https://orcid.org/0000-0003-0173-4332
Figures
Abstract
During infection, Mycobacterium tuberculosis (Mtb) encounters multiple environmental stressors, including nitric oxide (NO) and iron limitation, and an ability to mount an integrated response is essential for the bacterium’s adaptation and continued survival. Iron-containing prosthetic groups in key enzymes are critical for Mtb sensing and detoxification of NO, and there is significant overlap between NO- and low iron-responsive genes. However, how Mtb adapts to these two stressors concurrently is largely unknown. Here, we find that exposure to NO globally augments expression of low iron-responsive genes and vice versa, with a two gene operon, rv3839-rv3840, among the most highly upregulated. Deletion of rv3839-rv3840 resulted in increased growth under prolonged iron limitation and early exit of Mtb from an adaptive state of growth arrest induced upon exposure to NO/low iron. ∆rv3839-rv3840 Mtb exhibited an elongated cell morphology compared to wild type Mtb in NO/low iron conditions, indicating effects of this operon on cell growth and division under stress conditions, with Rv3839 as the key driver of this phenotype. Coproporphyrin III tetramethyl ester (TMC), a modified precursor molecule in the endogenous Mtb heme biosynthesis pathway, was found to accumulate in ∆rv3839-rv3840 Mtb under iron limiting conditions. Further, intrabacterial heme levels were increased in ∆rv3839-rv3840 Mtb under NO stress and iron limitation. Together, these findings reveal Rv3839-Rv3840 as proteins involved in the downregulation of heme biosynthesis under NO stress and iron limitation, and highlight the link between Mtb growth control in response to NO/low iron and endogenous heme biosynthesis.
Author summary
Slowed growth is a physiologic adaptation to key environmental cues important for host survival of Mycobacterium tuberculosis (Mtb), the bacterial cause of tuberculosis, which is the leading cause of death globally from a single infectious agent. Host-produced nitric oxide (NO) is one such signal, but while regulation of NO response by the Mtb DosRS(T) two-component system is well-studied, NO stress also provokes a broad transcriptional response outside of DosRS(T) regulation that overlaps with the transcriptional response to iron limitation. Here, we show that the mycobacterial proteins Rv3839-Rv3840 contribute to Mtb maintenance of NO and low iron-induced growth arrest, and that this inability to maintain growth arrest is connected to dysregulation of the endogenous heme biosynthetic pathway. Little is known about regulation of endogenous heme biosynthesis in Mtb and its role in Mtb survival under stress conditions, and our results reveal a previously unknown interplay between NO and iron limitation response regulation and heme homeostasis.
Citation: Quirk NF, Gregory KN, Morita YS, Tan S (2026) Rv3839-Rv3840 links the endogenous heme biosynthesis pathway with Mycobacterium tuberculosis adaptation to nitric oxide and iron limitation stress. PLoS Genet 22(6): e1012202. https://doi.org/10.1371/journal.pgen.1012202
Editor: Kai Papenfort, Friedrich-Schiller-Universitat Jena, GERMANY
Received: February 27, 2026; Accepted: June 3, 2026; Published: June 8, 2026
Copyright: © 2026 Quirk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: RNA sequencing data have been deposited in the NCBI GEO database (GSE319896). All other relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by grants from the National Institutes of Health (NIH) (R01 AI143768 and R21 AI168597) to ST, and by grants from the Mizutani Foundation for Glycoscience and University of Massachusetts Amherst Institute for Applied Life Sciences (IALS) Core Facilities Incentive Fund to YSM. NFQ was supported in part by training grant T32 GM007310 from the National Institutes of Health to the Tufts University Graduate School of Biomedical Sciences graduate program in Molecular Microbiology, and by a Dean’s Fellowship from the Tufts University Graduate School of Biomedical Sciences. KNG was supported by a fellowship from the University of Massachusetts Amherst as part of the Chemistry-Biology Interface Training Program (National Research Service Award T32 GM139789). The funders had no role in study design, data collection and analysis, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Tuberculosis remains a particularly difficult disease to treat in part because of the ability of Mycobacterium tuberculosis (Mtb) to persist in the face of host defense mechanisms [1]. Over the course of infection, Mtb encounters a range of environmental signals including nitric oxide (NO), hypoxia, and iron limitation. The environmental cues Mtb encounters varies depending on factors such as the infection stage (e.g., pre- or post-adaptive immune response onset) and its location within the host (e.g., necrotic core versus macrophage-rich cuff of a lesion) [2–4]. Sensing and integrating response to these various signals is critical for Mtb adaptation to its local environment, and its ability to do so is vital for the bacterium’s continued survival in vivo, as evidenced by the significant attenuation in host colonization of Mtb lacking key regulators [5–7]. However, while several key regulators have been identified [6–11], much remains unknown as to how Mtb integrates its response to different signals in its environment.
A key adaptive output of Mtb in response to environmental stress is alteration of its growth status. For example, Mtb growth is slowed at acidic pH [12,13], and the bacteria also enter an adaptive state of growth arrest upon extended exposure to NO or hypoxia [11,14,15]. Strikingly, a point mutation in a single two-component system (TCS) response regulator that resulted in faster growth of Mtb in vitro and failure to enter a state of growth arrest upon extended NO exposure conversely resulted in attenuation for in vivo host colonization [8]. This supports that slowed growth under certain environmental pressures is advantageous for Mtb during host colonization. NO stress, along with hypoxia, provokes a transcriptional response regulated by the DosRS(T) TCS in a set of 48 genes known as the “dormancy regulon” [6,10,11], and the two environmental signals have thus most often been studied together and in the context of DosR regulation. However, Mtb additionally encounters NO independently of hypoxia as part of the host adaptive immune response [16–19]. RNA sequencing (RNAseq) data indicate that as many as 100 genes are significantly differentially expressed in response to NO that are not responsive to hypoxia [8,20]. Therefore, the response of Mtb to NO and hypoxia is differentiated, despite traditionally being studied in the context of their co-regulation. Further, DosR regulates only a subset (48) of the genes differentially expressed in response to NO [6,8,10,11], indicating that our understanding of the regulatory network underlying the NO response of Mtb and how it may integrate with other aspects of Mtb cell biology is incomplete.
Notably, published transcriptional data show a significant overlap (46 genes) in Mtb transcriptional response to NO and iron limitation [8,21,22]. During infection, host cells sequester free iron to starve pathogens of iron, an essential cofactor for many cellular processes [23]. However, Mtb has an arsenal of iron binding, transport, and storage mechanisms, and can additionally utilize host heme as an iron source [24,25]. These strategies together allow Mtb to successfully compete with the host for iron [23,26] and are intertwined with the Mtb response to NO stress. For example, expression of much of the iron response machinery is controlled by the essential iron-dependent transcriptional regulator IdeR (Rv2711) [21,27–29], which has been shown to be required for resistance to reactive nitrogen intermediates [28]. NO exposure degrades iron-sulfur (Fe-S) clusters in key enzymes, and Mtb must therefore upregulate iron acquisition and Fe-S cluster biogenesis as part of its defense against NO stress [30]. Additionally, iron is required for heme production, which is involved in both the sensing and detoxification of NO stress. NO is directly sensed by the DosRS(T) TCS via heme prosthetic groups [31], and truncated hemoglobin N (HbN) detoxifies NO through its potent dioxygenase activity [32–34]. Together, these findings support the existence of critical links between the response of Mtb to simultaneous NO stress and iron limitation, two environmental cues that would be encountered together during infection.
