A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation

Rongsui Gao; Wenhui Wei; Bachar H Hassan; Jun Li; Jiaoyu Deng; Youjun Feng

doi:10.7554/eLife.51603

. 2019 Oct 9;8:e51603. doi: 10.7554/eLife.51603

A single regulator NrtR controls bacterial NAD⁺ homeostasis via its acetylation

Rongsui Gao ¹, Wenhui Wei ¹, Bachar H Hassan ², Jun Li ³, Jiaoyu Deng ⁴, Youjun Feng ^1,^5,^✉

Editors: Bavesh D Kana⁶, Gisela Storz⁷

PMCID: PMC6800001 PMID: 31596237

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) is an indispensable cofactor in all domains of life, and its homeostasis must be regulated tightly. 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, which negatively controls the de novo NAD⁺biosynthetic pathway. Binding of MsNrtR cognate DNA is finely mapped, and 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 by the enzymatic Pat/CobB pathway. In addition, the acetylation also occurs on the paralogs of NrtR in the Gram-positive bacterium Streptococcus and the Gram-negative bacterium Vibrio, suggesting that these proteins have a common mechanism of post-translational modification in the context of NAD⁺ homeostasis. Together, these findings provide a first paradigm for the recruitment of acetylated NrtR to regulate bacterial central NAD⁺ metabolism.

Research organism: Other

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is an indispensable cofactor of energy metabolism in all domains of life. It not only acts as an electron carrier in redox reactions (Belenky et al., 2007; Magni et al., 2004), but also functions as a co-substrate for a number of non-redox enzymes (DNA ligase [Wilkinson et al., 2001], NAD⁺-dependent de-acetylase CobB/Sir-2 [Schmidt et al., 2004] and ADP-ribose transferase [Domenighini and Rappuoli, 1996]). The intra-cellular level of NAD⁺ is dependent on the de novo synthesis pathway and/or its salvage or recycling route (Gazzaniga et al., 2009). Unlike NAD⁺ synthesis in eukaryotes, which begins with tryptophan as a primer (Kurnasov et al., 2003), NAD⁺ in most prokaryotes is produced de novo from the amino acid aspartate (Kurnasov et al., 2003). Also, certain species have evolved salvage pathway to produce NAD⁺ (Figure 1) by recycling its precursor metabolites ranging from nicotinic acid (Na) (Boshoff et al., 2008) to nicotinamide (Nam) (Boshoff et al., 2008) and nicotinamide riboside (RNam) (Rodionov et al., 2008a; Kurnasov et al., 2002).

Figure 1. — (A) The genetic context of *nrtR* and its signature in *Mycobacterium* compared with the NrtR-binding sequences in *Streptococcus* (B) and *Mycobacterium* (C). (D) NrtR acts as an auto-repressor and represses the transcription of the *nadA-nadB-nadC* operon that is responsible for the *de novo* synthesis of the NAD⁺ cofactor in *Mycobacterium*. (E) NAD⁺ homeostasis proceeds through cooperation of a salvage pathway with *de novo* synthesis in *Mycobacterium.* Designations: *nadA,* the gene encoding quinolinate synthase; *nadB,* gene encoding L-aspartate oxidase; *nadC*, gene encoding quinolinate phosphoribosyltransferase; PncA, nicotinamide deaminase; PncB, nicotinate phosphoribosyltransferase; NrtR, a bifunctional transcriptional factor involved in the regulation of NAD⁺ synthesis; ADP-R, ADP-ribose; Na, nicotinic acid; Nm, nicotinamide; Rib-P, ribose-5-phosphate; Asp, aspartate; NaMN, nicotinate mononucleotide; NAD⁺, nicotinamide adenine dinucleotide; CobB, an NAD⁺-consuming deacetylase.

Tight regulation of NAD⁺ homeostasis is needed to prevent the accumulation of harmful intermediates (Huang et al., 2009). In fact, three types of regulatory systems have been described for NAD⁺ biosynthesis and/or salvage. In addition to the two well-known regulatory proteins (NadR [Gerasimova and Gelfand, 2005; Raffaelli et al., 1999] and NiaR [Rodionov et al., 2008a]), a family of Nudix-related transcriptional regulators (NrtR) was initially proposed via bioinformatics (Rodionov et al., 2008b) and recently validated in Streptococcus suis (Wang et al., 2019). The paradigm NadR protein of Enterobacteriaceae is unusual in that it has three different functional domains (Grose et al., 2005): i) the N-terminal transcriptional repressor domain (Grose et al., 2005; Penfound and Foster, 1999); the central domain of a weak adenylyltransferase (Raffaelli et al., 1999; Grose et al., 2005), and the C-terminal domain of nicotinamide ribose kinase (Kurnasov et al., 2002; Grose et al., 2005). NadR is a NAD⁺ liganded regulator (Penfound and Foster, 1999), whereas NiaR is a nicotinic acid-responsive repressor in most species of Bacillus and Clostridium (Rodionov et al., 2008a). Although the prototypic NrtR possesses dual functions (Nudix-like hydrolase and DNA-binding/repressor) (Huang et al., 2009; Rodionov et al., 2008b), the NrtR homolog in S. suis seems to be an evolutionarily remnant regulator that lacks enzymatic activity (Wang et al., 2019). The phylogeny of NrtR suggests that it is widely distributed across diversified species (Huang et al., 2009; Rodionov et al., 2008b; Wang et al., 2019), and that its regulation of central NAD⁺ metabolism contributes to the virulence of an opportunistic pathogen, Pseudomonas aeruginosa (Okon et al., 2017).

Mycobacterium tuberculosis is a successful pathogen in that it exploits flexible metabolism to establish persistent infection within the host, resulting in the disease of tuberculosis (TB) (Bi et al., 2011; Shiloh and Champion, 2010). Together with an alternative salvage route, the de novo synthesis of NAD⁺ balances NAD⁺ metabolism (Boshoff et al., 2008; Bi et al., 2011) (Figure 1). An earlier microbial study by Vilcheze et al. (2005) indicated that the removal of ndhII, a type II NADH dehydrogenase-encoding gene, increases the intracellular NADH/NAD⁺ ratio, which results in phenotypic resistance to both the front-line anti-TB drug isoniazid (INH) and the related drug ethionamide (ETH). Subsequently, the de novo and salvage pathways of NAD⁺ have been proposed as potential targets for anti-TB drugs (Vilchèze et al., 2010). Lysine acetylation is an evolutionarily conserved, reversible post-translational modification in three domains of life (Weinert et al., 2013). In general, the acetyl moiety is provided via two distinct mechanisms: i) Pat-catalyzed acetylation (Starai and Escalante-Semerena, 2004) and CobB-aided deacetylation (Starai et al., 2002) with acetyl-CoA as the donor of the acetyl group; and ii) the non-enzymatic action of acetyl-phosphate (AcP) donated by glycolysis (Kakuda et al., 1994; Klein et al., 2007). Not surprisingly, the lysine acetylation is linked to central metabolism via acetyl-CoA synthetase (Xu et al., 2011) and the biosynthesis of siderophore, an intracellular iron chelator (Vergnolle et al., 2016) in Mycobacterium. A universal stress protein (USP) is acetylated with the cAMP-dependent Pat acetyltransferase (MSMEG_5458) in M. smegmatis (Nambi et al., 2010). Nevertheless, it remains largely unclear i) how the de novo NAD⁺ synthesis is regulated and ii) whether or not such regulation is connected with acetylation in Mycobacterium. Here, we report that this is the case. We illustrate a regulatory circuit of NAD⁺ homeostasis by NrtR in the non-tuberculosis relative, M. smegmatis. More importantly, we elucidate that a post-translational modification of NrtR, acetylation of K134 in the non-enzymatic AcP manner, is a pre-requisite for its regulatory role. This might represent a common mechanism that balances the central NAD⁺ metabolism.

Results

Discovery of NrtR in the context of the NAD⁺ biosynthetic pathway

Genome context analyses suggested that the genes that encode the enzymes involved in the initial three steps of NAD⁺ synthesis (nadA/nadB/nadC) are organized in a conserved manner as an operon and located adjacent to a Nudix related transcriptional regulator (nrtR) on the chromosome of Mycobacterium species (Figure 1A). We identified a 23-bp NrtR-binding palindrome conservatively located between the nrtR and nadA/B/C operons in mycobacteria (Figure 1C). The sequence of the NrtR-binding motif in Mycobacterium species [5′-GTTTTCGA-N7-TCGAAAAC-3′] is significantly different from that in Streptococcus [5′-ATA-N-TTTA-N3-TAAAA-N2-ATA-3′] (Wang et al., 2019) (Figure 1B). This may reflect the fact that these two NrtR homologs are not functionally exchangeable. Therefore, we anticipate that NrtR regulates NAD⁺de novo synthesis and coordinates it with the salvage pathway to maintain NAD⁺ homeostasis (Figure 1E). The most important clue is that de novo NAD⁺ synthesis is very conserved in different Mycobacterium species, enabling the use of Mycobacterium smegmatis as a model that can be used to study the regulatory mechanism for the de novo NAD⁺ synthesis pathway.

Phylogeny of NrtR

A maximum likelihood phylogenetic tree was constructed using 260 Nudix protein family representatives selected from diverse bacterial species (Figure 2A). The proteins carrying only a Nudix domain were removed, and 260 sequences coding for at least two protein domains were kept. Among these sequences, 38 with greater than 70% amino-acid identity were identified manually through literature mining and used for further analysis (Figure 2B). A common feature of the NrtR homologs is the invariant presence of the N-terminal Nudix domain (PF00293 or COG1051) fused with a characteristic C-terminal domain (PB002540), which is similar to the C-terminal part of proteins from the uncharacterized COG4111 family (Srouji et al., 2017). The data analyzed in this study suggest an evolutionary scenario for NrtR that includes the fusion of an ADPR-preferring Nudix hydrolase to a diversified DNA-binding domain. The phylogenetic groups found in this study show that these variable DNA-binding domains could result from the duplication of this domain and its subsequent substitution with a domain originating from a prototypical Nudix like Tlet_0901 in Thermotoga lettinagae (Zhaxybayeva et al., 2009). Moreover, some bacterial genomes encode many Nudix proteins. For example, two probable Nudix proteins were found in Mycobacterium (purple clade). Interestingly, these two homologs were distributed into different phylogenetic subgroups, indicating that they have distinct origins (Figure 2A).

