Localization of EccA3 at the growing pole in Mycobacterium smegmatis

Abstract

Background: Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA₃) and five membrane components (EccB3 - EccE3 and MycP₃). In this study we sought to identify the sub-cellular location of EccA₃ of the ESX-3 secretion system in mycobacteria.

Results: Fluorescently tagged EccA₃ localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA₃, suggesting that EccA₃ unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA₃ associated proteins which may contribute to the localization of EccA₃ at the growing pole. EccA₃ co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified.

Conclusions: Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA₃ with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA₃ is located at the site of active cell growth.

Keywords: ESX-3; EccA3; Mycobacterium; Polar localization; Type VII secretion.

Fig. 1

Unipolar localization of EccA₃ in Mycobacterium smegmatis. Fluorescent microscopy demonstrated no GFP fluorescence of WT_MS (a) and ΔESX-3_MS (d) cells. GFP fluorescence was detected throughout the cell in positive control WT_MS::pDMNI (b) and ΔESX-3_MS::pDMNI (e) M. smegmatis. EccA₃ was found to reside at a single pole within WT_MS::pDMNI0615 (c) and ΔESX-3_MS::pDMNI0615 (f) cells

Fig. 2

Localization of EccA₃ within a population of Mycobacterium smegmatis. Three independent clonal populations were used for imaging flow cytometry experiments. a Imaging flow cytometry was used to demonstrate that more than 60% of clonal populations of WT_MS::pDMNI0615 and ΔESX-3_MS::pDMNI0615 had a GFP focus at least 0.5 μm from the mid-cell when compared to no GFP (WT_MS and ΔESX-3_MS) and GFP positive controls (WT_MS::pDMNI and ΔESX-3_MS::pDMNI). Unipolar localisation was significant following a Kruskal-Wallis test with a p-value of 0.0036 for both WT_MS and ΔESX-3_MS (indicated with **). b The majority of GFP positive WT_MS::pDMNI0615 and ΔESX-3_MS::pDMNI0615 cells with a fluorescent focus at least 0.5 μm from the mid-cell had only one fluorescent focus. c The histogram shows distance of a fluorescent foci from the mid-cell of populations WT_MS (red), WT_MS::pDMNI (green), WT_MS::pDMNI0615.1 (blue), WT_MS::pDMNI0615.2 (blue-purple), WT_MS::pDMNI0615.3 (purple). d The distance of fluorescent foci from the mid-cell of ΔESX-3_MS (red), ΔESX-3_MS::pDMNI (green), ΔESX-3_MS::pDMNI0615.1 (blue), ΔESX-3_MS::pDMNI0615.2 (blue-purple), ΔESX-3_MS::pDMNI0615.3 (purple) populations. A population with GFP fluorescence located at least 0.5 μm from the mid-cell, represented by the red line called Delta, was used to score the number of fluorescent foci per cell

Fig. 3

EccA₃ localizes at the growing pole in Mycobacterium smegmatis. a Time-lapse imaging of eight independent clonal populations demonstrated that EccA₃ localized at the old pole in 52 of the 73 cells imaged. b A single WT_MS::pDMNI0615 cell with a fluorescent focus at the pole (i, white arrow) can be seen dividing into two cells (ii) with a red arrow at the septum. A second fluorescent foci representing EccA₃-GFP can be seen at the old pole (iii, white arrow) before a third fluorescent foci (iv, white arrow) is detected following cell division (iv, red arrow at the septum). One of the fluorescent foci detected previously at the old pole (iii, white arrow) migrated to be polar adjacent (iv, white arrow)