Here, we find that the Mtb Rv3839-Rv3840 proteins, which are encoded together in an operon and highly expressed upon exposure to both NO stress and iron limitation, limit Mtb growth under iron-limiting conditions and contribute to the maintenance of NO and iron stress-induced growth arrest. Iron limitation resulted in elongated Mtb cells, and while wild type (WT) Mtb lost this phenotype when acute NO stress was introduced, ∆rv3839-rv3840 Mtb continued to exhibit an elongated morphology. Further, deletion of rv3839-rv3840 resulted in accumulation of coproporphyrin III tetramethyl ester (TMC) under iron-limiting stress and increased intracellular heme. Together, these data reveal a previously unknown aspect of NO and iron limitation response regulation in Mtb and support an intrinsic interplay between Mtb adaptation to NO stress, iron limitation, and heme homeostasis.
Results
Transcriptional response of Mtb to NO stress is augmented in the presence of iron limitation and vice versa
To first understand the intersection between Mtb response to iron limitation and NO stress, we sought to define the global transcriptional response to simultaneous NO stress and iron limitation via RNA sequencing (RNAseq) analysis with Mtb exposed to conditions of iron limitation, NO stress, or both. Notably, the RNAseq dataset showed augmentation in the induction of 94 out of 135 NO-responsive genes when NO stress is compounded with iron limitation (genes differentially expressed log2-fold change ≥1 in the NO condition; log2-fold change ≥0.6 between NO + low iron and NO conditions; p < 0.05, FDR < 0.01 in both sets) (Fig 1A and S1 Table). Similarly, induction of 75 out 142 low iron-responsive genes was augmented in the simultaneous presence of NO stress (genes differentially expressed log2-fold change ≥1 in the low iron condition; log2-fold change ≥0.6 between NO + low iron and low iron conditions; p < 0.05, FDR < 0.01 in both sets) (Fig 1B and S2 Table). Overall, 780 genes were differentially expressed (log2-fold change ≥1) under simultaneous NO stress and iron limitation, with 579 genes specifically differentially expressed in the dual condition, but not in either single condition alone (Fig 1C and S1-S3 Tables). qRT-PCR analysis on genes exhibiting the largest expression changes under simultaneous NO stress and iron limitation versus the single conditions validated the results observed in the RNAseq dataset (Fig 1D). These data support the concept that NO stress exacerbates the iron limitation experienced by Mtb and vice versa.
Log-phase WT Mtb was exposed for 4 hours to: (i) iron-depleted minimal media + 150 µM FeNO3 (control), (ii) iron-depleted minimal media + 150 µM FeNO3 + 100 µM DETA NONOate (NO), (iii) iron-depleted minimal media + 100 µM 2’2’-dipyridyl (low iron), or (iv) iron-depleted minimal media + 100 µM 2’2’-dipyridyl + 100 µM DETA NONOate (NO + low iron), before RNA was extracted for RNAseq (A and B) or qRT-PCR (D). For RNAseq data, log2-fold change compares gene expression in each indicated condition to the control condition. Genes marked in red were the most highly augmented genes in NO + low iron compared to NO (A) or low iron (B) respectively (p < 0.05, FDR < 0.01 in both sets, with log2-fold change ≥1 in NO or low iron respectively). For qRT-PCR data shown in (D), fold change is as compared to the control condition. sigA was used as the control gene and data are shown as means ± SEM from 3-4 experiments. p-values were obtained with a one-way ANOVA with Tukey’s multiple comparisons for each gene. N.S. not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) illustrates overlap in differentially regulated genes in the different conditions as revealed by RNAseq. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
Rv3839-Rv3840 is important for maintenance of NO and iron stress-induced growth arrest
From the RNAseq data, the rv3839-rv3840 operon stood out as it was among the most highly increased in expression in the dual NO + iron limitation condition compared to NO stress alone (Fig 1A and 1D) and robustly upregulated under iron limitation (Fig 1B). rv3840, encoding a gene annotated as a transcription factor (although it lacks a DNA-binding domain), is downstream of rv3839, which encodes a conserved hypothetical protein containing a domain of unknown function (DUF2470) found in heme utilization proteins [35,36]. Of note, rv3839-rv3840 expression is induced by iron-limiting conditions in an IdeR-dependent manner, with a putative IdeR binding site identified upstream of rv3839 [21,27]. bfrB (rv3841), encoding bacterioferritin B, an iron storage protein, is located immediately downstream of this operon. However, the function of Rv3839 and Rv3840 in Mtb is unknown. To determine if Rv3839-Rv3840 plays a role in regulating the Mtb iron limitation response, we generated a Δrv3839-rv3840 Mtb mutant and tested its growth under iron-limiting conditions. In standard 7H9 rich medium, Δrv3839-rv3840 Mtb grew indistinguishably from WT Mtb (Fig 2A). In contrast, under iron-limiting conditions, Δrv3839-rv3840 Mtb exhibited increased growth compared to WT Mtb, which was most clearly observed with continued sub-culturing in iron-limiting conditions (Figs 2A, 2B, and S1A). Complementation of the rv3839-rv3840 operon (rv3839-rv3840*) partially returned growth to WT levels (Figs 2A, 2B, and S1A). These data suggest that Rv3839-Rv3840 acts to limit growth under prolonged iron limitation, a phenomenon likely adaptive for Mtb survival, similar to the slowed growth also observed with other stressors such as acidic pH and NO [11–15].
(A and B) Δrv3839-rv3840 Mtb fails to limit growth under iron limitation. Growth curves (A) and day 18 timepoint (B) of WT, Δrv3839-rv3840, and rv3839-rv3840* (complemented strain) Mtb sequentially cultured in 7H9, pH 7.0 media (control) or iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron). Bacterial growth was tracked by OD600 every 3 days. At day 12, the strains were sub-cultured into the same medium. Data are shown as means ± SEM from 4 experiments. p-values were obtained with unpaired t-tests with Welch’s correction in (A), comparing ∆rv3839-rv3840 to WT Mtb in the low iron condition. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test in (B). N.S. not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001. (C and D) Δrv3839-rv3840 Mtb prematurely exits NO and low iron stress-induced growth arrest. WT, Δrv3839-rv3840, and rv3839-rv3840* Mtb were grown in aerated conditions in 7H9, pH 7.0 and sub-cultured in either 7H9, pH 7.0 ± 6 doses of 100 µM DETA NONOate (C) over 30 hours (shaded area), or in iron-depleted minimal media with 100 µM 2’2’-dipyridyl and treated with 6 doses of 100 µM DETA NONOate (D) over 30 hours (shaded area). Bacterial growth was tracked by OD600 every day for 20-23 days. Data are shown as means ± SEM from 3 experiments. p-values in (D) were obtained with unpaired t-tests with Welch’s correction, comparing ∆rv3839-rv3840 to WT Mtb. * p < 0.05, ** p < 0.01, *** p < 0.001. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
Given the augmented induction of rv3839-rv3840 in the simultaneous presence of NO stress and iron limitation (Fig 1), we next investigated whether deletion of rv3839-rv3840 also affected Mtb growth under the dual conditions of extended NO exposure and iron limitation. Mtb cultures were treated with 6 doses of 100 µM DETA NONOate, a NO donor with a half-life of ~20 hours, over the course of 30 hours, to drive Mtb entry into a state of growth arrest [8]. In the presence of extended NO exposure alone, Δrv3839-rv3840 Mtb exited growth arrest at the same time as WT Mtb (Fig 2C). In contrast, Δrv3839-rv3840 Mtb strikingly exited growth arrest induced by simultaneous iron and extended NO stress significantly earlier than WT Mtb, with complementation partially restoring the WT Mtb outcome (Figs 2D and S1B). This result indicates that Rv3839-Rv3840 impacts the ability of Mtb to maintain growth arrest under NO stress and iron limitation.