Figure 2. — (A) The unrooted radial phylogeny of Nudix-like proteins. A variety of distinct subclades involve homologs containing a Nudix domain alone, a Nudix domain combined with DNA-binding domains or zinc finger domains, a Nudix domain combined with a CTP-transf (Cytidylyltransferase family) domain, Nudix+Nudix_N (Nudix located at N-terminal), or a Nudix pyrophosphate hydrolase with ADP-ribose substrate preference (YjhB, Nudix+YjhB superfamily). These distinct subclades seem to coincide with known taxonomic groups with few exceptions. NrtR candidates in *Mycobacterium*, *Vibrio* and *Streptococcus* species are indicated with purple, green and orange text, respectively. (B) Hierarchical tree of NrtR homologs. Several distinct sub-clades are clustered in a pattern that is generally consistent with bacterial taxonomic groups. The protein-sequence-based phylogeny of NrtR homologs was inferred using the maximum likelihood method and the WAG substitution model. The evolutionary distance for each node is shown next to the branches. Gene locus tags and strain names corresponding to the protein sequences used are indicated in the figure.

Binding of M. smegmatis NrtR to cognate DNA

Using the NrtR-DNA complex (PDB: 3GZ6) as a template, structural modeling allowed us to probe the interaction of MsNrtR with its cognate DNA targets (Figure 3A). In total, six residues in its DNA-binding domain (namely D167, T169, N170, R173, K179, and R196) were predicted to be crucial for its DNA-binding ability (Figure 3A–B). Prior to biochemical analyses, the recombinant form of MsNrtR was purified to homogeneity (Figure 3—figure supplement 1A), and validated with both a chemical cross-linking assay (Figure 3—figure supplement 1B) and mass spectrometry (Figure 3—figure supplement 1C).

Figure 3. — (A) Structural analysis of the predicted DNA-binding motif through structural modeling of *M. smegmatis* NrtR (http://swissmodel.espasy.org/). The image shows the superposition of *M. smegmatis* NrtR with the *Shewanella oneidensis* NrtR-DNA complex (PDB: 3GZ6). MsNrtR is highlighted in cyan and soNrtR is indicated in gray. Double-stranded DNA is denoted by two orange lines. (B) Structural prediction of the critical DNA-binding residues of the *M. smegmatis* NrtR. The six residues (D167, T169, N170, R173, K179, and R196) that are implicated in direct or indirect contact with cognate DNA are labeled in red. (C) Electrophoretic mobility shift assay (EMSA)-based visualization of the interaction of MsNrtR with the *nrtR* probe. The amount of NrtR protein incubated with the DNA probe is in each lane is (left to right): 0, 0.5, 1, 2, 5, 10, 20, and 40 pmol. (D) Surface plasmon resonance (SPR) measurements of *M. smegmatis* NrtR binding to the *nrtR* promoter. NrtR protein at various concentrations (typically 15.625–500 nM) were injected over the immobilized DNA probe comprising of the NrtR palindrome of *nrtR* gene. KD, kd/ka, ka, association constant; kd, dissociation constant; RU, response units.

Figure 3—figure supplement 1. — (A) Structural analysis of the predicted DNA-binding motif through structural modeling of *M. smegmatis* NrtR (http://swissmodel.espasy.org/). The image shows the superposition of *M. smegmatis* NrtR with the *Shewanella oneidensis* NrtR-DNA complex (PDB: 3GZ6). MsNrtR is highlighted in cyan and soNrtR is indicated in gray. Double-stranded DNA is denoted by two orange lines. (B) Structural prediction of the critical DNA-binding residues of the *M. smegmatis* NrtR. The six residues (D167, T169, N170, R173, K179, and R196) that are implicated in direct or indirect contact with cognate DNA are labeled in red. (C) Electrophoretic mobility shift assay (EMSA)-based visualization of the interaction of MsNrtR with the *nrtR* probe. The amount of NrtR protein incubated with the DNA probe is in each lane is (left to right): 0, 0.5, 1, 2, 5, 10, 20, and 40 pmol. (D) Surface plasmon resonance (SPR) measurements of *M. smegmatis* NrtR binding to the *nrtR* promoter. NrtR protein at various concentrations (typically 15.625–500 nM) were injected over the immobilized DNA probe comprising of the NrtR palindrome of *nrtR* gene. KD, kd/ka, ka, association constant; kd, dissociation constant; RU, response units.

In our gel shift assays, the nrtR probe refers to a DNA fragment that contains a putative NrtR-recognizable palindrome [5′-GTTTTCGA-N7-TCGAAAAC-3′] (Figure 1C). The electrophoresis mobility shift assay (EMSA) confirmed that MsNrtR binds specifically to the nrtR probe (Figure 3C), rather than to an irrelevant DNA probe (such as the promoter of vprA, which encodes a response regulator of V. cholerae, Figure 3—figure supplement 2). This binding appears to be protein-dose-dependent (Figure 3C), which is consistent with the scenario suggested by the assay of surface plasmon resonance (SPR). In addition, SPR evaluated the binding affinity of MsNrtR to the cognate DNA probe (i.e., KD, the equilibrium dissociation constant, was around 1530 nM, Figure 3D). Then, six point-mutant versions of MsNrtR (D167A, T169A, N170A, R173A, K179A and R196A; Figure 3—figure supplement 3A–B) were also subjected to EMSA-based functional assays. In contrast to the wild-type protein (Figure 3—figure supplement 3C), each of these MsNrtR mutants (D167A [Figure 3—figure supplement 3D], T169A [Figure 3—figure supplement 3E], N170A [Figure 3—figure supplement 3F], R173A [Figure 3—figure supplement 3G], R196A [Figure 3—figure supplement 3H], and K179A [Figure 3—figure supplement 3I]) consistently lost their DNA-binding ability in our gel shift assays. Therefore, these six residues are indispensable for the efficient binding of MsNrtR to the cognate target gene.

ADP-ribose disrupts interplay between NrtR and DNA

MsNrtR is an ADP-ribose pyrophosphohydrolase that belongs to the Nudix hydrolase family. Multiple sequence alignment revealed that the Nudix motif of NrtR is less conserved than is the traditional Nudix hydrolase (Figure 3—figure supplement 4A). Consistent with this alignment, we could not detect any apparent ADPR pyrophosphohydrolase activity in the presence of Mg²⁺ or Mn²⁺ (Figure 3—figure supplement 4E). ADP-ribose is an intermediary metabolite that is produced by glycol-hydrolytic cleavage of NAD⁺ (Figure 1E), and it has been considered a highly reactive and potentially toxic molecule (Jacobson et al., 1994). Moreover, ADP-ribose is also a putative messenger in both eukaryotes and prokaryotes (Huang et al., 2009; Rodionov et al., 2008b; Li et al., 1998; Heiner et al., 2006). Although MsNrtR has lost its catalytic activity as an ADP-ribose pyrophosphohydrolase, we hypothesized that it retains an ability to interact with ADP-ribose. As expected, we observed that 50 mM of ADP-ribose can significantly release MsNrtR from DNA (Figure 3—figure supplement 5). Obviously, ADP-ribose is a ligand for the MsNrtR regulator.

In vivo role of MsNrtR in NAD⁺ synthesis

The results of PCR combined with reverse transcriptional PCR (RT-PCR) proved that the three adjacent genes nadA, nadB and nadC are transcribed in an operon (Figure 4A). Subsequently, real-time quantitative PCR (RT-qPCR) demonstrated that the removal of nrtR provides a 2- to 6-fold increase of the expression of the nadA/B/C operon compared with that in the WT (Figure 4B). In accordance with the qRT-PCR results (Figure 4B), the levels of intracellular NAD⁺ and NADH in the ΔnrtR mutant are higher than those in the WT, suggesting that NrtR acts as a repressor for homeostasis of the NAD⁺(NADH) pool (Figure 4C–D). To test whether or not NrtR is an auto-regulator, we fused a promoter-less LacZ to the nrtR promoter, giving the nrtR-lacZ transcriptional fusion (Figure 4—figure supplement 1A). Consequently, we found that deletion of nrtR causes a dramatic increase in the LacZ activity of nrtR-lacZ on agar plates (Figure 4—figure supplement 1A). Subsequently, we selected bacterial cultures at different growth stages (lag phase, log phase, and stationary phase, in Figure 4—figure supplement 1B) to compare the β-gal level of the nrtR promoter (Figure 4—figure supplement 1C–E). Intriguingly, the amplitude for auto-repression of nrtR is constantly around 8- to 10-fold, regardless of the growth stage of the bacteria (ranging from lag phase in Figure 4—figure supplement 1C and mid-log phase in Figure 4—figure supplement 1D, to equilibrium stage in Figure 4—figure supplement 1E). Thus, we concluded that the auto-repressor, NrtR, negatively regulates the expression of nadABC to maintain the homeostasis of the NAD⁺(NADH) pool in Mycobacterium.

Figure 4. — (A) Genetic organization and transcriptional analyses of the *nrtR* and its neighboring *de novo* NAD⁺ synthesis genes. The arrows represent open reading frames, and the numbered short lines (1 to 7) represent the specific PCR amplicons that were observed in the following PCR and RT-PCR assays (in the bottom panels). PCR and RT-PCR were applied to analyze the transcription of the putative NAD⁺*de novo* synthesis loci. The primer numbering was identical to that shown in the top panel. CK (control) denotes the 16S rDNA. (B) RT-qPCR analyses of *nad* operon expression in the wild-type strain and in the Δ*nrtR* mutant and *nrtR* complementary strains. RT-qPCR experiments were performed at least three times and the data were expressed as means ± standard deviations (SD). The p-value was calculated using one-way ANOVA along with Tukey's test. *p<0.05 and **p<0.01. Comparison of the intra-cellular level of NAD⁺ (C) and NADH (D) among the WT, Δ*nrtR* and CΔ*nrtR* strains. Each dark circle or triangle represents an independent experiment. The data are shown as means ± SD. The statistical significance of differences among WT, Δ*nrtR* and CΔ*nrtR* was determined by Student’s t test and by ANOVA with heterogeneous variances. ***p<0.001; ns, no significant difference.

