GlnR-Mediated Regulation of Short-Chain Fatty Acid Assimilation in Mycobacterium smegmatis

Abstract

Assimilation of short-chain fatty acids (SCFAs) plays an important role in the survival and lipid biosynthesis of Mycobacteria. However, regulation of this process has not been thoroughly described. In the present work, we demonstrate that GlnR as a well-known nitrogen-sensing regulator transcriptionally modulates the AMP-forming propionyl-CoA synthetase (MsPrpE), and acetyl-CoA synthetases (MsAcs) is associated with SCFAs assimilation in Mycobacterium smegmatis, a model Mycobacterium. GlnR can directly activate the expression of MsprpE and Msacs by binding to their promoter regions based upon sensed nitrogen starvation in the host. Moreover, GlnR can activate the expression of lysine acetyltransferase encoding Mspat, which significantly decreases the activity of MsPrpE and MsAcs through increased acylation. Next, growth curves and resazurin assay show that GlnR can further regulate the growth of M. smegmatis on different SCFAs to control the viability. These results demonstrate that GlnR-mediated regulation of SCFA assimilation in response to the change of nitrogen signal serves to control the survival of M. smegmatis. These findings provide insights into the survival and nutrient utilization mechanisms of Mycobacteria in their host, which may enable new strategies in drug discovery for the control of tuberculosis.

Nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis

“…4b-f. These results indicate that both nitrogen atoms from Gln and Asn are utilised by M. tuberculosis and, specifically that: (i) glutamate synthase is converting the δ-15 N from Gln into α-15 N-Glu (Fig 4b); this explains the incorporation of δ- 15 N from Gln into the α-15 N-Asp, via direct transamination from α-15 N-Glu ( Fig. 4d); (ii) direct transamination between α-15 N-Glu, the other product of the glutamate synthase reaction, and α-15 N-Asp is clearly observed (Fig.…”

“…Label incorporation from 15 N Glu, 15 N2-Gln, 15 N-Asp, 15 N2-Asn and 15 NH4Cl obtained under metabolic steady-state, over the course of 17 hours, revealed several important features of M. tuberculosis nitrogen metabolism, including different kinetics of 15 N labelling ( Fig. 4g, 3h, S4).…”

“…4a). The most direct chemical reactions producing 5 key amino acids (namely Glu, Gln, Asp, Asn and Ala) and the label incorporation data obtained from doubly and position-specific labelled 15 N-Gln and 15 N-Asn are shown in Fig. 4b-f.…”

“…Co-catabolism of carbon sources is a metabolic feature highly unusual in bacteria, which usually catabolise carbon sources sequentially, displaying biphasic (diauxic) growth kinetics. To investigate nitrogen source co-metabolism in M. tuberculosis, we grew cells in media containing the following combinations of nitrogen sources: 15 N-Glu+ 14 N-Gln, 14 N-Glu+U 15 N-Gln, 15 N-Asp+ 14 N-Asn, or 14 N-Asp+U 15 N-Asn. All nitrogen source combinations lead to robust labelling of Glu, Gln, Asp, Asn (Fig.…”

“…S1). The transcriptional factor GlnR does not perform canonical functions in mycobacteria 14,15 . Nitrogen utilisation by different mycobacterial species appears to be very different, not only due to inherent growth rate differences but also lag phase and final biomass achieved ( Fig.…”