Deletion of rv3839-rv3840 alters Mtb cell length in the presence of NO and iron limitation
Remodeling of the Mtb cell envelope is thought to accompany the shift into growth arrest driven by NO and hypoxia [37–40]. In addition, iron deprivation reduces cell wall thickness and increases susceptibility to cell membrane targeting antibiotics in Mycobacterium smegmatis [41,42]. Notably, rv3840 encodes one of four LytR-CpsA-Psr (LCP) proteins in Mtb, others of which act in bacterial cell envelope maintenance by catalyzing the crosslinking reaction between arabinogalactan (AG) and peptidoglycan (PG) [43–47], and influence Mtb virulence and antibiotic resistance [43,45–47]. However, Rv3840 differs from the three characterized LCP proteins in Mtb (Rv3484, Rv3267, Rv0822c) in that it lacks the N-terminal transmembrane domain and C-terminal LytR_C domain found in canonical LCP proteins [44,48] and has only the central catalytic domain. We therefore next sought to examine if exposure to iron limitation and NO stress and deletion of rv3839-rv3840 affected Mtb morphology. A first observation was that WT Mtb were more elongated upon exposure to iron-limiting conditions as compared to 7H9, pH 7.0 control media (3.64 ± 0.33 µm versus 2.10 ± 0.04 µm, p < 0.0001) (Fig 3A and 3B). Interestingly, when NO stress was present together with iron limitation, WT Mtb lost this elongated phenotype (2.67 ± 0.09 µm versus 3.64 ± 0.33 µm, p < 0.05) (Fig 3A and 3B). Δrv3839-rv3840 Mtb was elongated compared to WT Mtb under both iron limitation (4.60 ± 0.07 µm versus 3.64 ± 0.33 µm, p < 0.05) and NO stress + iron limitation (4.56 ± 0.16 µm versus 2.67 ± 0.09 µm, p < 0.0001), with length restored to WT levels by complementation (Fig 3A and 3B). These results are in accord with Mtb modulation of its growth and/or division in response to NO and iron limitation as environmental stresses. The continued presence of an elongated phenotype for Δrv3839-rv3840 Mtb in NO + iron limitation further supports that Rv3839-Rv3840 contributes to appropriate adaptation of Mtb growth and/or division when both environmental stressors are experienced concurrently.
(A and B) Δrv3839-rv3840 Mtb exhibit increased cell length in response to iron limitation and NO stress. WT, Δrv3839-rv3840, and rv3839-rv3840* (complemented mutant) Mtb carrying a constitutive smyc’::mCherry reporter were sub-cultured into 7H9, pH 7.0 (control) or iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron), ± 100 µM DETA NONOate for 3 days. (A) shows representative images, with lengths quantified in (B). Data are shown from 3 experiments. Each point represents a single bacterium and median values for each replicate are represented by the larger symbol. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. ** p < 0.01, **** p < 0.0001. (C-E) Rv3839 regulates cell elongation under prolonged iron limitation and NO stress. (C) shows a schematic representation of the sequential low iron and NO exposure experiment. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb carrying a constitutive smyc’::mCherry reporter were sub-cultured in 7H9, pH 7.0 (control) or iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron). After 3 days, strains in the low iron media were sub-cultured into low iron medium ± 100 µM DETA NONOate for an additional 3 days. Flask image provided by Servier Medical Art (https://smart.servier.com), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). (D) shows bacterial lengths quantified after the initial 3 days of growth. (E) shows bacterial lengths quantified after the additional 3 days of growth in low iron media ± NO. Data are shown from 3-4 experiments. Each point represents a single bacterium and median values for each replicate are represented by the larger symbol. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. N.S. not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
To begin to delineate the role of Rv3839 versus Rv3840, we restored only one gene at a time to Δrv3839-rv3840 Mtb by complementation (rv3839* or rv3840*). Additionally, here we introduced NO stress only after an initial 3 days of iron limitation (Fig 3C), rather than simultaneously (as in Fig 3B), to determine the impact of subsequent NO stress exposure on the elongated phenotype observed under iron limitation. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were grown in either 7H9, pH 7.0 medium (control), or in iron-limited medium to induce the elongated phenotype (Fig 3D). Strains in the iron-limited condition were then sub-cultured into iron-limited medium ± 100 μM DETA NONOate (Fig 3C). All strains remained elongated when sub-cultured from iron-limiting conditions into low iron medium again (Fig 3E, left side of graph). However, subsequent introduction of NO stress decreased WT Mtb length (3.69 ± 0.09 µm, “low iron→ low iron + NO”, versus 4.61 ± 0.06 µm, “low iron→ low iron”, p < 0.001) (Fig 3E). In contrast, Δrv3839-rv3840 Mtb remained elongated in all conditions where iron limitation was present (4.66 ± 0.27 µm, “low iron→ low iron”), including upon introduction of NO stress (4.57 ± 0.07 µm, “low iron→ low iron + NO”) (Fig 3E). These results suggest that Δrv3839-rv3840 Mtb is unable to appropriately respond to NO stress under iron-limiting conditions, in accord with the observed premature exit from growth arrest in NO + iron-limiting conditions (Figs 2D and S1B). Surprisingly, complementation with rv3839 alone restored cell length to WT levels under NO stress and iron limitation (3.90 ± 0.24 µm compared to 3.69 ± 0.09 µm), while complementation with rv3840 did not (4.66 ± 0.24 µm compared to 3.69 ± 0.09 µm, p < 0.01) (Fig 3E). These data support that Rv3839 function affects Mtb morphology under NO stress and iron limitation, not Rv3840, as may have been expected a priori given its association with the LCP family of proteins.
Endogenous heme biosynthesis contributes to maintenance of growth arrest in response to iron limitation
Rv3839 contains a domain of unknown function (DUF2470) found in heme-related proteins in bacteria and eukaryotes and is thought to be a heme-binding regulatory domain (Fig 4A) [49]. For example, DUF2470 is found in HugZ, a heme oxygenase involved in iron release and uptake in Helicobacter pylori [36], and in GluBP, a regulatory protein that enhances heme synthesis in Arabidopsis thaliana by binding glutamyl-tRNA reductase [50,51]. Phylogenetic analysis has shown that genes encoding DUF2470-containing proteins are often found near genes related to iron homeostasis, suggesting a conserved role in iron and heme homeostasis [49]. Given the primary role of Rv3839 observed above and that Rv3839 contains DUF2470, we thus next pursued further study of the role of the operon in endogenous heme biosynthesis. In Mtb, heme is synthesized via the coproporphyrin-dependent (CPD) pathway [52,53]. The initial steps of this pathway, 5-aminolevulinic acid (ALA) to coproporphyrinogen III, are shared with the protoporphyrin-dependent heme biosynthesis pathway used by most Gram-negative bacteria (Fig 4B) [53,54]. To produce heme by the CPD pathway, coproporphyrinogen III is oxidized to coproporphyrin III, and then iron is inserted to form coproheme, which then undergoes double oxidative decarboxylation to yield heme (Fig 4B) [53,54]. Given the requirement of iron in this process, we hypothesized that rv3839-rv3840 is upregulated to dampen heme biosynthesis and prevent the production of heme when there is insufficient iron available.