Figure 4—figure supplement 1. — (A) Genetic organization and transcriptional analyses of the *nrtR* and its neighboring *de novo* NAD⁺ synthesis genes. The arrows represent open reading frames, and the numbered short lines (1 to 7) represent the specific PCR amplicons that were observed in the following PCR and RT-PCR assays (in the bottom panels). PCR and RT-PCR were applied to analyze the transcription of the putative NAD⁺*de novo* synthesis loci. The primer numbering was identical to that shown in the top panel. CK (control) denotes the 16S rDNA. (B) RT-qPCR analyses of *nad* operon expression in the wild-type strain and in the Δ*nrtR* mutant and *nrtR* complementary strains. RT-qPCR experiments were performed at least three times and the data were expressed as means ± standard deviations (SD). The p-value was calculated using one-way ANOVA along with Tukey's test. *p<0.05 and **p<0.01. Comparison of the intra-cellular level of NAD⁺ (C) and NADH (D) among the WT, Δ*nrtR* and CΔ*nrtR* strains. Each dark circle or triangle represents an independent experiment. The data are shown as means ± SD. The statistical significance of differences among WT, Δ*nrtR* and CΔ*nrtR* was determined by Student’s t test and by ANOVA with heterogeneous variances. ***p<0.001; ns, no significant difference.

Acetylation of K134 in MsNrtR

Unexpectedly, Western blotting elucidated that the recombinant MsNrtR is constantly acetylated, regardless of whether Escherichia coli or M. smegmatis was used as the expression host (Figure 5A). To further consolidate this observation, the MsNrtR protein was subjected to peptide mass fingerprinting through LC/MS (LTQ Orbitrap Elite) analysis. Of the five acetylated lysine sites that we identified, the Lys134 (K134) acetylation site is highly conserved in NrtR homologs from different species (Figure 5B and Figure 5—figure supplement 1A). Structural analysis suggested that K134 is located at the junction between the N-terminal Nudix domain and the C-terminal HTH domain (Figure 5B and Figure 5—figure supplement 1A). This is distinct from the other six residues that have direct contact with cognate DNA (Figure 3B and Figure 3—figure supplement 3B). In addition, it seems likely that acetylation of K134 is a prevalent form because the K134A mutant protein cannot be acetylated efficiently (Figure 5C). Moreover, Western blot assays informed us that acetylation is present in the NrtR paralogs of Vibrio and Streptococcus (other than Mycobacterium, Figure 5—figure supplement 2).

Figure 5. — (A) Use of Western blot to probe the acetylation of recombinant MsNrtR protein in both *E. coli* and *M. smegmatis*. The two forms of recombinant NrtR protein were purified from *E. coli* BL21 and *M. smegmatis,* and analyzed by western blotting using both anti-acetyl-lysine antibody (α-Acetyl) and poly anti-MsNrtR rabbit serum. The bigger version of MsNrtR is produced by the pET28 expression plasmid in *E. coli*, whose N-terminus is fused to the 6xHis-containing tag of 23 residues (Supplementary file 1). By contrast, the smaller version of MsNrtR is generated by pMV261 in *M. smegmatis*, which is only tagged with C-terminal 6xHis. The altered molecular mass (~2 kDa) is the reason why the migration rate of protein electrophoresis differs slightly for the two MsNrtR versions. A representative result is given from three independent trials. (B) The discovery of a unique Lys134 acetylation site in MsNrtR. A LC/MS spectrum reveals that a charged peptide (LVAkLSYTNIGFALAPK) of MsNrtR bears an acetylated lysine (K134^Ac). The sequence depicted in the yellow box illustrates the K134 site of acetylation in the context of the modeled structure of MsNrtR-DNA. (C) The mutation of K134A results in reduced acetylation of MsNrtR in *M. smegmatis* MC²155 (Magni et al., 2004). A representative result from three independent experiments is given.

Figure 5—figure supplement 1. — (A) Use of Western blot to probe the acetylation of recombinant MsNrtR protein in both *E. coli* and *M. smegmatis*. The two forms of recombinant NrtR protein were purified from *E. coli* BL21 and *M. smegmatis,* and analyzed by western blotting using both anti-acetyl-lysine antibody (α-Acetyl) and poly anti-MsNrtR rabbit serum. The bigger version of MsNrtR is produced by the pET28 expression plasmid in *E. coli*, whose N-terminus is fused to the 6xHis-containing tag of 23 residues (Supplementary file 1). By contrast, the smaller version of MsNrtR is generated by pMV261 in *M. smegmatis*, which is only tagged with C-terminal 6xHis. The altered molecular mass (~2 kDa) is the reason why the migration rate of protein electrophoresis differs slightly for the two MsNrtR versions. A representative result is given from three independent trials. (B) The discovery of a unique Lys134 acetylation site in MsNrtR. A LC/MS spectrum reveals that a charged peptide (LVAkLSYTNIGFALAPK) of MsNrtR bears an acetylated lysine (K134^Ac). The sequence depicted in the yellow box illustrates the K134 site of acetylation in the context of the modeled structure of MsNrtR-DNA. (C) The mutation of K134A results in reduced acetylation of MsNrtR in *M. smegmatis* MC²155 (Magni et al., 2004). A representative result from three independent experiments is given.

As an important post-translational modification of protein, acetylation can adjust protein activity (e.g., DNA-binding) by affecting protein charge (and/or its conformation) in E. coli (Castaño-Cerezo et al., 2014; Li et al., 2010) and Salmonella (Sang et al., 2016; Sang et al., 2017; Ren et al., 2016). This prompted us to investigate the physiological role of K134 acetylation in MsNrtR. To mimic the non-acetylated form, K134 of MsNrtR was designed to mutate into arginine (R), glutamine (Q), or alanine (A), giving three mutants of MsNrtR protein (K134A, K134Q, and K134R, Figure 5—figure supplement 1B). Similar to the wild-type of MsNrtR, all of the mutant proteins can be purified to homogeneity and eluted at the position of dimer in our gel filtration (Figure 5—figure supplement 1B). This ruled out the possibility that the acetylation of K134 associated with the dimeric configuration of MsNrtR. However, the EMSA experiments verified that the DNA-binding abilities of the aforementioned three protein mutants (K134R [Figure 5—figure supplement 1D], K134Q [Figure 5—figure supplement 1E] and K134A [Figure 5—figure supplement 1F]) are impaired to varying degrees when compared with their parental version (Figure 5—figure supplement 1C–G). This hints at a possibility that an acetylation of K134 might have a physiological role in NAD⁺ synthesis.

Dependence of non-enzymatic AcP in MsNrtR acetylation

In general, the reversible acetylation of MsNrtR falls into one of two categories: enzymatic action by the acetyltransferase Pat and a non-enzymatic acetyl phosphate (AcP)-dependent m (Figure 6A) (Ren et al., 2016; Ren et al., 2019). Of note, both of these processes can be reversed by the deacetylase CobB (Figure 6A) (Ren et al., 2016; Ren et al., 2019). To address possible origin of K134 acetylation, we integrated an in vitro chemical assay and a genetic exploration in vivo (Figure 6B–I). To test the relevance of K134 acetylation to the enzymatic action of Pat/CobB, we deleted the pat (MSMEG_5458)/cobB (MSMEG_5175) gene (Figure 6A) from M. smegmatis by homologous recombination. As expected, the USP (universal stress protein, MSMEG_4207) is validated as the positive control that requires Pat for its enzymatic acetylation (Figure 6B). By contrast, we detected no difference in the acetylation levels of the MsNrtR proteins of WT, Δpat and ΔcobB (Figure 6C). This might rule out the possibility of Pat-catalyzed acetylation of MsNrtR. Then, we wondered whether or not K134 acetylation of MsNrtR proceeds via AcP-dependent route (Figure 6D–I). First, we established an in vitro system in which MsNrtR was incubated with AcP (Figure 6D–E), an intermediate product of glycolysis (Figure 6J). This showed clearly that the percentage of acetylated NrtR increased in a time-dependent manner (Figure 6D and F). This acetylation also occurred in an AcP-dose-dependent manner (Figure 6E and G). Obviously, these findings constitute in vitro evidence that AcP donates the acetyl group for the post-translational acetylation of NrtR (Figure 6D–G). Second, we secured in vivo evidence by knocking-out the AcP-pathway-encoding genes ackA and pta (Figure 6A and J). The acetylation level of MsNrtR in the double mutant of M. smegmatis (ΔackA+Δpta), is reduced 4–5-fold when compared with its parental strain (Figure 6H–I). This finding is similar to those of Weinert et al. (2013) working on NrtR acetylation in E. coli under different growth conditions (induced with glucose or acetate). In M. smegmatis, the removal of a single ackA only slightly repressed the acetylation of MsNrtR growing under inducing conditions of either 0.2% glucose or 1.0% acetate (Figure 6H–I). Along with the in vitro data, the in vivo evidence allowed us to conclude that the K134 acetylation of MsNrtR is physiologically dependent on the AckA/Pta-containing route for AcP formation (Figure 6J).

Figure 6. — (A) Genetic context of the two types of acetylation pathways in *M. smegmatis*. The two genes *pat* (MSMEG_5458) and *cobB* (MSMEG_5175) are responsible for the reversible enzymatic route of acetylation. The two loci *ackA* (MSMEG_0784) and *pta* (MSMEG_0783) participate in the non-enzymatic AcP pathway. Of note, *ackA* and *pta* are two overlapping loci that appear as an operon. (B) The acetylation levels of USP are dependent on the Pat/CobB-requiring enzymatic route in *M. smegmatis.* USP denotes the universal stress protein (MSMEG_4207). Using the recombinant plasmid pMV261-*usp*, the 6 × His tagged USP protein was expressed in wild-type *M. smegmatis* and its derivatives (Δ*cobB* and Δ*pat*). As a result, the acetylation levels of the purified USP proteins were detected with the pan anti-acetyl lysine antibody (α-Acetyl) and an anti-6 ×his antibody was used as a loading control. A representative result for three independent experiments displayed. (C) The acetylation levels of NrtR are not distinguishable in the three strains of *M. smegmatis* (wild-type, Δ*cobB* and Δ*pat*). 6 × His tagged MsNrtR was expressed in the three strains described for panel (B) using pMV261-*nrtR*. The acetylation levels of the purified MsNrtR proteins were determined using the α-Acetyl antibody, and anti-NrtR antiserum (α-NrtR) acted as a loading control. Western blots were conducted in triplicates. (D) Western-blot-based detection of the *in vitro* non-enzymatic acetylation of MsNrtR using AcP as the phosphate donor. Acetylation of MsNrtR by AcP (10 mM) was measured by incubating MsNrtR and AcP for 0, 0.5, 1, 2 and 4 hr at 37°C. The concentration of NrtR was determined by Western blot with anti-NrtR serum as a primary antibody (lower panel). (E) Acetylation of MsNrtR is AcP dose-dependent. MsNrtR was incubated with different levels of AcP (0, 2, 5 and 10 mM) for 2 hr at 37°C. (F) Altered acetylation of MsNrtR as incubation progresses over time with constant AcP. Acetylation was quantified using Image J software and normalized to the signal at 0 hr. (G) MsNrtR acetylation at various levels of AcP. Data were measured with Image J software and normalized to the signal at 0 mM AcP. Data are shown as mean ± standard deviation (SD). (H) *In vivo* evidence that the AcP pathway is associated with NrtR acetylation. In addition to the parental strain, a single mutant (Δ*ackA*) and double mutant (Δ*ackA*+Δ*pta*) were used to prepare the recombinant MsNrtR proteins with varied levels of acetylation. Of note, the bacterial growth conditions were supplemented with an inducer of 0.2% glucose or 1.0% acetate recommended by Weinert et al. (2013). The Western blot was performed as described for panels (B) and (C). Representative results of three or more independent experiments are shown. (I) Contribution of the AckA and Pta-requiring AcP pathway to NrtR acetylation. The acetylation signal was quantified using Image J software, and the density in the WT was normalized as 1. Each dot denotes a Western blot experiment. (J) Working model for non-enzymatic acetylation of MsNrtR in a metabolic context, and the working model for the enzymatic acetylation of USP. Abbreviations: MsNrtR, *M. smegmatis* NrtR; AcP, Acetyl-phosphate; AcAMP, Acetyl-AMP; Ac-CoA, acetyl-CoA; USP (MSMEG_4207), universal stress protein (130aa); Pta (MSMEG_0783), phosphate acetyltransferase (692aa); and AckA (MSMEG_0784), acetate kinase (376aa).

