This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.
Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.
The field of bacterial cell biology has been significantly enhanced by recent advancements in microscopy techniques1,2. Among other instruments, microscopes that are capable of conducting timelapse fluorescence microscopy experiments remain a valuable tool. Investigators can monitor various physiological events in real-time using fluorescent proteins such as, green fluorescent protein (GFP)-based transcriptional and translational reporter fusions, fluorescent D-amino acids (FDAA)3, or use other stains for labeling the cell wall, membrane and DNA. It is therefore of no surprise that fluorescence microscopy remains popular among microbial cell biologists. In addition to simply showing the end phenotypes, providing information as to how the observed phenotypes arise using timelapse microscopy could add significant value to the findings and potentially offer clues as to what cellular processes are being targeted by potential drug candidates4.
The protocols to conduct high-resolution imaging using a fully motorized, inverted, wide-field fluorescence microscope (see the Table of Materials) are provided in this article. These protocols could be adapted to suit the needs of other fluorescence microscopes that are capable of conducting timelapse microscopy. Although the software discussed here corresponds to the specific manufacturer-supplied software as indicated in the Table of Materials, software commonly supplied by other microscope manufacturers or the freely available ImageJ5, have equivalent tools for analyzing microscopy data. For conditions where timelapse is not conducive, time-course experiments could be conducted as described in this article. The protocols described here provide a detailed guide to study the phenotypic changes in two different bacterial species: B. subtilis and S. aureus. See Table 1 for strains used.
1. General growth conditions
2. Sample preparation
3. Imaging
4. Image processing
GpsB phenotypes
Previously we have shown that Sa-GpsB is an essential protein as depletion of GpsB using an antisense RNA results in cell lysis9. Here we describe how the emergence of various cell division phenotypes and changes in protein localization could be captured using the timelapse microscopy protocol described in this article. For this purpose, S. aureus strains RB143 [SH1000 harboring pEPSA5 (empty vector)] and GGS8 [SH1000 harboring pGG59 (Pxyl-gpsBantisense bla cat)] reported previously9, were grown as follows. Strains RB143 and GGS8 were inoculated in 2 mL of tryptic soy broth (TSB) supplemented with 5 µg/mL chloramphenicol (chlor) in a 15 mL test tube and were incubated overnight at 22 °C while shaking. The overnight cultures were diluted 1:20 in 10 mL of fresh TSB + chlor in a 125 mL flask and grown at 37 °C with shaking until mid-logarithmic phase (OD600 = 0.5). The inducer, 1% xylose, was added to the culture medium to trigger the expression of antisense RNA of gpsB and the culture was grown for another 3 h. Cells were then stained with fluorescent dye FM4-64 (membrane stain), where required, by the addition of 0.5 µL of a 10 µg/mL stock of FM4-64 directly onto the 5 µL aliquot of culture on the microscope dish as described in the protocol section. As shown in Figure 1 and Video 1, addition of xylose to induce GGS8 strain resulted in a “sick” cell phenotype, as described previously9, while empty vector control (RB143) appeared similar to our control—cells grown in the absence of inducer.
Our group also reported that overproduction of S. aureus GpsB (Sa-GpsB) disrupts cell division in B. subtilis9. We use this overexpression phenotype as an example to demonstrate the protocol described here. To this end, a B. subtilis strain GG9 (amyE::Phyperspank-gpsBSa spc; ftsAZ::ftsAZ-gfpΩerm) was used9. Subcellular localization of fluorescently-labeled FtsZ, a key cell division protein which marks the cell division sites10,11, was used to monitor the status of cell division. The sample for microscopy was prepared as follows. A single colony of GG9 was inoculated in 2 mL Luria-Bertani (LB) medium and incubated overnight at 22 °C in an incubator shaker. The overnight cultures were 1:20 in 10 mL of fresh LB, and grown at 37 °C with shaking until mid-logarithmic phase (OD600 = 0.5). GG9 cells (5 µL aliquot) to be imaged were placed on the bottom of a glass bottom culture dish and covered with a 1% agarose pad made with LB supplemented with 250 µM (final concentration) of isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the expression of Sa-gpsB (Figure 2 and Video 2). Timelapse microscopy and cell length quantification were performed as described in the protocol section.