(A) Schematic representation of rv3839-rv3840 operon. Bacterioferritin B (encoded by bfrB) is located downstream of rv3839-rv3840. (B) Schematic representation of Mtb endogenous heme biosynthesis pathway. (C) Succinylacetone (SA) treatment inhibits elongation of Δrv3839-rv3840 Mtb under NO + low iron conditions. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb carrying a constitutive smyc’::mCherry reporter were sub-cultured in iron-depleted minimal media with 100 µM 2’2’-dipyridyl and 100 µM DETA NONOate (NO + low iron), ± 500 µM SA for 3 days. Fixed samples were analyzed via microscopy and bacterial cell length quantified. Data are shown from 3-4 experiments. Each point represents a single bacterium and median values for each replicate are represented by the larger symbol. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. N.S. not significant, ** p < 0.01. (D) Increased growth of Δrv3839-rv3840 Mtb under iron limitation is inhibited by SA treatment. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb strains were cultured in iron-depleted minimal media with 100 µM 2’2’-dipyridyl ± 250 µM SA. At day 12, the strains were sub-cultured in the same medium. OD600 of the bacterial cultures at day 18 are shown. Data are shown as means ± SEM from 3-4 biological replicates. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. N.S. not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
To study the impact of heme homeostasis on Mtb growth under NO stress and iron limitation, and the related role of Rv3839-Rv3840, we perturbed the endogenous heme biosynthesis pathway in Mtb with the inhibitor succinylacetone (SA). This compound blocks the activity of porphobilinogen synthase (PbgS) that converts ALA to porphobilinogen, one of the early steps in heme synthesis (Fig 4B) [55,56]. We reasoned that if Rv3839-Rv3840 activates heme biosynthesis, WT Mtb would behave like Δrv3839-rv3840 Mtb if the pathway is blocked via SA treatment. If instead Rv3839-Rv3840 represses heme synthesis, blocking the pathway would restore Δrv3839-rv3840 Mtb cell length and growth to that of WT Mtb. We first investigated whether the elongated phenotype observed in Δrv3839-rv3840 Mtb under NO stress and iron limitation was dependent on increased flux through the heme biosynthesis pathway. Treatment of Δrv3839-rv3840 Mtb with SA resulted in a decrease in cell length under NO stress and iron limitation, compared to untreated samples (3.85 ± 0.14 µm compared to 4.75 ± 0.11 µm, p < 0.001) (Fig 4C). The cell lengths of WT and the complemented mutant Mtb strains were not impacted by SA treatment (Fig 4C). These data support that endogenous heme biosynthesis impacts Mtb cell length and suggests that Rv3839-Rv3840 inhibits this pathway under NO stress and iron limitation.
We next investigated whether dysregulated heme biosynthesis in Δrv3839-rv3840 Mtb is a contributing factor to its inability to limit growth under iron limitation. Under iron-limiting conditions, Δrv3839-rv3840 Mtb exhibited increased growth compared to WT Mtb (Figs 2A, 4D and S1A). Complementation with rv3839, but not rv3840, largely returned growth to WT levels (Fig 4D). Likewise, in NO + low iron conditions, complementation with rv3839 alone was sufficient to phenocopy the growth profile of the full rv3839-rv3840* complemented Mtb strain, while rv3840 complementation alone largely did not change the growth phenotype of ∆rv3839-rv3840 Mtb (S2 Fig). Blocking endogenous heme biosynthesis by treatment with SA under iron limitation resulted in growth inhibition of Δrv3839-rv3840 Mtb, restoring growth to levels more similar to WT Mtb in iron-limited medium (Fig 4D). These data suggest that reduced heme biosynthesis contributes to Mtb growth arrest under iron limitation and that Rv3839 may act as an inhibitor of heme biosynthesis to maintain an adaptive growth-arrested state.
Deletion of rv3839-rv3840 results in accumulation of coproporphyrin III trimethyl ester in iron-limiting conditions
If Rv3839-Rv3840 indeed represses the heme biosynthesis pathway when iron is unavailable for coproheme production, we would expect a buildup of the upstream intermediates of the pathway in Δrv3839-rv3840 Mtb under iron limitation. We capitalized on the natural fluorescence of porphyrins to measure flux through the heme biosynthesis pathway in Δrv3839-rv3840 Mtb under iron limitation [57]. Δrv3839-rv3840 Mtb exhibited strongly increased levels of free porphyrins when grown under iron limitation compared to WT Mtb (2696.67 ± 340.84 AFU versus 283.50 ± 14.91 AFU, p<0.0001) (Fig 5A). Porphyrin levels were dampened in Δrv3839-rv3840 Mtb under simultaneous NO stress and iron limitation, although levels were still elevated compared to WT Mtb (947.50 ± 45.05 AFU versus 287.17 ± 8.40 AFU, p<0.0001) (Fig 5A). Interestingly, either rv3839 or rv3840 was sufficient for complementation of the Δrv3839-rv3840 Mtb phenotype in this case (Fig 5A), indicating that an increase in free porphyrins is not the sole driver of the elongated cell and growth phenotypes observed in Δrv3839-rv3840 Mtb. Together, these results support that under iron limitation, Rv3839-Rv3840 prevents the buildup of porphyrin intermediates.