Physiological roles of K134 acetylation

To further investigate the in vivo role of K134 acetylation in the regulatory function of NrtR (Figure 7), we engineered M. smegmatis mutants carrying point-mutations of K134 (namely K134A, K134Q, and K134R) on chromosomal nrtR (Figure 5—figure supplement 3A–C). All the point-mutants of K134 were confirmed with direct DNA sequencing (Figure 5—figure supplement 3C). Also, Western blot was applied to prove that the mutated proteins are well expressed in vivo (Figure 5—figure supplement 3D). As expected, the removal of nrtR (positive control) gave around a ten-fold increase in the β-gal level of nrtR-lacZ transcriptional fusion (Figure 7A). In general agreement with the positive control, the K134Q mutation led to a three-fold upregulation in nrtR transcription (Figure 7A), Similar scenarios were also observed for the other two point-mutants, K134A and K134R, despite the lower level of (close to two-fold) of regulatory dysfunction in the control of nrtR transcription (Figure 7A). RT-qPCR assays showed that functional impairment of the K134 acetylation site can increase the transcriptional level of the nadABC operon 2–3-fold (Figure 7B). More importantly, the pool of intra-cellular NAD⁺in the K134 point-mutants accumulated to a level that was 2–3-fold that in the wild-type strain (Figure 7C). A similar observation occurs with NADH (Figure 7D).

Figure 7—figure supplement 1. — (A) Genetic assays for the auto-repression of *nrtR* using PnrtR-lacZ transcriptional fusion. These results suggest that functional impairments in K134 acetylation lead to de-repression of *nrtR*, as does the removal of *nrtR*. (B) RT-qPCR analyses of the transcription of the *nad* operon in the mutant carrying a point-mutation of K134 in *nrtR* (K134A, K134R and K134Q). Levels of intracellular NAD⁺ (C) and NADH (D) in the WT *nrtR* strain and its point-mutants (K134A, K134R and K134Q). All of the experiments were performed at least three times, and the data are presented as means ± SD. The p-values were calculated using one-way ANOVA along with Tukey's test.

Given that i) the K134 acetylation of NrtR involves the control of the cytosolic NAD⁺ pool (Figure 7) and ii) the acetylation of NrtR is AcP-dependent (Figure 6), we hypothesized that the AckA/Pta-including AcP pathway contributes to the NrtR-mediated regulation of the cytosolic NAD⁺ pool (Figure 8A). As anticipated, inactivation of the AcP pathway (especially in the Δpta+ΔackA double mutan having AcP level of ~10 μM, largely lower than that of the parental strain, ~460 μM) led to a significant increase in the β-gal level of nrtR-lacZ transcriptional fusion (Figure 8B). In particular, the cytosolic pools of both NAD⁺ (Figure 8C) and NADH (Figure 8D) were increased 2.5–3-fold in the double mutant (Δpta+ΔackA), which is deficient in the AcP pathway. Therefore, the data suggest that the AcP-dependent acetylation of K134 is necessary for NrtR to regulate the homeostasis of NAD⁺ in Mycobacterium.

Figure 8. — (A) Scheme for the maintenance of NAD⁺ homeostasis by AcP-dependent NrtR acetylation (B) The removal of *ackA* and *pta* from *M. smegmatis* increases the β-gal level of Pnrt-lacZ transcriptional fusion in the presence of 0.2% glucose in the growth medium. (C) The level of the cytosolic NAD⁺ pool is elevated in the double mutant of *M. smegmatis* (Δ*ackA* + Δ*pta*) in the growth condition with 0.2% glucose added. (D) The inactivation of the AcP path gives an increase of ~3 fold in the cytosolic NADH pool Three strains of *M. smegmatis* (WT, Δ*ackA*, and Δ*ackA* + Δ*pta*) were cultivated in 7H10 medium supplemented with 0.2% glucose. No less than three independent measures were carried out, and the values presented here are averages ± SD. *, p<0.001; ns, no significance.

Discussion

It has been estimated that 17% of the enzymes of central metabolism that are essential for the survival of M. tuberculosis require the NAD⁺ cofactor (Beste et al., 2007), regardless of the organism's state of latency or active-replication (Rodionova et al., 2014). It is rational that the NAD⁺ metabolic pathway and its regulatory mechanism have been recognized as an attractive target for the development of new anti-TB therapeutics (Rodionova et al., 2014). Although NrtR, as the third regulator of NAD⁺ metabolism (Rodionov et al., 2008b), has been described in vitro in different species such as Shewanella (Huang et al., 2009), the data we show here provide the first relatively full picture of the regulatory circuits of NAD⁺ synthesis involving NrtR, an evolutionarily distinct regulator, in a non-pathogenic M. smegmatis (Figure 1). Although it structurally comprises an N-terminal Nudix domain and a C-terminal Helix-Turn-Helix motif (Huang et al., 2009), MsNrtR retains only the ability to bind cognate DNA (Figure 3) and loses its ADP-ribose hydrolase activity (Figure 3—figure supplement 3). There are mutations at three residues (GX₅EX₇RQUXEKXDU) (Carreras-Puigvert et al., 2017) in the Nudix motif of MsNrtR that we attempted to reverse, but we were unable to recover the enzymatic function of the protein (Figure 3—figure supplement 4). It seems likely that natural selection/evolution has rendered the hydrolyzing ADP-ribose NrtR inactive in order to avoid functional redundancy. This prediction is in part (if not entirely) explained by the genome-wide distribution of 29 predicted proteins of the Nudix hydrolase family in M. smegmatis (Supplementary file 3).

A similar scenario in which the S. suis NrtR has an inactive Nudix hydrolase domain (Wang et al., 2019) allowed us to further hypothesize that most members of the Nudix-related regulator family might initially recruit ancient Nudix hydrolase as a signaling module, and then un-necessitate its enzymatic function while retaining its regulatory role as an evolutionary relic (Wang et al., 2019). The diversity of genomic organization of nrtR and its neighboring regulatory loci highlights that this gene is being subjected to dynamic domestication (Rodionov et al., 2008b). Evidently, the nrtR is integrated into the ‘nadR-pnuC-nrtR’ cluster in the human pathogen S. suis 2, assuring a regulated salvage/recycling pathway (Rodionov et al., 2008b; Wang et al., 2019). By contrast, the nrtR on the opposite strand is adjacent to an operon of nadA/B/C (the intergenic region of which contains a NrtR-recognizable site; Figures 1A and 4A), guaranteeing the control of de novo NAD⁺ synthesis (Figure 1D–E). Indeed, the NrtR is somewhat promiscuous because it modulates xylose (e.g., xylBAT of Bacteroides) and arabinose (e.g., araBDA of Flavabacterium) utilization in rare microorganisms (Rodionov et al., 2008b). Consistent with earlier descriptions (Rodionov et al., 2008b), the NrtR orthologs of S. suis (Wang et al., 2019) and M. smegmatis (Figure 3—figure supplement 5) proved to be antagonized by ADP-ribose in the binding to cognate DNA targets. By contrast, the NrtR (also named NdnR) of Corynebacterium glutamicum, a close relative of Mycobacterium, has surprisingly been found to exhibit more affinity to binding the DNA probe in the presence of NAD⁺ (Teramoto et al., 2012). This is probably explained by the varied configuration of signaling module within different NrtR orthologs. Because the NrtR of Pseudomonas participates in the fitness and virulence of this pathogen within mice (Okon et al., 2017), it is very interesting to wonder how NrtR and its regulated route of NAD⁺ synthesis contribute to the survival and chronic infection of Mycobacterium within the host environment.

Protein acetylation is a ubiquitous form of post-translational modification in prokaryotes (Ren et al., 2017), which is implicated in central metabolism and even bacterial pathogenicity (Ren et al., 2019; Sang et al., 2016). A global acetylome analysis of M. tuberculosis by Ge and coworkers (Liu et al., 2014) identified almost 137 unique acetylated proteins that are involved in diverse biological processes, some of which had undergone lysine acetylation. In this study, we are first to discover the lysine acetylation (K134) at the junction between the N-terminal Nudix domain and the C-terminal wHTH domain of MsNrtR (Figure 5). It is unusual, but not without any precedent. In fact, our research group very recently affirmed the presences of such a modification of K47 in the M. smegmatis BioQ that regulates biotin metabolism (Tang et al., 2014; Wei et al., 2018). Given that K134 is conserved in NrtR orthologs of different origins (Figure 7—figure supplement 1A), we hypothesized that it is a common hallmark for NrtR. However, this requires further experimental demonstration. Evidently, it is likely that a single lysine acetylation maintains the pools of two distinct cofactors (biotin and NAD⁺) by modifying a certain regulator. In light of the facts that i) mycobacterial NAD⁺ metabolism is regarded as an promising drug target (Bi et al., 2011; Rodionova et al., 2014) and ii) the synthesis and utilization of biotin is necessary for survival and infectivity of intracellular pathogens (Park et al., 2015; Bockman et al., 2015; Woong Park et al., 2011), we hypothesized that lysine acetylation plays an indispensable role in bacterial virulence. This is generally consistent with the K201 acetylation of PhoP, a response regulator of the two-component system in Salmonella virulence (Ren et al., 2016; Ren et al., 2019). Given that the acetylation of NrtR paralogs is also detected in two additional pathogenic species, the Gram-negative V. cholerae and Gram-positive S. suis (Figure 5—figure supplement 2), it is possible that the lysine acetylation of NrtR represents an evolutionarily conserved mechanism by which a group of pathogens develop successful infections within the nutrition-limited tough host niche.

