Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics

Abstract

Background: Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acidassociated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA-and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results: AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification.Conclusions: Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA-and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase βsubunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.

Lsr2 and Its Novel Paralogue Mediate the Adjustment of Mycobacterium smegmatis to Unfavorable Environmental Conditions

“…These NAPs include HupB, mycobacterial integration host factor (mIHF), and Lsr2, which are structural or functional homologs of the wellknown Escherichia coli proteins HU, IHF, and histone-like nucleoid-structuring protein (H-NS), respectively; of them, some exhibit unique features (e.g., HupB, Lsr2) and/or are essential (e.g., mIHF) (7,8). Recently, two novel mycobacterial NAPs were identified; NapM is a conserved protein in mycobacteria, while MSMEG_1060 is a putative paralogue of Lsr2 that is found in M. smegmatis but absent from M. tuberculosis (9,10). While NapM has been relatively well characterized (9,11), almost no data are available regarding the function of MSMEG_1060, except that immunoprecipitation of FLAGtagged proteins suggested that it may be associated with the chromosome (10).…”

“…Recently, two novel mycobacterial NAPs were identified; NapM is a conserved protein in mycobacteria, while MSMEG_1060 is a putative paralogue of Lsr2 that is found in M. smegmatis but absent from M. tuberculosis (9,10). While NapM has been relatively well characterized (9,11), almost no data are available regarding the function of MSMEG_1060, except that immunoprecipitation of FLAGtagged proteins suggested that it may be associated with the chromosome (10).…”

Localization of EccA3 at the growing pole in Mycobacterium smegmatis

“…PCR amplified eccA 3 was cloned into the commercial plasmid CloneJet (ThermoFisher Scientific) prior to subcloning into pDMNI at restriction sites EcoRI and HindIII, in frame with 3′ gfp to create pDMNI0615. To identify possible interacting proteins of EccA 3 the plasmid pNFLAG0615 was constructed as described [ 49 ]. Gene sequence integrity of all plasmid constructs was verified using Sanger sequencing at the Central Analytical Facilities (CAF), Stellenbosch University, South Africa.…”

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

“…Here, chemical crosslinking and affinity purification of the RNA polymerase β-subunit led to the global identification of 275 transcription-associated proteins [ 153 ]. Of these proteins, 20 were not previously associated with nucleic acids [ 153 ]. The function of these transcription associated proteins could then be confirmed by coupling genetics and transcriptomics approaches as indicated above.…”

“…Recent work in M. smegmatis used an affinity purification-mass spectrometry (AP-MS) approach to globally identify transcription-associated proteins. Here, chemical crosslinking and affinity purification of the RNA polymerase β-subunit led to the global identification of 275 transcription-associated proteins [153]. Of these proteins, 20 were not previously associated with nucleic acids [153].…”