(A) Δrv3839-rv3840 Mtb has elevated levels of porphyrins under iron limitation. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were sub-cultured and grown for 3 days in 7H9, pH 7.0 or iron-depleted minimal media with 100 µM 2’2’-dipyridyl, ± 100 µM DETA NONOate. Samples were tested for intrabacterial porphyrin levels. Data are shown as means ± SEM from 3 experiments. AFU = arbitrary fluorescence unit. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. (B) Reflective positive MALDI-TOF spectra of WT, Δrv3839-rv3840, and rv3839-rv3840* grown in iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron) for 3 days. Accumulation of a m/z 711.757 ion in Δrv3839-rv3840 Mtb is indicated. AU = arbitrary units. (C) Reverse phase LC-MS ion chromatogram of Δrv3839-rv3840 Mtb grown in low iron. Total ion chromatogram is shown in the top panel. Ion chromatogram of species matching m/z 711.3 (± 100 ppm), showing a single elution peak at 15.38 min, is shown in the bottom panel. (D) Positive-mode mass spectrum of Δrv3839-rv3840 at retention time of 15.38 min, showing the accumulating peak of m/z 711.3. (E) Identification of the m/z 711.3 ion as coproporphyrin III tetramethyl ester (TMC). Higher-energy collisional dissociation spectrum of the precursor ion (m/z 711.3) from Δrv3839-rv3840 Mtb fragmented by higher collisional dissociation at 55 V (top) compared to the fragmentation pattern of the TMC standard (bottom). (F) Chemical structure of TMC with the masses of expected fragments. (G) TMC accumulation in Δrv3839-rv3840 Mtb under iron limitation can be blocked by succinylacetone (SA) treatment. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were grown 7H9, pH 7.0 (control), or iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron) ± 500 µM SA for 3 days, and samples analyzed via MALDI-TOF-MS. TMC was quantified using a standard curve generated using TMC standards (inset standard curve graph). Data are shown as mean ± SEM from 3 experiments. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
To elucidate the identity of the porphyrin-containing compound that accumulates in ∆rv3839-rv3840 Mtb in iron-limiting conditions, WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were grown in iron-limited medium and samples extracted for analysis by positive ion mode MALDI-TOF mass spectroscopy (MS). This analysis revealed a strong signal with an m/z value of 711.757 in Δrv3839-rv3840 Mtb (Fig 5B). This m/z closely matched with 711.34, the monoisotopic mass of the protonated form of coproporphyrin III tetramethyl ester (TMC). To determine its identity, we analyzed lipid extracts from WT and Δrv3839-rv3840 Mtb using reverse phase LC-MS. The 711 peak eluted at 15.38 min (Fig 5C), and the mass spectrum at 15.38 min indicated the 711 peak as a major peak from the Δrv3839-rv3840 Mtb extract (Fig 5C and 5D). To further confirm the identity of this peak, we fragmented the parental ion from the Δrv3839-rv3840 Mtb sample (Fig 5D); this analysis demonstrated that the patterns of the fragmentation were nearly identical to those of an authentic TMC (Fig 5E and 5F). Having determined its identity, we used MALDI-TOF MS for high-throughput analysis. First, we determined the dose-response curve of TMC in MALDI-TOF MS, demonstrating a linear range between 0.25 – 2500 ng per spot (Fig 5G, inset). Using this standard curve, we quantified TMC abundance in lipid extracts from WT, Δrv3839-rv3840 and the various complementation Mtb strains, which showed high accumulation of TMC in iron-limiting conditions in Δrv3839-rv3840 Mtb (234.22 ± 40.77 ng/mg pellet) compared to WT (72.48 ± 6.88 ng/mg pellet) (Fig 5G). TMC levels were complemented by either rv3839 or rv3840, and SA treatment lowered TMC levels in Δrv3839-rv3840 Mtb to WT levels (Fig 5G), indicating that Rv3839-Rv3840 regulates the heme biosynthesis pathway upstream of porphobilinogen synthase.
Rv3839-Rv3840 regulates Mtb heme homeostasis
Often, elevated intracellular porphyrin levels correspond with increased heme production. However, given the modification of coproporphyrin III to TMC, it was unclear whether TMC abundance was indicative of elevated heme production or whether this modification is a strategy for diverting excess precursors to prevent excess heme production under iron limiting conditions. To distinguish between these two possibilities, we measured total intracellular heme in WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb grown in iron-limiting conditions, with or without NO stress. WT Mtb showed a trend towards decreased heme levels under iron limitation compared to control media, and a further reduction in total heme with the addition of NO stress, although this did not reach statistical significance (Fig 6A). In contrast, Δrv3839-rv3840 Mtb showed increased heme levels compared to WT Mtb under iron limitation (5146.83 ± 438.32 AFU versus 1088.00 ± 70.36 AFU, p < 0.0001) and increased heme levels compared to WT Mtb under NO stress and iron limitation (3240.83 ± 349.66 versus 859.17 ± 78.05 AFU, p < 0.0001) (Fig 6A). Total heme levels were reduced in Δrv3839-rv3840 Mtb under the dual NO + iron limitation condition compared to iron limitation alone (Fig 6A). Total heme levels were complemented by rv3839 or rv3839-rv3840, but complementation with rv3840 only partially reduced total heme levels compared to Δrv3839-rv3840 (Fig 6A).
(A) Total cellular heme levels are elevated in Δrv3839-rv3840 Mtb under iron limitation ± NO stress. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were sub-cultured and grown for 3 days in 7H9, pH 7.0 (control) or iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron) ± 100 µM DETA NONOate. Total cellular heme is calculated as fluorescence of treated samples with background porphyrin fluorescence subtracted. Data are shown as means ± SEM from 3-4 experiments. AFU = arbitrary fluorescence units. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. ** p < 0.01, **** p < 0.0001. (B) Schematic representation of intrabacterial heme reporter design. Heme binding to cytochrome b562 (Cyt b562) results in quenching of eGFP fluorescence but does not impact mKATE2 fluorescence. (C) Intrabacterial labile heme levels are elevated in Δrv3839-rv3840 Mtb under iron limitation ± NO stress. WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were sub-cultured and grown for 3 days in iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron) ± 100 µM DETA NONOate. Intrabacterial labile heme is reported as the ratio of mKATE2/eGFP fluorescence, and data are shown as means ± SEM from 3-4 experiments. p-values were obtained with a two-way ANOVA with Tukey’s multiple comparisons test. N.S. not significant, **** p < 0.0001. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
Total intracellular heme is composed of both inert heme tightly bound to hemoproteins and a smaller fraction of kinetically labile heme that is more available for exchange and biological processes [57,58]. As an independent manner to assess intrabacterial heme levels, we introduced a labile heme reporter into Mtb, where fluorescence of green fluorescent protein (GFP) is quenched upon binding of labile heme to cytochrome b562, with a constitutively expressed mKATE2 enabling ratiometric analysis (Fig 6B) [57,59]. As with total intrabacterial heme, Δrv3839-rv3840 Mtb showed increased intrabacterial labile heme levels compared to WT Mtb under both iron limitation (0.108 ± 0.003 versus 0.081 ± 0.002 mKATE2/eGFP signal, p < 0.0001) and NO stress and iron limitation combined (0.140 ± 0.004 versus 0.103 ± 0.003 mKATE2/eGFP signal, p < 0.0001) (Fig 6C). The increase in labile heme in Δrv3839-rv3840 Mtb was lower than the increase in total intrabacterial heme observed, likely due to labile heme representing a lower overall proportion of the heme present in the bacteria. This difference could also reflect differences in the amount of heme binding proteins present in WT versus ∆rv3839-rv3840 Mtb. Interestingly, WT Mtb showed elevated labile heme levels under NO stress and iron limitation compared to iron limitation alone (0.103 ± 0.003 versus 0.081 ± 0.002 mKATE2/eGFP signal, p < 0.0001) (Fig 6C), suggesting that NO stress induces increased de novo synthesis of heme, even as total intrabacterial heme levels decrease (Fig 6A). Labile heme levels were complemented by rv3839 or rv3839-rv3840, but complementation with rv3840 did not reduce labile heme levels compared to Δrv3839-rv3840 Mtb (Fig 6C). Together, these data support that the abundance of TMC in Δrv3839-rv3840 Mtb under iron limitation corresponds with increased total intracellular heme, and demonstrate a role for Rv3839-Rv3840 as a repressor of heme synthesis that is important in Mtb adaptation to NO stress and iron limitation.
