Dataset Information

Investigating the protein-protein interactions of EccA3 in Mycobacterium smegmatis

ABSTRACT: Mycobacterium smegmatis encodes three ESX secretion systems, ESX-1, -3 and -4. Previous research has suggested localisation of the ESX-1 secretion system at the growing polar region of M. smegmatis. In a seperate study, we demonstrated unipolar localisation of EccA3 from ESX-3 at the growing polar region of M. smegmatis. Here we demonstrate that affinity purification of N-terminally FLAG-tagged MSMEG_0615 (EccA3) followed by mass spectrometry resulted in the co-purification proteins previously identified in the membrane fractions of M. tuberculosis. Furthermore, we demonstrate that EccA3 co-purified with proteins known to localise at the growing polar region of asymmetrically growing M. smegmatis, including Wag31 and CwsA.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycobacterium Smegmatis (strain Atcc 700084 / Mc(2)155) Bacteria

DISEASE(S): Tuberculosis

SUBMITTER: Nastassja Kriel

LAB HEAD: Samantha Sampson

PROVIDER: PXD018978 | Pride | 2022-06-09

REPOSITORIES: Pride

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Localization of EccA<sub>3</sub> at the growing pole in Mycobacterium smegmatis.

Kriel Nastassja L NL Newton-Foot Mae M Bennion Owen T OT Aldridge Bree B BB Mehaffy Carolina C Belisle John T JT Walzl Gerhard G Warren Robin M RM Sampson Samantha L SL Gey van Pittius Nico C NC

BMC microbiology 20220519 1

<h4>Background</h4>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 (EccA<sub>3</sub>) and five membrane components (EccB3 - EccE3 and MycP<sub>3</sub>). In this study we sought to identify the sub-cellular location of EccA<sub>3</sub> of the ESX-3 secretio ...[more]

PMID: 35590245

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

			Action	DRS
	B1_0615_AB.raw	Raw
	B1_0615_MYH.raw	Raw
	B1_FLAG_AB.raw	Raw
	B1_WT_AB.raw	Raw
	B1_WT_noAB.raw	Raw