Inhibition of FtsZ
FtsZ, being a protein essential for cell division, is considered an attractive drug target and multiple groups are developing FtsZ inhibitors as a way to develop new antibiotics12. Localization patterns of FtsZ or one of the proteins associated with it, such as ZapA, can be used as a reporter to study and/or identify novel antimicrobial compounds. We use the protocol provided here to demonstrate this approach using S. aureus RB197 [SH1000 harboring pRB42 (PCd-zapASa-gfp bla erm)]9 and B. subtilis PE92 (ftsAZ::ftsAZ-gfpΩerm)13 strains. RB197 and PE92 strains were grown as described above in TSB (containing 5 µg/mL erythromycin; and 1.25 µM CdCl2 to induce the expression of zapA-gfp) and LB respectively. At mid-logarithmic phase, a well-characterized FtsZ inhibitor, PC19072314,15, was added at 2 µg/mL final concentration and its effect on the S. aureus and B. subtilis cells were monitored using microscopy at different time intervals (Figure 3 and Figure 4). Quantification of cell diameter of S. aureus and cell length of B. subtilis was performed as described in the protocol section.
Figure 1: High-resolution micrograph of S. aureus cells displaying sick phenotype. Fluorescence micrographs of S. aureus strains harbouring either empty vector (left; RB143) or an inducible copy of antisense RNA of gpsBSa (right; GGS8) in the presence and absence of 1% xylose (inducer). Cells awere stained with FM4-64 membrane stain (stocks dissolved in sterile water) and imaged using TRITC filter set. Scale bar: 1 µm. Please click here to view a larger version of this figure.
Figure 2: Representative data showing cell division inhibition in B. subtilis. (A) Timelapse micrographs of B. subtilis strain GG9 with images acquired at 20-min intervals for 120 min using the DIC/FITC channels. Fluorescence data of FtsZ-GFP (green) are shown. Arrows follow one cell throughout the experiment. Scale bar: 1 µm. (B) Quantification of cell lengths at all time points. Average cell length with error bars indicating standard deviation (n = 50) are shown. Please click here to view a larger version of this figure.
Figure 3: Time course investigation of cell division inhibition in S. aureus. (A) Mid-logarithmic phase cells of strain RB197 untreated (top) or treated (bottom) with FtsZ inhibitor (PC190723), subsequent to 30 min growth, aliquots of growing cultures were taken every 10 min for 90 min and imaged using DIC and FITC filter sets. Fluorescence from ZapA-GFP is shown. Scale bar: 1 µm. (B) Quantification of microscopy data. Average cell width with error bars indicating standard deviation (n = 50) and percentage of cells (n = 50) displaying proper ZapA-GFP localization (mid-cell and periphery) are shown. Data points of cells treated with inhibitor are shown in red. Shapes with green outline corresponds to the right Y-axis. Please click here to view a larger version of this figure.
Figure 4: Investigation of cell division inhibition by a synthetic inhibitor in B. subtilis. B. subtilis strain PE92 was either untreated or treated with an FtsZ inhibitor (PC190723) at mid-logarithmic phase and were monitored for the subsequent 90 min. Aliquots of growing cultures were taken every 10 min for microscopy and images were acquired using DIC/FITC channels. Fluorescence from FtsZ-GFP is shown. Scale bar: 1 µm. (B) Quantification of microscopy data. Average cell length with error bars indicating standard deviation (n = 50) and percentage of cells (n = 50) displaying proper mid-cell FtsZ-GFP localization are shown. Data points of cells treated with inhibitor are shown in red. Shapes with green outline corresponds to the right Y-axis. Please click here to view a larger version of this figure.
Video 1: Timelapse microscopy of S. aureus cells developing sick phenotype. Strain GGS8 (gpsB antisense) treated with 1% xylose. Cells were stained with FM4-64 membrane stain and imaged at 10 min intervals for 60 min using the TRITC channel as described in the protocol. Please click here to download this video.
Video 2: Overexpression of Sa-gpsB leads to inhibition of cell division in B. subtilis. Timelapse video showing filamentation and change in FtsZ-GFP localization in GG9. Images were taken at 20 min intervals for 120 min using DIC and FITC channels. Please click here to download this video.