Discussion
The ability of Mtb to sense and integrate its response to multiple environmental cues is critical for its survival in the host environment. Here, we show that the response of Mtb to NO stress is augmented in the simultaneous presence of iron limitation and vice versa, and reveal an important role of Rv3839-Rv3840 in the regulation of heme biosynthesis and the adaptive response of the bacterium to iron limitation and NO stress. We propose a model wherein the rv3839-rv3840 operon is upregulated under NO stress and iron limitation to repress heme biosynthesis in Mtb due to the reduced availability of iron for heme production (Fig 7). Loss of this regulation upon deletion of rv3839-rv3840 leads to the accumulation of modified porphyrin intermediates and intracellular heme, resulting in an elongated cell phenotype and premature exit from NO and iron limitation-induced adaptive growth arrest (Fig 7).
Under NO stress and low iron, WT Mtb (left side of the model) regulates heme biosynthesis to balance the requirement for heme in the NO response with the limitation imposed by the lack of iron. rv3839/rv3840 are expressed and regulate heme biosynthesis early in the pathway, upstream of porphobilinogen synthase, and by modulating buildup of porphyrin intermediates respectively. Blue circles represent glutamyl-tRNA and other precursor molecules. This regulation of heme biosynthesis allows Mtb to maintain adaptive growth arrest. Deletion of rv3839-rv3840 (right side of the model) results in increased flux through the heme biosynthetic pathways, resulting in the buildup of modified porphyrin intermediates and intracellular heme. The dysregulation of the pathway results in elongated cell morphology and premature exit from an adaptive growth arrest state.
Our observation that Mtb exhibits elongated cell morphology under iron-limiting conditions is in accord with previous work showing that iron starvation inhibits late-stage cytokinesis in Escherichia coli [60]. We thus posit that the elongated cell morphology observed in Mtb upon iron limitation reflects a similar inhibition of cytokinesis; introduction of NO stress halts growth entirely and results in a shorter cell length, while deletion of rv3839-rv3840 prevents appropriate sensing of NO stress in the context of iron limitation. It has been reported previously that host stress conditions, including NO stress and iron limitation, can alter the regulation of peptidoglycan synthesis through an Fe-S cluster-dependent mechanism, linking cell envelope-related processes with the status of iron-containing prosthetic groups [61]. Interestingly, our results show that it is Rv3839, not Rv3840, that is a driver of the elongated cell phenotype. As noted above, Rv3840 is an atypical LCP protein as it lacks the N-terminal transmembrane domain and C-terminal LytR_C domain found in the canonical LCP protein domain structure (Fig 4A) [46,48]. In addition to the three canonical LCP proteins, Mtb encodes two LytR_C domain-only proteins, VirR (Rv0431) and Cei (Rv2700). Cei contributes to Mtb cell envelope integrity and virulence [62], and loss of VirR function results in increased vesiculogenesis [63,64], a phenotype also associated with iron limitation [65]. The existence of these two LytR_C domain-only proteins alongside Rv3840 raises the intriguing possibility that these proteins interact to reconstitute a “complete” LCP protein with function at the Mtb cell envelope. Additionally, LCP proteins are known to exhibit functional redundancy in Mtb [43] and other organisms [43,66,67], and have been shown to be essential for septum formation and cell separation in Staphylococcus aureus and Streptococcus pneumoniae [67,68]. Future studies analyzing possible interplay between Rv3840 and VirR and/or Cei, as well as the impact of NO stress and iron limitation on bacterial growth and cell division, will shed light on the involvement of LCP proteins in these processes.
One of the striking phenotypes associated with deletion of rv3839-rv3840 is its premature exit from NO and iron limitation-induced growth arrest. Although TMC has been shown to accumulate in dormant M. smegmatis under acidified conditions [69,70], M. smegmatis is a fast-growing mycobacterial species and has not been shown to enter into NO-induced growth arrest [71]. It is also unclear why coproporphyrin III is methylated in Δrv3839-rv3840 Mtb and what purpose this modification serves. Given that WT Mtb does not normally accumulate TMC under NO and iron limitation stress, it seems unlikely that modification of coproporphyrin III and its accumulation would be protective. Indeed, SA treatment of the rv3840* complement Mtb strain under iron limitation does not dampen growth to the same degree as in Δrv3839-rv3840 Mtb, although the treatment does reduce TMC levels back to those of WT Mtb. Notably, our results show that Δrv3839-rv3840 Mtb is unable to properly respond to the introduction of NO stress in the context of iron limitation as evidenced by its elongated cell morphology. Mtb has multiple heme enzymes with important roles, including the NO/hypoxia sensors DosS and DosT [31], truncated hemoglobin (TrHbN) [32–34], catalase peroxidase (KatG) [72], and cytochrome P450s (CYPs) [73], all of which may be impacted by the dysregulation of intrabacterial heme synthesis in Δrv3839-rv3840 Mtb, resulting in premature exit from an adaptive state of growth arrest. Mtb also encodes a heme-containing bacterioferritin, BfrA, which is required for efficient storage of iron under iron limiting conditions, and previous work has demonstrated that the heme-bound form of BfrA exhibits enhanced iron release compared to heme-free variants [74,75]. Increased intrabacterial heme levels in Δrv3839-rv3840 Mtb may therefore cause increased release of iron from storage proteins, resulting in enhanced growth under NO stress and iron limitation.
Another outstanding question from this work is the separate functional roles of Rv3839 versus Rv3840 and the precise mechanism by which they regulate heme biosynthesis. In Corynebacterium glutamicum, it has been shown that transcription of heme synthesis proteins is controlled in part by the iron-dependent regulator DtxR [52,76,77]. In mycobacteria, recent work has shown that the terminal heme synthetic enzyme, coproheme decarboxylase (ChdC), exhibits decreased expression under iron limitation and acts as a negative regulator of heme uptake and utilization, demonstrating that there is coordination between heme synthesis and uptake and iron availability [78]. Glutamate semialdehyde aminomutase (GsaM) and ferrochelatase (CpfC), two other enzymes in the heme biosynthesis pathway, also exhibit decreased expression in iron-limited medium [26]. However, porphobilinogen synthase (PbgS) was upregulated under those same conditions [26]. How iron or other factors in Mtb may regulate heme biosynthesis thus remains to be fully elucidated. In our work, we have seen no evidence that deletion of rv3839-rv3840 impacts the transcript levels of heme biosynthesis proteins, suggesting that Rv3839-Rv3840 may instead impact protein levels or activity state. Our results show that complementation with rv3839 alone is sufficient to restore Δrv3839-rv3840 Mtb cell length, intrabacterial heme levels, and growth to that of WT Mtb, supporting Rv3839 as the main driver of the NO stress and iron-limitation-related phenotypes. Further, regulation of the heme biosynthesis pathway by Rv3839 likely occurs upstream of PbgS, as inhibition with SA dampens the growth of Δrv3839-rv3840 under iron limitation. We posit that Rv3839 regulates the activity of glutamyl-tRNA reductase (GtrR), which serves as a regulatory node for the heme biosynthesis pathway in multiple organisms [52,76,77,79,80] and has been shown to be regulated by other DUF2470-containing proteins [50,51]. In contrast, the possible functional role of Rv3840 is less clear. Complementation with rv3840 is not sufficient to restore the Δrv3839-rv3840 Mtb intrabacterial heme and growth phenotypes, nor the elongated length phenotype in NO + low iron conditions, to that of WT Mtb. However, Rv3840 is sufficient to restore TMC levels to that of WT Mtb, supporting a functional role for Rv3840 in preventing the buildup of precursor molecules in the heme biosynthesis pathway. We hypothesize that Rv3840 either: (i) regulates the modification of coproporphyrin III to TMC, or (ii) regulates the removal of TMC from the cell. Future studies aimed at defining the role of Rv3840 in modulating TMC buildup may also clarify the relationship between bulk population growth rate, cell length, and heme biosynthesis. Overall, we propose a model in which Rv3839 functions to repress heme biosynthesis early in the pathway, while Rv3840 acts as a secondary layer of regulation downstream to prevent the buildup of porphyrin intermediates (Fig 7). Further experiments will be required to delineate the molecular mechanisms underlying how Rv3839 and Rv3840 regulate heme biosynthesis.
