Nicotinamide adenine dinucleotide (NAD+) is an indispensable cofactor in all domains of life, the homeostasis of which requires tight regulation. Here we report that a Nudix-related transcriptional factor, designated MsNrtR (MSMEG_3198), controls the de novo pathway of NAD+ biosynthesis in M. smegmatis, a non-tuberculosis Mycobacterium. The integrated evidence in vitro and in vivo confirms that MsNrtR is an auto-repressor, and negatively controls the de novo NAD+ biosynthetic pathway. Binding of MsNrtR cognate DNA is finely mapped, which can be disrupted by an ADP-ribose intermediate. Unexpectedly, we discover that the acetylation of MsNrtR at Lysine 134 participates in the homeostasis of intra-cellular NAD+ level in M. smegmatis. Furthermore, we demonstrate that NrtR acetylation proceeds via the non-enzymatic acetyl-phosphate (AcP) route rather than the enzymatic Pat/CobB pathway. In addition, the acetylation of NrtR also occurs in its paralogs of Gram-positive bacterium Streptococcus and Gram-negative bacterium Vibrio, suggesting a common mechanism of post-translational modification in the context of NAD+ homeostasis. Together, it represents a first paradigm for the recruitment of acetylated NrtR to regulate bacterial central NAD+ metabolism.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Youjun Feng
- Youjun Feng
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Bavesh D Kana, University of the Witwatersrand, South Africa
© 2019, Gao et al.
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Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.
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