In summary, the functional definition of K134 acetylation in MsNrtR updates our understanding of the homeostasis of NAD⁺, an indispensable coenzyme. This evidence provides an alternative paradigm for the development of anti-TB virulence lead drugs that can impair crosstalk between the nutritional/restricted virulence factor (NAD⁺) and NrtR by disrupting the acetylation-requiring regulatory system (Figure 8A).

Materials and methods

Bacterial strains, plasmids and growth conditions

The bacterial species used in this study include E. coli and M. smegmatis (Supplementary file 1). Strains were cultured as described previously (Tang et al., 2014; Wei et al., 2018). pET28a-nrtR and pMV261-nrtR were constructed as follows. nrtR was cloned into the pET-28a expression vector between BamHI and XbaI restriction sites. The resulting plasmid contained the nrtR gene fused to a hexahistidine-tag sequence at the N-terminus and was transformed into E. coli BL21(DE3) for heterologous expression of NrtR. The mycobacterial expression plasmid pMV261-nrtR was constructed by cloning the nrtR gene fused to a His-tag sequence at the C-terminus via BamHI and SalI sites. This recombinant plasmid was then electro-transformed into M. smegmatis MC²155 (Magni et al., 2004) for endogenous production of NrtR. Overlap PCR was utilized for site-directed mutagenesis of the nrtR gene using specific primers (Supplementary file 2).

Protein expression, purification and identification

Wild-type MsNrtR and its point mutants were overexpressed in E. coli or M. smegmatis MC²155 (Magni et al., 2004). The expression of proteins in E. coli (BL21) was induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Gao et al., 2017). For protein purification, the cells were harvested by centrifugation and lysed by sonication. The clarified lysate was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column (Qiagen) and eluted with 150 mM imidazole (Gao et al., 2017). The protein preparation was further purified by gel filtration through a Superdex 75 10/300 column (GL, GE Healthcare) and the protein purity was judged with 12% SDS-PAGE.

The recombinant MsNrtR expressed in E. coli and M. smegmatis was separated by SDS-PAGE gel and subjected to peptide mass fingerprinting with Liquid Chromatography (LC)-mass spectrometry (MS) (Wei et al., 2018; Gao et al., 2016a). The resultant polypeptides were separated by the EASY-nLC HPLC system (Thermo Scientific, USA) and detected using a Thermo Fisher LTQ orbitrap elite mass spectrometer (Thermo Scientific, USA). The MS spectrum containing the possible site of acetylation was detected and assigned by Mascot 2.2 (Ren et al., 2016). To further visualize the solution structure, 6 × His tagged MsNrtR protein was subjected to chemical cross-linking assays with the cross-linker of ethylene glycol bis-succinimidylsuccinate (Pierce) as we earlier described (Feng and Cronan, 2010).

Electrophoretic mobility shift assays

The interaction of MsNrtR with its DNA target was specified by gel shift assay (Gao et al., 2017). The DNA probe containing an MsNrtR-recognizable palindrome (designated nrtR) was generated by annealing two complementary primers (nrtR-probe-F and nrtR-probe-R) in TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl [pH 8.0]) (Gao et al., 2016b). The DNA probe was mixed with purified NrtR protein in EMSA buffer (50 mM Tris-HCl [pH 7.5]; 10 mM MgCl₂; 1 mM DDT; 100 mM NaCl) and incubated at room temperature for about 30 min (Wei et al., 2018). The DNA–protein complexes were separated on native 8% native polyacrylamide gels, and the shifted DNA bands were visualized by staining with ethidium bromide (EB) (Gao et al., 2017).

Surface plasmon resonance

To evaluate the parameters of binding between NrtR and its DNA target, surface plasmon resonance (SPR) was employed using a Biacore3000 instrument (GE Healthcare) at 25°C. A biotinylated nrtR probe was injected onto the flow cells of a SA sensor chip at a flow rate of 10 μl/min until the calculated amount of DNA had been bound, giving a 34 RU maximum. All of the SPR experiments were conducted in the running buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.005% (v/v) Tween 20) at a flow rate of 30 μl/min (Gao et al., 2017). A series of dilutions of protein samples were injected and passed over the chip surface for 2 min. The dissociation phase was followed for 3 min in the same buffer, and the surface was then regenerated with 0.025% SDS for 24 s. Kinetic parameters were analyzed using a global data analysis program (BIA evaluation software).

Assays for β-gal activity

Transcriptional levels were measured using lacZ transcriptional fusions carried on plasmid pMV261 in M. smegmatis (WT and ΔnrtR). Cells from log-phase cultures grown in LB broth containing 0.2% glycerol, 0.05% Tween-80, and 50 μg/ml kanamycin were collected by centrifugation, washed twice with RB medium supplemented with 0.05% Tween-80. and re-suspended in Z-buffer for measurement of β-gal activity (Miller, 1992; Feng and Cronan, 2009a; Feng and Cronan, 2009b). Data were obtained from three independent trials and presented as a means and standard deviations (SD).

Generation of chromosomal knock-out and knock-in strains

The knock-out and knock-in strains of the M. smegmatis nrtR (MSMEG_3198) gene were generated using the homologous recombination method as described before (Tang et al., 2014; Yang et al., 2012). A suicide plasmid was constructed by cloning the knock-out or knock-in fusion PCR products (Supplementary file 1) into pMind between NheI and PacI, followed by the insertion of a sacB-lacZ cassette as a selection marker at the PacI site (Supplementary file 1). The recombinant plasmid was electroporated into competent cells of M. smegmatis (wild-type or nrtR deletion mutant) and plated on LB medium containing 100 μg/ml X-gal and 50 mg/L kanamycin for screening of single-crossover mutant strains. Single colonies were picked and inoculated into kanamycin-free LB broth, 37°C, 220 rpm for 24 hr. The incubated culture was plated on LB medium containing 100 μg/ml X-gal and 10% sucrose. The white colonies representing allelic-exchange mutants were picked and identified by multiplex-PCR and direct DNA sequencing. The mutants of M. smegmatis include an in-frame deletion mutant (ΔnrtR) and three point-mutants of K134 on the chromosomal nrtR (namely K134A, K134R, and K134Q, in Supplementary file 1). A similar approach was applied to delete the four acetylation pathway genes in M. smegmatis, namely pat (MSMEG_5458), cobB (MSMEG_5175), ackA (MSMEG_0784), and pta (MSMEG_0783). The resultant mutants denote three single mutants (Δpat, ΔcobB, and ΔackA) and a double mutant (ΔackA+Δpta) (Supplementary file 1).

Western blot

Polyclonal anti-serum against MsNrtR was generated by immunizing a rabbit with purified MsNrtR as shown recently (Gao et al., 2017). The specificity and sensitivity of the acquired polyclonal antibody was evaluated by western blot and ELISA with pre-immune sera used as a negative control. Subsequently, western blot was conducted routinely (Gao et al., 2017). To probe the possible acetylation of the MsNrtR, the anti-acetyl-lysine antibody (Abcam, ab61257) acted as primary antibody. To normalize the relative level of NrtR acetylation, the NrtR concentrations were measured with western blot in which an anti-MsNrtR polyclonal serum was introduced as a primary antibody, as described recently but with minor alteration (Ren et al., 2016).

Non-enzymatic acetylation of NrtR in vitro

The MsNrtR protein (10 μg) was incubated with different concentrations of acetyl phosphate (AcP) in Tris-HCl buffer (50 mM [pH 8.0] containing NaCl (150 mM) at 37°C for 2 hr; Weinert et al., 2013; Wang et al., 2017). Following the separation of the reaction mixture by SDS-PAGE (12%), the acetylated form of MsNrtR protein was detected using western blot with an anti-acetyl-lysine antibody (Abcam, ab61257).

Determination of intracellular NAD⁺ and NADH concentrations

Prior to the quantification of the bacterial NAD⁺/NADH pool, cell counts per optical density at wavelength 600 (OD600) were determined via bacterial plating. In brief, the log-phase cultures of M. smegmatis (wild-type and its mutants) were adjusted to OD600 of 1.0, and serially diluted in 10⁵-folds with fresh 7H9 broth. The resultant bacterial suspension (100 μl) was plated on LB medium, and kept for around three days at 37°C. Finally, colony counting (each at OD600) was determined to be about 1.0 × 10⁸ CFU/ml (Figure 7—figure supplement 1). The intracellular level of both NAD⁺ and NADH was determined using a NAD⁺/NADH kit (Sino Best Biological Technology). Wild-type and mutant strains were collected when their OD₆₀₀ reached around 1.0 (about 1.0 × 10⁸ CFU/ml, in Figure 7—figure supplement 1). Aliquots of bacterial cultures (2–4 ml) were harvested by centrifugation at 4°C for 10 min at 4000 rpm. After discarding the supernatant, the bacterial pellets were washed twice with 1 ml fresh 7H9 medium. Then NAD⁺ and NADH concentrations in bacterial pellets were extracted and calculated as recommended by the manufacturer.

RNA isolation, RT-PCR and real-time quantitative RT-PCR

Mid-log phase cultures of M. smegmatis and its mutants grown in LB media or 7H9 media were collected for total bacterial RNA preparations. TRIzol reagent (Life Technologies) was used to isolate total RNA. The RNA quality was detected to avoid trace genomic DNA contamination (Feng and Cronan, 2010; Feng and Cronan, 2009b). First-strand cDNAs were synthesized using a PrimeScript RT reagent Kit with gDNA Eraser (Takara). The final cDNAs were diluted and served as the template for PCR amplification of the nad operon-related DNA fragments using specific primers (Supplementary file 2). Real-time PCR analysis was performed using SYBR Green Master Mix Reagent (Takara). The 16S rDNA gene served as internal reference and the relative expression levels were calculated using the 2^-∆∆CT method (Livak and Schmittgen, 2001).