NO stress and iron limitation are two major environmental stressors Mtb encounters during infection, and the bacterium mounts a robust transcriptional response in order to adapt and survive. Our findings here emphasize the integrated nature of the NO and iron limitation stress responses and identify Rv3839 and Rv3840 as key factors involved in the regulation of heme biosynthesis in response to these two signals. Mtb response to NO stress requires iron-containing prosthetic groups, including heme, for proper sensing and response, even while NO simultaneously degrades factors such as Fe-S clusters. Thus, NO stress likely exacerbates iron limitation, as available iron is needed for multiple aspects of Mtb NO response and adaptation. Therefore, the allocation of these resources is likely tightly regulated by the bacteria, with Rv3839-Rv3840 acting as one such layer of regulation. Our work adds to the understanding of how heme biosynthesis is regulated, and we propose that further studies investigating how the integration of NO stress, iron limitation, and heme biosynthesis impact Mtb cell division and growth will continue to reveal important insight into how Mtb environmental response is coordinated with its replication control.
Materials and methods
Mtb strains and culture
Mtb CDC1551 was used as the parental strain for all assays, and Mtb strains were cultured and maintained as previously described, with 7H9 broth supplemented with 10% OADC, 0.2% glycerol, 0.05% Tween 80, and 100 mM MOPS used for buffering to pH 7.0 [12]. Iron-depleted minimal media was made as previously described [8,26]. All antibiotics were added as appropriate at the following concentrations: 100 μg/ml streptomycin, 50 μg/ml hygromycin, 50 μg/ml apramycin, and 25 μg/ml kanamycin. Δrv3839-rv3840 Mtb and its complements were constructed with methods as previously described [4], with the Δrv3839-rv3840 mutation generated by homologous recombination and consisting of a deletion beginning at nucleotide 107 of the rv3839 open reading frame through the rv3840 stop codon. The rv3839-rv3840 complement (rv3839-rv3840*) consisted of a region beginning 830 bp upstream of the rv3839 start codon and included both the rv3839 and rv3840 open reading frames. The same promoter region was used to drive expression of rv3839 or rv3840 in the single gene complements (rv3839* and rv3840*). All complements were constructed in the integrative plasmid pMV306 (integration at the attB site) and are thus expressed in single copy on the chromosome. The smyc’::mCherry [4] and intrabacterial heme (HS1) reporters [57,59,81] introduced to indicated strains were previously described.
RNA sequencing and qRT-PCR analysis
For RNA sequencing (RNAseq) and qRT-PCR analyses, log-phase Mtb cultures (OD600 ~ 0.6) grown in aerated conditions were used to inoculate filter-capped T75 flasks laid flat at an OD600 = 0.3, containing 12 ml: (i) iron-depleted minimal medium + 150 µM FeNO3 (control condition), (ii) iron-depleted minimal medium + 150 µM FeNO3 + 100 µM DETA NONOate (NO condition), (iii) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl (low iron condition), or (iv) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl + 100 µM DETA NONOate (NO + low iron condition). A wash step with iron-depleted minimal medium was performed prior to inoculation of the culture into the various test conditions. After four hours of exposure, Mtb samples were collected and RNA extracted as previously described [82]. For RNAseq, three biological replicates were prepared for each condition, and library preparation was performed by the Tufts University Genomics Core Facility using the Illumina stranded total RNA with Ribo-Zero Plus kit. Barcoded samples were pooled and sequenced on an Illumina NovaSeq X Plus (single-end 100 bp reads). RNAseq data were analyzed as previously described [83]. A log2-fold change ≥1 was used as the cutoff for the list of genes differentially expressed in various conditions in WT Mtb. A cutoff of log2-fold change ≥0.6 was used when comparing the effect of combined NO + low iron exposure versus either single condition (i.e., NO or low iron). qRT-PCR experiments were carried out and analyzed as previously described [84].
Mtb growth assays
For low iron growth assays, log-phase Mtb strains were used to inoculate 10 ml of 7H9, pH 7.0 or iron-depleted minimal medium + 100 µM 2’2’-dipyridyl at a starting OD600 = 0.05 in standing, filter-capped T25 flasks. A wash step was conducted prior to inoculation as described above, and OD600 was measured at the indicated timepoints. After 12 days of growth, the strains were sub-cultured to an OD600 = 0.05 in the same media type. For growth assays testing the impact of succinylacetone (4,6-dioxoheptanoic acid, Sigma) treatment, strains were treated with 250 µM succinylacetone at days 0 and 12.
NO and low iron growth arrest assays were conducted as previously described [8]. In brief, log-phase Mtb cultures grown in aerated conditions were sub-cultured to OD600 = 0.1 in 12 ml 7H9, pH 7.0 or iron-depleted minimal medium + 100 µM 2’2’-dipyridyl, with a wash step as described above, in filter-capped T75 flasks laid flat. Cultures were then treated with 100 µM DETA NONOate 6 times across 30 hours, and growth was tracked over time via OD600 measurement or plating of serial dilutions on 7H10 agar plates for CFU quantification.
Mtb length measurements
To examine Mtb cell lengths, each of the strains carrying a constitutively expressed smyc’::mCherry reporter were grown under aerated conditions before inoculation at an OD600 = 0.1 in filter-capped T25 flasks laid flat containing 4 ml of: (i) 7H9, pH 7.0 (control condition), (ii) 7H9, pH 7.0 + 100 µM DETA NONOate (NO condition), (iii) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl (low iron condition), or (iv) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl + 100 µM DETA NONOate (NO + low iron condition). A wash step was carried out as described above prior to inoculation. Strains were grown for 3 days before samples were fixed in 4% paraformaldehyde (PFA) overnight and resuspended in phosphate-buffered saline (PBS) + 0.1% Tween 80.
For sequential exposure experiments, Mtb strains each carrying a constitutively expressed smyc’::mCherry reporter were grown under aerated conditions before inoculation, with a wash step, at an OD600 = 0.1 in filter-capped T25 flasks laid flat containing 4 ml of 7H9, pH 7.0 or iron-depleted minimal medium + 100 µM 2’2’-dipyridyl. After 3 days growth, the strains were sub-cultured to an OD600 = 0.1 in the same media type ± 100 µM DETA NONOate. After an additional 3 days growth, samples were fixed in 4% PFA overnight and resuspended in PBS + 0.1% Tween 80.
