stained cells were analyzed and type of staining was recorded for about 100 cells from each strain. Data were plotted in excel.

Fig. 4

GFP-DnaA fluorescent fusion protein localization in M. smegmatis. Actively growing M. smegmatis Pami::gfp-dnaA cells were induced with acetamide for 1 hr and examined by fluorescent microscopy (A). Top panels are brightfield images and bottom panels are fluorescent images showing localization of GFP-DnaA. (B) Approximately 100 cells in brightfield images were measured for length and the corresponding fluorescence images were scored for the type of GFP-DnaA localizations.

Fig. 5

Viability of M. smegmatis gfp-dnaA strains. Cultures were grown to an optical density (OD600) of 0.4, then serially diluted and spread on agar plates. Colonies were counted after 3 days of growth and viability was calculated as the number of CFU per mL of 0.1 OD600