Name: live-cell fluorescence microscopy to investigate subcellular protein localization and cell morphology changes in bacteria
Uploaded: 2019-11-23T14:00:00
Description: Watch this Scientific Journal Video about Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria at JoVE.com

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).

1. General growth conditions
<ol>
	<li>Inoculate 2 mL of appropriate growth medium supplemented with antibiotics (where required) with a single colony of the strain(s) to be imaged. Incubate these seed cultures overnight at 22 &#176;C in a shaking incubator. 
	NOTE: The specific bacterial growth conditions used in this article are provided under the representative results section.</li>
	<li>Dilute overnight cultures 1:20 in fresh media in a 125 mL flask, supplement with antibiotics (where required).</li>
	<li>Grow...

<ol>
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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...

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.

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Fisher BioReagents</td>
			<td>BP160-100</td>
			<td>Molecular Biology Grade - Low EEO</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T3166</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Fisher BioReagents</td>
			<td>BP1755-10</td>
			<td>Dioxane-free</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>GE</td>
			<td>DeltaVision Elite</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>PC190723</td>
			<td>MilliporeSigma</td>
			<td>3445805MG</td>
			<td>FtsZ inhibitor</td>
		</tr>
		<tr>
			<td>SoftWorx</td>
			<td>GE</td>
			<td></td>
			<td>Manufacturer-supplied imaging software</td>
		</tr>
	</tbody>
</table>

live-cell fluorescence microscopy to investigate subcellular protein localization and cell morphology changes in bacteria

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.

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 (P...

Watch this Scientific Journal Video about Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria at JoVE.com

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

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 ...

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