Comprehensive Proteomic Analysis of Nitrogen-Starved Mycobacterium smegmatisΔpupReveals the Impact of Pupylation on Nitrogen Stress Response

Pupylation is a bacterial ubiquitin-like protein modification pathway, which results in the attachment of the small protein Pup to specific lysine residues of cellular targets. Pup was shown to serve as a degradation signal, directing proteins toward the bacterial proteasome for turnover. Recently, it was hypothesized that pupylation and proteasomal protein degradation support the survival of Mycobacterium smegmatis(Msm) during nitrogen starvation by supplying recycled amino acids. In the present study we generated a Pup deletion strain to investigate the influence of pupylation on Msmproteome in the absence of nitrogen sources. Quantitative proteomic analyses revealed a relatively low impact of Pup on MsmΔpupproteome immediately after exposure to growth medium lacking nitrogen. Less than 5.4% of the proteins displayed altered cellular levels when compared to Msmwild type. In contrast, post 24 h of nitrogen starvation 501 proteins (41% of the total quantified proteome) of Msm pupdeletion strain showed significant changes in abundance. Noteworthy, important players involved in nitrogen assimilation were significantly affected in MsmΔpup. Furthermore, we quantified pupylated proteins of nitrogen-starved Msmto gain more detailed insights in the role of pupylation in surviving and overcoming the lack of nitrogen.