Enzymatic assays

The Nudix hydrolase activities of NrtR and NrtR^{Q54E&K58E&D60E} were purified to homogeneity. The reaction mixture (150 μl) contained 50 mM HEPES (pH 8.2), together with either 5 mM MgCl₂ or 0.2 mM ADP-ribose and an appropriate amount of NrtR_ms or NrtR^{Q54E&K58E&D60E}. After 30 min incubation at 37°C, the reaction was stopped by adding 75 μl of cold 1.2 M HClO₄. Reaction products were assayed using HPLC with a column (DiKMA C18-T, 4.6 × 250 mm, 5 μm particle size) at 16°C. The elution conditions were the same as those used before (Wang et al., 2019).

Phylogenetic analysis

NrtR proteins were collated from Vibrio cholerae, Mycobacterium smegmatis and Streptococcus suis. BLASTp (Johnson et al., 2008) was used to identify homologs with identity 30% and coverage 30% as cut-offs. 750 homologs of NrtR were found and manually curated in the 9434 RefSeq-archived genomes as of April, 2018, including bacterial and archaeal sequences. To verify the Nudix domain in these homologs, the Protein Families database (Pfam, available at pfam.xfam.org) and the Clusters of Orthologous Groups (COG, available at ncbi.nlm.nih.gov/COG) database were used to identify conserved functional domains (Tatusov et al., 2000). Next, protein homologs were further filtered by examining their conserved domain. The proteins carrying only the Nudix domain were removed, and 260 sequences coding for at least two protein domains were kept (Unrooted phylogeny). Subsequently, 38 sequences were further analyzed on the basis of 70% identity at amino-acid sequence level by manual collation (Hierarchical tree). Multiple sequence alignments of protein sequences were produced by the Clustal W program (Chenna et al., 2003). The MEGA software was used for construction of a maximum likelihood phylogenetic tree for the NrtR protein family, including bootstrapping with 1000 replicates and drawing of a consensus tree. The number of the corresponding phylogenetic clade reflects the putative evolutionary distance for each node.

Bioinformatics

The protein sequences of MsNrtR and its homologs from different species were aligned by Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/), and the final output of the multiple sequence alignments was given processed by the program ESPript 2.2 http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Structural modeling for the MsNrtR-DNA was processed using Swiss-Model with SoNrtR-DNA as a structural template (PDB: 3GZ6). The resultant result was given in ribbon structure via PyMol (https://pymol.org/2).

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31830001, 81772142 and 31570027, YF) and the National Key R & D Program of China (2017YFD0500202, YF). Dr Feng is a recipient of the national ‘Young 1000 Talents’ Award of China.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Youjun Feng, Email: fengyj@zju.edu.cn.

Bavesh D Kana, University of the Witwatersrand, South Africa.

Gisela Storz, National Institute of Child Health and Human Development, United States.

Funding Information

This paper was supported by the following grants:

National Natural Science Foundation of China 31830001 to Youjun Feng.
National Natural Science Foundation of China 81772142 to Youjun Feng.
National Natural Science Foundation of China 31570027 to Youjun Feng.
Ministry of Science and Technology of the People's Republic of China 2017YFD0500202 to Youjun Feng.
Thousand Talents Plan to Youjun Feng.

Additional information

Competing interests

No competing interests declared.

Author contributions

Data curation, Formal analysis, Investigation, Methodology, Writing—original draft.

Data curation, Software, Formal analysis.

Resources, Software, Formal analysis, Visualization, Methodology, Writing—review and editing.

Data curation, Software, Formal analysis, Visualization, Methodology.

Resources, Software, Formal analysis, Visualization, Methodology, Writing—review and editing.

Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing.

Additional files

Supplementary file 1. Strains and plasmids used in this study.

elife-51603-supp1.doc^{(116KB, doc)}

DOI: 10.7554/eLife.51603.020

Supplementary file 2. Primers used in this study.

elife-51603-supp2.doc^{(87KB, doc)}

DOI: 10.7554/eLife.51603.021

Supplementary file 3. Nudix family protein in M. smegmatis MC² 155 (Magni et al., 2004).

elife-51603-supp3.doc^{(60KB, doc)}

DOI: 10.7554/eLife.51603.022

Transparent reporting form

elife-51603-transrepform.docx^{(245.9KB, docx)}

DOI: 10.7554/eLife.51603.023

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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eLife. doi: 10.7554/eLife.51603.025

Decision letter

Editor: Bavesh D Kana¹

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "A Single Regulator NrtR Maintains Bacterial NAD⁺ Homeostasis via Its Acetylation" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The reviewers agreed that the study of NAD biosynthesis and its regulation represents and important, broad reaching area of bacterial metabolism. In general, the experiments are carefully conducted and presented well. However, The role of acetylation of NrtR in regulation of NAD biosynthesis remains unclear in vivo. Also there is insufficient evidence presented that the acetyl donor for this process is acetyl-phosphate in vivo. These, and other shortfalls mentioned by the reviewers, weaken the manuscript and cast doubt on the conclusions

Reviewer #1:

In most prokaryotes, NAD⁺ is produced either by de novo biosynthesis from tryptophan or through salvage of NAD metabolites. These biosynthetic processes are regulated by three distinct mechanisms, one of which include the activity of a family of Nudix-related transcriptional regulators (NrtRs). The authors set out to characterise this mode of regulation in mycobacteria.

Key findings:

1) Using computational approaches, the authors identify an NrtR binding palindromic site proximal to the locus encoding the first three enzymes in the NAD⁺ biosynthesis pathway. This site is located between the nrtR gene and the nadA/B/C operon.

2) They describe the phylogenetic distribution of NrtR-like homologues and highlight some differences in domain organisation and possible functional consequences. This is followed by homology modelling of NrtR bound to DNA and description of the resiu.

3) They purify the Mycobacterium smegmatis NrtR (confirmed by SDS-PAGE, chemical cross-linking and MS) and demonstrate that it binds in a concentration-dependent manner to the putative regulatory sequence. They further construct six point mutations and demonstrate that these residues are individually essential for NrtR DNA binding activity.

4) They demonstrate that ADP-ribose, a toxic breakdown product of NAD⁺, inhibits NrtR DNA binding activity, although, NrtR does not have an ADP-ribose pyrophosphohydrolase activity.

5) A mutant lacking NrtR displayed increased expression of nadA and elevated levels of NAD⁺/NADH – confirming NrtR as a negative repressor of NAD biosynthesis, regardless of growth phase. NrtR also autoregulates its own biosynthesis.

6) The authors demonstrate that NrtR is acetylated, predominantly at the K134 residue, mutagenesis of this residue to remove the acetylation effect resulted in variances in DNA binding – not sure what this means, not quantified by SPR, as with their earlier experiment.

7) Acetylation by a non-enzymatic acetyl phosphate-dependent mechanism seems to be responsible for acetylation.

8) in vivo, acetylation was required for NrtR-dependent regulation of the nadABC operon and NAD+/NADH biosynthesis.

Generally the study is conducted well, with a careful experimental approach. The following should be addressed.

1) Much of the conclusions described in the bioinformatics sections about domains and putative function appear to be speculation as there are no references to experimental validation. This section should be shortened.

2) All electrophoretic gel mobility assays are missing cold competitive controls. This is necessary to ensure specificity and should be done.

3) For Figure 5—figure supplement 1, some graphic representation of the westerns is required to make comparisons. Density scans of the bands should be carried out over multiple experiments.

4) Whilst not essential to the entire story, the dimerization of acetylation defective mutants should have been assessed. Perhaps acetylation affects protein-protein interactions.

Reviewer #2:

The regulation of NAD biosynthesis in bacteria occurs through the NrtR and/or NadR regulators. The NrtR transcriptional regulator regulates the expression of the NAD biosynthetic genes in organisms such as Corynebacterium. In mycobacteria there are both NadR and NrtR homologs, depending on the species and the regulation of mycobacterial NAD biosynthesis by NrtR has been discussed based on work done in other bacteria (see Bi et al., 2010. In this work the authors confirm that NrtR regulates expression of the nad genes in M. smegmatis and that, similar to the situation in some NrtR homologs, ADP-ribose binds to the protein, affecting DNA binding, but is not hydrolyzed by it. Deletion of the nrtR homolog in M. smegmatis modestly affects NAD(H) and nadA transcript levels. The really novel aspect of this work is the finding that recombinantly expressed protein is acetylated. The acetyl donor is speculated to be acetyl-phosphate but there is little evidence to support this except for the fact that this reactive metabolite, at high concentrations, can acetylate to proteinin vitro. The role of acetylation is speculated to be regulation of protein binding but it should be noted that there is no evidence provided in this work that protein acetylation is ever regulated. Recombinantly expressed protein is always found to be acetylated whereas mutants of the reactive lysine, although they seem to bind cognate DNA in EMSA experiments (contrary to what the authors state), seem to be inactive in cells returning levels of transcript and NAD(H) to that found in knockout mutant cells.

Overall, the experiments are elegantly performed with well-done biochemistry. It certainly is interesting that the protein is always acetylated but the relevance of this acetylation to regulation of NAD biosynthesis is not known.

Reviewer #3:

Goal: demonstrate that the Nudix-related transcriptional regulator NrtR (MSMEG_3198) controls NAD homeostasis in M. smegmatis via acetylation of a lysine group at position 134.

The authors found a 23 bp palindromic sequence between nrtR and nadABC, assert that it binds to NrtR yet do not shown evidence to support this claim.

The authors constructed a phylogenetic tree of 260 Nudix protein family representatives including only 2 Nudix proteins from M. smegmatis. It would have been useful to include the Nudix proteins from other mycobacterial species such as M. tuberculosis, M. avium, M. leprae, or M. marinum.

The authors tested whether NrtR had the predicted ADP-ribose pyrophosphohydrolase activity but could not detect any.

The authors constructed a nrtR KO in M. smegmatis and showed by RT-PCR that deletion of nrtR increased nadA expression by 2-6 fold. The authors also measured NAD+ and NADH in wt and nrtR KO strains and observed less than 2-fold increase in the cofactor levels. This experimental design lacks the requisite precision to draw a meaningful conclusion as they reported levels of NAD+ and NADH as pmol/106 CFU but they did not plate for CFUs in their experiment. They only estimated CFUs based on OD while admitting that there could be a 2-fold variation in their CFU estimation. Therefore, the results cannot support the conclusion that NrtR regulates NAD+ and NADH levels. Additionally, nrtR deletion does not modify the NADH/NAD+ ratio as both cofactors are altered at the same rate further indicating that NrtR has no role in maintaining the redox status of mycobacterial cells.