Species | Strain | Genotype | Reference | |
S. aureus | RB143 | SH1000 pEPSA5, bla, cat | Eswara et al, 2018 | |
S. aureus | GGS8 | SH1000 pGG59 (pEPSA5 backbone) Pxyl-gpsBantisense, bla, cat | Eswara et al, 2018 | |
S. aureus | RB197 | SH1000 pRB42 (pJB67 backbone) PCd-zapASA-gfp, bla, cat | Eswara et al, 2018 | |
B. subtilis | GG9 | amyE::Phyperspank-gpsBSA spc; ftsAZΩftsAZ-gfp erm | Eswara et al, 2018 | |
B. subtilis | PE92 | ftsAZ::ftsAZ-gfp Ωerm | Brzozowski et al, 2019 |
Table 1: Strains used.
Microscopy has remained a mainstay in studies pertaining to microbial organisms. Given their micron-scale cell size, single-cell level studies have traditionally relied on electron microscopy (EM). Although EM has become quite a powerful technique in recent years, it has its own intrinsic limitations in addition to limited user access16. Improvements in fluorescence microscopy techniques and development of different fluorescent probes, such as FDAA3, have provided microbial cell biologists with a vast array of tools to study various cellular processes in live cells. Researchers are also actively building fluorescent tools to monitor changes, for example in the level of signaling molecules such as c-di-GMP among others, in living cells17,18. In addition, high-resolution timelapse fluorescence microscopy allows investigators to monitor changes as they happen and to study relevant phenotypes.
We have provided detailed protocols to conduct microscopy experiments with a high-resolution fluorescence microscope (see Table of Materials). However, the steps in the protocols could be altered to fit the needs of the user and the microscope used. We use S. aureus and B. subtilis as our model organisms to show how to monitor various cell division phenotypes, track the changes in protein localization, and quantify the data. In addition, for cases where timelapse is not conducive, we show with the help of an FtsZ inhibitor, how to set up a time course experiment.
The inherent limitation with fluorescence microscopy is the resolution set by the diffraction limit, which could be overcome to some extent with the aid of advanced super-resolution microscopy techniques19,20. Other issues such as phototoxicity and photobleaching could be circumvented by collecting fewer Z-stacks or minimizing the duration and/or frequency of exposure to laser. Other guidance materials specific to live-cell microscopy are available21. Apart from Gram-positive organisms B. subtilis and S. aureus, using this set up, we have successfully imaged Gram-negative bacterium Escherichia coli, yeast Saccharomyces cerevisiae, and nematode Caenorhabditis elegans.
In addition to the experiments described here, similar methodologies could be used to identify compounds that target specific cellular processes in a high-throughput fashion. Algorithms that automate the quantification process can also be incorporated for large datasets22,23. There is an immense need to study different bacterial species to address the antibiotic-resistance crisis and more studies are warranted to understand the mechanisms of basic biological processes and to identify novel therapeutic compounds. Various fluorescence microscopy techniques have gained the power and momentum to aid researchers in addressing these challenges among others.
The authors have nothing to disclose.
We thank our lab members for their comments on this article. This work was funded by a start-up grant from the University of South Florida (PE).
Agarose | Fisher BioReagents | BP160-100 | Molecular Biology Grade – Low EEO |
DAPI | Invitrogen | D3571 | Microscopy |
FM4-64 | Invitrogen | T3166 | Microscopy |
Glass bottom dish | MatTek | P35G-1.5-14-C | Microscopy |
IPTG | Fisher BioReagents | BP1755-10 | Dioxane-free |
Microscope | GE | DeltaVision Elite | Customized Olympus IX-71 Inverted Microscope Stand; Custom Illumination Tower and Transmitted Light Illuminator Module. Objectives: PLAPON 60X (N.A. 1.42, WD 0.15 mm); OLY 100X OIL (N.A. 1.4, WD 0.12 mm); DIC Prism Nomarski for 100X Objective; CoolSnap HQ2 camera; SSI Assembly 7-color; Environmental control chamber – opaque. |
PC190723 | MilliporeSigma | 3445805MG | FtsZ inhibitor |
SoftWorx | GE | Manufacturer-supplied imaging software |