The authors demonstrated that M. smegmatis NrtR K134 lysine residue is acetylated. They constructed M. smegmatis mutants where the lysine residue was replaced by an alanine, a glutamine or an arginine group. These mutants had similar phenotypes as the nrtR KO strain. The authors concluded that "acetylation of K134 is a prerequisite for NrtR to regulate homeostasis of NAD+ in Mycobacterium". This conclusion is not substantiated by the data provided.

The authors should have compared the data obtained from this study with NadR, a known regulator of NAD biosynthesis. I would not recommend this for publication.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Congratulations, we are pleased to inform you that your article, "A single regulator NrtR controls bacterial NAD⁺ homeostasis via its acetylation", has been accepted for publication in eLife.

Prokaryotic NAD+ biosynthesis is regulated by three distinct mechanisms, one of which includes the activity of a family of Nudix-related transcriptional regulators (NrtRs). Your work describes the role of NrtR in modulating the activity of the biosynthetic operon for NAD+ biosynthesis in mycobacteria. The finding that NrtR is acetylated by acetyl-phosphate and that this acetylation status affects NAD+ homeostasis is novel and has important implications for studies on NAD+, and related cofactor, biosynthesis in other prokaryotes. Metabolic enzymes/pathways in bacteria are fast gaining traction as novel drug targets, and how these pathways are regulated is central to the discovery of new antimicrobials. Hence, your work also has important implications for bacterial, target-driven drug discovery approaches. Further dissection of the interplay between this regulatory mechanism and other modalities of NAD biosynthesis should form an important component of future work in this area of bacterial metabolism.

Reviewer #1:

The revised manuscript by Gao and colleagues attempts to improve upon the earlier submission and address concerns that were raised during review. This manuscript reports a study that was aimed to investigate the regulation of NAD⁺ biosynthesis in bacteria, with a focus on the Nudix-related transcriptional regulators (NrtRs). In mycobacteria, the authors reported the presence of palindromic binding sites for a regulator upstream of the nadA/B/Coperon and demonstrated that the regulator, NrtR, bound this in a somewhat sequence specific manner that is affected by ADP-ribose, a toxic breakdown product of NAD⁺. The authors also demonstrated that NrtR is acetylated by a non-enzymatic acetyl phosphate-dependent mechanism. The specific concerns that were raised and how these have been addressed are outlined below.

1) Long and complicated exposition of bioinformatics: This has been shortened and is more succinct. In general, some of the writing still needs attention. Tenses are mixed up. In the Results section and in the Abstract, results need to be reported in the past tense. The phyologenetics has also been appropriately revised.

2) EMSA controls: They attempted to address this through adding non-specific controls. This is acceptable but needs to be displayed with the specific positive controls on the same gel. Figure 3—figure supplement 2 needs to be amended to add a lane of specific DNA, or it should be removed. Binding of NrtA to its own promoter is now convincingly shown in Figure 3—figure supplement 3C.

3) Quantification of westerns: This is addressed in an acceptable manner, please add statistics to the figure.

4) Dimerization status of mutant proteins: Addressed.

5) High concentrations of ADP-ribose: The authors make a compelling argument, based on evidence from the literature.

6) Acetylation (mechanism and role in vivo): This was a substantive comment from all reviewers. The authors have attempted to address. They mutate the enzymatic mechanism of acetylation through deletion of the AcP pathway in Mycobacterium smegmatis and confirm acetylation status. The evidence is not definitive but does bring greater clarity. The conclusions regards acetylation could be stated more carefully, in cognisance of the knowledge gaps that still exist.

Language has improved substantively but it can still be polished further. The last sentence of the Abstract is not clear. What does "it" refer to? Most likely the mechanism of regulation but a more careful statement of the overall conclusions is required. Another round of careful reading and editing should address these minor issues.

Reviewer #2:

The authors have justified their conclusion that levels of acetylation of NtrR by acetyl-phosphate affect NAD homeostasis in Mycobacterium. Thus, the finding that NtrR is acetylated and that the level of acetylation modestly affects transcription of the NAD biosynthetic genes and NAD levels is novel and an important finding.

eLife. 2019 Oct 9;8:e51603. doi: 10.7554/eLife.51603.026

Author response

In this revision, we have invited an expert in Mycobacteriumacetylation, Prof. Jiaoyu Deng to help improve this manuscript. More importantly, we have addressed all the questions appropriately, modified the manuscript extensively, and added all the new data (in vitro and in vivo) requested by three referees.

In brief, all the new data are included as follows: 1) phylogenetic tree has been replaced, including NrtR homologs of different Mycobacterium species; 2) the negative control of EMSA has been provided; 3) we added the relative quantification results of NrtR mutants in the altered DNA-binding ability; 4) we provided our platting data used to measure CFU, which is helpful in declaring the misunderstanding of referee 3 on this issue; 5) we expanded the generality of NrtR acetylation by examining NrtR from two more microbes, Gram-negative Vibrio cholerae and Gram-positive Streptococcus suis); 6) we constructed mutants of M. smegmatis whose AcP route is inactivated, namely a single mutant of ΔackA and a double mutant of ΔackA Δpta. As Weinert et al stated (Weinert et al., 2013), we found that the NrtR acetylation is significantly impaired in the double mutant on the certain growth condition (such as the addition of exogenous acetate or glucose, Figs 6H and I). It constitutes in vivo evidence that NrtR is acetylated via Acp-dependence (Figures 6H and I), rather than the enzymatic action of Pat/CobB (Figures 6B-G).

Reviewer #1:

In most prokaryotes, NAD+ is produced either by de novo biosynthesis from tryptophan or through salvage of NAD metabolites. These biosynthetic processes are regulated by three distinct mechanisms, one of which include the activity of a family of Nudix-related transcriptional regulators (NrtRs). The authors set out to characterise this mode of regulation in mycobacteria.

Key findings:

1) Using computational approaches, the authors identify an NrtR binding palindromic site proximal to the locus encoding the first three enzymes in the NAD+ biosynthesis pathway. This site is located between the nrtR gene and the nadA/B/C operon.

2) They describe the phylogenetic distribution of NrtR-like homologues and highlight some differences in domain organisation and possible functional consequences. This is followed by homology modelling of NrtR bound to DNA and description of the resiu.

3) They purify the Mycobacterium smegmatis NrtR (confirmed by SDS-PAGE, chemical cross-linking and MS) and demonstrate that it binds in a concentration-dependent manner to the putative regulatory sequence. They further construct six point mutations and demonstrate that these residues are individually essential for NrtR DNA binding activity.

4) They demonstrate that ADP-ribose, a toxic breakdown product of NAD+, inhibits NrtR DNA binding activity, although, NrtR does not have an ADP-ribose pyrophosphohydrolase activity.

5) A mutant lacking NrtR displayed increased expression of nadA and elevated levels of NAD+/NADH – confirming NrtR as a negative repressor of NAD biosynthesis, regardless of growth phase. NrtR also autoregulates its own biosynthesis.

6) The authors demonstrate that NrtR is acetylated, predominantly at the K134 residue, mutagenesis of this residue to remove the acetylation effect resulted in variances in DNA binding – not sure what this means, not quantified by SPR, as with their earlier experiment.

7) Acetylation by a non-enzymatic acetyl phosphate-dependent mechanism seems to be responsible for acetylation.

8) In vivo, acetylation was required for NrtR-dependent regulation of the nadABC operon and NAD+/NADH biosynthesis.

We are thankful to reviewer 1 for detailed summary of key findings in this study. As for the 6^th concern raised by reviewer 1, it is because that i) two different approaches (SPR and EMSA) are found to be effective in assaying the NrtR-DNA interaction; ii) EMSA is much cheaper and convenient method when compared to that of SPR.

Generally the study is conducted well, with a careful experimental approach. The following should be addressed.

1) Much of the conclusions described in the bioinformatics sections about domains and putative function appear to be speculation as there are no references to experimental validation. This section should be shortened

We have rephrased this section appropriately. Also, references are cited accordingly.

2) All electrophoretic gel mobility assays are missing cold competitive controls. This is necessary to ensure specificity and should be done.

It is a good suggestion. Technically, cold probe is only suitable in the EMSA with isotope-labeled DNA probe. However, our assay is based on EB staining of PAGE. That is why cold probe can’t work here. Equivalently, we introduced an unrelated DNA probe, vprA promoter as a negative control, which effectively verify the specificity of NrtR-cognate interaction (new Figure 3—figure supplement 2).

3) For Figure 5—figure supplement 1, some graphic representation of the westerns is required to make comparisons. Density scans of the bands should be carried out over multiple experiments.

As reviewer 1 suggested, no less than 3 independent trials of western blot have been calculated. Here, relative quantification results of NrtR protein in the DNA-binding affinities were plotted (new Figure 5—figure supplement 1G).

4) Whilst not essential to the entire story, the dimerization of acetylation defective mutants should have been assessed. Perhaps acetylation affects protein-protein interactions.

It is a good point, because that acetylation can affect protein-protein interaction in certain cases. However, our results of gel filtration experiments revealed that all the K134 mutant NrtR proteins (K134A, K134Q, and K134R) still possess the ability to form dimer in solution, ruling out this possibility (new Figure 5—figure supplement 1B).

Reviewer #2:

[…] Overall, the experiments are elegantly performed with well-done biochemistry. It certainly is interesting that the protein is always acetylated but the relevance of this acetylation to regulation of NAD biosynthesis is not known.

We do appreciate reviewer 2 for the positive overall judgement on this work. In the revision, we have added new data, that is solid in vivo evidence that such kind of acetylation is dependent on the non-enzymatic AcP action, rather than the enzymatic form of Pat/CobB. It is unusual, but not without precedent, because that AcP-dependent acetylation seems common in E. coli as Weinert and coworkers described (Weinert et al., 2013). In fact, protein acetylation has been well known as a prevalent form of post-translational modifications and plays multiple roles in bacterial physiology, augmenting cross-talks amongst different metabolisms at multiple levels. NAD⁺ refers to an essential vitamin involving in central metabolic activities. Evidently, the deacetylase CobB also requires NAD⁺ for its enzymatic activity (Sang et al., 2016) and links to the signaling of the second messenger c-di-GMP (Xu et al., 2019). To the best (but not limited to) of our knowledge, NAD⁺ has already been connected with protein acetylation in mammals. Not only does cytosolic NAD⁺ level involve protein acetylation (Marcu et al., 2014), but its depletion of NAD⁺ connects with p65 acetylation via the elevation of nuclear factor-kappaB transcription (Kauppinen et al., 2013). In Saccharomyces cerevisiae, it has been elucidated that a functional link occurs between NAD⁺ homeostasis NatB-mediated protein acetylation (Croft et al., 2018). Together with the aforementioned literature, we can conclude that i) the observation of NAD⁺ homeostasis in connection with NrtR acetylation is physiologically reasonable; 2) it represents a first example that acetylation of NrtR controls NAD⁺ metabolism at least in the prokaryotic domain of life. Finally, we hope reviewer 2 can understand it and respect the novelty on this standing.

Reviewer #3:

Goal: demonstrate that the Nudix-related transcriptional regulator NrtR (MSMEG_3198) controls NAD homeostasis in M. smegmatis via acetylation of a lysine group at position 134.

The authors found a 23 bp palindromic sequence between nrtR and nadABC, assert that it binds to NrtR yet do not shown evidence to support this claim.

Technically, a target palindrome sequence is too short to directly be applied in the in vitro EMSA experiment. In general, a DNA probe containing this palindrome is applied in EMSA assays. As described by different groups (Huang et al., 2009; Rodionov et al., 2008; Wang et al., 2019) with little change, we used a short probe 57bp covering the 23 palindrome in our SPR (Figure 3D) and EMSA assays (Figure 3C and new Figures 3—figure supplement 3C and Figure 5—figure supplement 1C). Two different approaches convincingly confirm this DNA-NrtR interaction. This binding is specific in that NrtR can’t bind to its unrelated vprA promoter (new Figure 3—figure supplement 2). More importantly, this physical binding of NrtR and cognate DNA has physiological role in the regulated expression nad operon and level of cytosolic NAD⁺ (NADH) pool (Figure 4). Therefore, we believe our result is solid and convincing, which is fully agreement with those reported by others (Huang et al., 2009; Rodionov et al., 2008; Wang et al., 2019).

The authors constructed a phylogenetic tree of 260 Nudix protein family representatives including only 2 Nudix proteins from M. smegmatis. It would have been useful to include the Nudix proteins from other mycobacterial species such as M. tuberculosis, M. avium, M. leprae, or M. marinum.

In fact, four NrtR-like proteins, Rv1593c [Mycobacterium tuberculosis H37Rv], MAP4_2560 [Mycobacterium aviumsubsp. paratuberculosis MAP4], ML1224 [Mycobacterium leprae TN], MMAR_2390 [Mycobacterium marinum M] have been included in the unrooted tree (Figure 2A). In addition, we found that their sequences were highly similar to MSMEG_3198 [Mycolicibacterium smegmatis MC² 155] and MSMEI_3116 [Mycobacterium smegmatisstr. MC² 155] via BLASTp-based assays. For example, Rv1593c is 79% identical to MSMEG_3198. Based on this scenario, they were compactly grouped into a clade.

Next, we concluded these four protein sequences to remove redundant homologs for the inferred phylogeny shown in old Figure 2B (cut-offs: 70% identity). However, we fully agreed with the reviewer’s critical suggestion, considering the important representativeness of M. avium, M. leprae, M. marinum and M. tuberculosis forMycobacterium strains.

Here, we followed reviewer 3’s comment to re-introduce them into our revised phylogenetic tree (new Figure 2B).

The authors tested whether NrtR had the predicted ADP-ribose pyrophosphohydrolase activity but could not detect any.

Yes, we did. Unfortunately, we can’t detect enzymatic activity. This is an evolutionary relic (Huang et al., 2009; Rodionov et al., 2008; Wang et al., 2019). In fact, a similar scenario is seen with NrtR of Streptococcus suis (Wang et al., 2019).

The authors constructed a nrtR KO in M. smegmatis and showed by RT-PCR that deletion of nrtR increased nadA expression by 2-6 fold. The authors also measured NAD+ and NADH in wt and nrtR KO strains and observed less than 2-fold increase in the cofactor levels. This experimental design lacks the requisite precision to draw a meaningful conclusion as they reported levels of NAD+ and NADH as pmol/10⁶ CFU but they did not plate for CFUs in their experiment. They only estimated CFUs based on OD while admitting that there could be a 2-fold variation in their CFU estimation. Therefore, the results cannot support the conclusion that NrtR regulates NAD⁺ and NADH levels.

First, we would like to thank reviewer 3 for the summary of nrtR KO results. It seems likely that misunderstanding or omitting the plating experiment by the reviewer 3 is due to my simplified statement in the Materials and methods section in last version. In this revision, we have added detailed information on how I do the bacterial cell plating (subsection “Determination of intracellular NAD+ and NADH concentrations”). The plating counts appeared in new Figure 7—figure supplement 1. We thereby expect that reviewer 3 can consider it on this standing, and our result is reasonable.

Additionally, nrtR deletion does not modify the NADH/NAD⁺ ratio as both cofactors are altered at the same rate further indicating that NrtR has no role in maintaining the redox status of mycobacterial cells.

We have read careful several literatures related to NAD metabolism in Mycobacterium from the research group of reviewer 3. We found them informative and cited them appropriately in the section of Introduction. It was described as follows: “Earlier microbial study by Vilcheze et al. (Vilcheze et al., 2005) indicated that the removal of ndhII, a type II NADH dehydrogenase-encoding gene, enhances the intracellular NADH/NAD⁺ ratio, giving phenotypic resistance to the front-line anti-TB drug isoniazid (INH) and its related drug ethionamide (ETH). Subsequently, the de novo and salvage pathways of NAD⁺ is proposed to exhibit potential of being anti-TB drug targets (Vilcheze et al., 2010)“.

Presumably, the checkpoint of NrtR is the mixed pool of cytosolic NAD⁺ and NADH, rather than the ratio of NAD⁺ /NADH (Huang et al., 2009; Rodionov et al., 2008; Wang et al., 2019). In contrast, the ratio of NAD⁺ /NADH in E. coli is determined by the reversable reduction reaction of NAD. This reaction is catalyzed by PntA/B and UdhA in E. coli (Anderlund et al., 1999; Boonstra et al., 1999; Sauer et al., 2004). In M. smegmatis, the homologs appear as follows: MSMEG_0110 (75% similarity) for pntA, MSMEG_0109 (77.7% similarity) for pntB, and MSMEG_2748 (58.1% similarity) for udhA. However, the aforementioned three genes are not cognate targets of NrtR at all. Therefore, the available literature and reasonable analysis argues against the statement by reviewer 3. Obviously, the ratio of NADH/NAD⁺ ratio should be the checkpoint of the reaction by the three enzymes of PntA, PntB and UdhA.

The authors demonstrated that M. smegmatis NrtR K134 lysine residue is acetylated. They constructed M. smegmatis mutants where the lysine residue was replaced by an alanine, a glutamine or an arginine group. These mutants had similar phenotypes as the nrtR KO strain. The authors concluded that "acetylation of K134 is a prerequisite for NrtR to regulate homeostasis of NAD⁺ in Mycobacterium". This conclusion is not substantiated by the data provided.

The authors should have compared the data obtained from this study with NadR, a known regulator of NAD biosynthesis. I would not recommend this for publication.

We do respect the criticism of reviewer 3. However, to the best of our knowledge, our in vitro and in vivo data have reached the high standing level in the acetylation filed. Moreover, we added new in vivo data (i.e., removal of AcP pathway-encoding genes Pta/AckA), supporting that NrtR proceeds in AcP-dependent acetylation (new-Figure 6). This is completely consistent with our former conclusion. As for the suggestion of “comparison with NadR, a known regulator”, we did it. However, it is not practically feasible, because the only available two literatures of NadR from E. coli and Salmonella is an in vitro EMSA description without any physiological data of NAD⁺ level (Penfound and Foster, 1999; Raffaelli et al., 1999).

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file 1. Strains and plasmids used in this study.

elife-51603-supp1.doc^{(116KB, doc)}

DOI: 10.7554/eLife.51603.020

Supplementary file 2. Primers used in this study.

elife-51603-supp2.doc^{(87KB, doc)}

DOI: 10.7554/eLife.51603.021

Supplementary file 3. Nudix family protein in M. smegmatis MC² 155 (Magni et al., 2004).

elife-51603-supp3.doc^{(60KB, doc)}

DOI: 10.7554/eLife.51603.022

Transparent reporting form

elife-51603-transrepform.docx^{(245.9KB, docx)}

DOI: 10.7554/eLife.51603.023

Data Availability Statement

All data generated or analysed during this study are included in the manuscript and supporting files.

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PERMALINK

A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation

Rongsui Gao

Wenhui Wei

Bachar H Hassan

Jun Li

Jiaoyu Deng

Youjun Feng

Roles

Abstract

Introduction

Figure 1. Working model for the regulation of NAD homeostasis by NrtR in Mycobacterium.

Results

Discovery of NrtR in the context of the NAD+ biosynthetic pathway

Phylogeny of NrtR

Figure 2. Phylogeny of NrtR proteins.

Binding of M. smegmatis NrtR to cognate DNA

Figure 3. Structural and functional insights into the binding of MsNrtR to its cognate DNA target.

Figure 3—figure supplement 1. Characterization of the M. smegmatis NrtR.

Figure 3—figure supplement 2. The M. smegmatis NrtR cannot bind to an unrelated DNA, the vprA probe.

Figure 3—figure supplement 3. Mapping of NrtR-DNA interactions.

Figure 3—figure supplement 4. Identification of the ADPR pyrophosphatase activity of the M. smegmatis NrtR and its mutant (Q54E, K58E and D60G).

ADP-ribose disrupts interplay between NrtR and DNA

In vivo role of MsNrtR in NAD+ synthesis

Figure 4. NrtR is a repressor for the nadABC operon that is responsible for NAD+ and NADH concentration in M. smegmatis.

Figure 4—figure supplement 1. In vivo evidence that MsNrtR is an auto-repressor.

Acetylation of K134 in MsNrtR

Figure 5. The discovery of acetylation of K134 in MsNrtR.

Figure 5—figure supplement 1. Dependence on K134 acetylation in the binding of MsNrtR to a cognate DNA target.

Figure 5—figure supplement 2. Acetylation is ubiquitous in three bacterial NrtR proteins.

Figure 5—figure supplement 3. Construction and identification of NrtR K134 point mutants on the M. smegmatis chromosome.