To test the impact of succinylacetone treatment on bacterial cell length, strains carrying a constitutively expressed smyc’::mCherry reporter were grown under aerated conditions before inoculation, with a wash step, at an OD600 = 0.1 in filter-capped T25 flasks laid flat containing 4 ml of iron-depleted minimal medium + 100 µM 2’2’-dipyridyl + 100 µM DETA NONOate (NO + low iron) ± 500 µM succinylacetone. Strains were grown for 3 days before samples were fixed in 4% PFA overnight and resuspended in PBS + 0.1% Tween 80.
For all length measurements, samples were mounted using ProLong Glass antifade (Invitrogen) and imaged as previously described [85]. Bacterial lengths were measured in Volocity, with a median of 259 cells (minimum 163 cells) total measured per strain per condition across 3–4 experiments.
Mass spectrometric analysis of lipid extracts
Log-phase Mtb strains grown in aerated conditions were sub-cultured to OD600 = 0.1 in 12 ml of: (i) 7H9, pH 7.0, (ii) 7H9, pH 7.0 + 100 µM DETA NONOate (NO condition), (iii) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl (low iron condition), or (iv) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl + 100 µM DETA NONOate (NO + low iron condition). Strains were grown for 3 days before pelleting and extraction with 100% methanol. Lipids were subsequently extracted from Mtb cell pellets by chloroform/methanol and purified by 1-butanol/water partitioning as previously described [86]. Final butanol phase was dried on a speed-vac concentrator and resuspended at 1 mg wet cell pellet equivalent per µl of water-saturated 1-butanol.
For matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we first spotted 0.5 µl of matrix (a saturated solution of α-cyano-4-hydroxycinnamic acid prepared in 70% acetonitrile with 0.1% trifluoroacetic acid in water) on a polished steel MTP target plate (Bruker #8280781) and allowed it to air-dry. We then spotted 0.5 µl of the lipid extract on top of the dried matrix and allowed it to air-dry. Another 0.5 µl of the matrix solution was then placed on top of the lipid sample and allowed to air-dry. Mass spectra were acquired using a UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) operated in reflective positive-ion mode with the laser intensity set at 80%. Spectra were collected by averaging 2000 laser shots per sample. The standard coproporphyrin III tetramethyl ester (Sigma-Aldrich, C7157) was used to identify and quantify the mass peak of tetramethyl coproporphyrin.
For LC-MS, lipid extract (3 µl in water-saturated 1-butanol, purified from 1.5 mg wet cell pellet) was dried by a speed-vac concentrator and resuspended in 10 µl of 10% (v/v) acetonitrile in water (solvent A). The lipid suspension was transferred to an autosampler vial. HPLC separation and MS were performed using an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo) with a Waters Acquity BEH C18 reversed-phase column (1.7 µm x 2.1 mm x 50 mm). 3 µl was injected, and the column was eluted at 0.100 ml/min with a binary gradient from 0% to 100% solvent B (100% acetonitrile): 2–12 min (0 →100% B); 12–14 min (100 → 0% B); 14–18 min (0% B). Spectra were collected in positive ion mode from m/z 197–2,000 at 1 spectrum/s. Internal calibration was performed with the EASY-IC system integral to the instrument. Fragmentation was performed on the peak of interest using higher energy collisional dissociation (HCD) at energies between 45–65 V. Fragmentation data in Thermo Fisher Compound Discoverer were used to determine the molecular identity.
Intrabacterial porphyrin and total heme measurements
Intrabacterial porphyrin and total heme measurements were conducted as previously described [57], with some modifications. Briefly, log-phase Mtb strains grown in aerated conditions were sub-cultured to OD600 = 0.1 in 12 ml of: (i) 7H9, pH 7.0 (control condition), (ii) 7H9, pH 7.0 + 100 µM DETA NONOate (NO condition), (iii) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl (low iron condition), or (iv) iron-depleted minimal medium + 100 µM 2’2’-dipyridyl + 100 µM DETA NONOate (NO + low iron condition). After 3 days growth, samples were normalized to an OD600 = 2 and washed twice, first with UltraPure H2O (Invitrogen) + 0.1% Tween 80, then with UltraPure H2O, before resuspension in 1 ml PBS. After resuspension, 500 µl of cells were pelleted and frozen at -80°C overnight. For the assay, cell pellets were thawed and resuspended in 500 µl of 20 mM oxalic acid and stored at 4°C overnight. Then, 500 µl of 2 M oxalic acid was added to each cell suspension, and the sample divided into two 500 µl aliquots. One was stored in the dark at room temperature as a blank and to obtain the intrabacterial porphyrin measurements. The other set was boiled at 100°C for 30 minutes covered (total heme measurements). Both sets were centrifuged at 21,100 x g for 2 minutes to remove cell debris. For each sample, 200 µl of supernatant was plated in technical duplicate in a 96-well black flat-bottom plate, and fluorescence measured using a Biotek Synergy Neo2 multi-mode microplate reader with excitation at 400 nm and emission at 608 nm. Heme levels were calculated by subtracting fluorescence of intrabacterial porphyrin (blank samples) from fluorescence of boiled samples. Values are reported as arbitrary fluorescence units (AFUs).
Intrabacterial heme reporter assay
Labile intrabacterial heme was measured using Mtb strains carrying the HS1 reporter as described previously [57,59,81]. Briefly, strains were grown as in the intrabacterial porphyrin and total heme assay. After 3 days growth, samples were normalized to an OD600 = 1 in 500 µl PBS. To measure fluorescence, 200 µl of supernatant was plated in technical duplicate in 96-well black flat-bottom plate, and fluorescence was measured using a Biotek Synergy Neo2 multi-mode microplate reader. Excitation was 480 nm and emission at 510 nm for eGFP, with excitation at 580 nm and emission at 620 nm for mKATE2. Three reads were taken over 10 min to account for variability in fluorescence over time and averaged as one ratio.
Supporting information
S1 Fig. Colony forming unit (CFU) counts corroborate the importance of Rv3839-Rv3840 for maintenance of NO and iron stress-induced growth arrest of Mtb.
(A) Growth of Δrv3839-rv3840 Mtb is less restricted under iron limitation than WT Mtb. WT, Δrv3839-rv3840, and rv3839-rv3840* (complemented strain) Mtb were cultured in 7H9, pH 7.0 media (control) or iron-depleted minimal media with 100 µM 2’2’-dipyridyl (low iron) for 12 days. At day 12, the strains were sub-cultured at OD600 = 0.05 into the same medium. Aliquots from the low iron condition were plated for CFUs at day 12 (after the sub-culture to the same starting OD600 = 0.05 for all strains) or 18, and from the control condition at day 18. Data are shown as means ± SEM from 3-4 experiments. (B) Δrv3839-rv3840 Mtb prematurely exits NO and low iron stress-induced growth arrest. WT, Δrv3839-rv3840, and rv3839-rv3840* Mtb were grown in aerated conditions in 7H9, pH 7.0 and sub-cultured in either 7H9, pH 7.0 (control) or in iron-depleted minimal media with 100 µM 2’2’-dipyridyl and treated with 6 doses of 100 µM DETA NONOate over 30 hours (NO + low iron). Aliquots from the NO + low iron condition were plated for CFUs at 30 hours (after the last dose of DETA NONOate) or day 12, and from the control condition at day 12. Data are shown as means ± SEM from 3-4 experiments. p-values in both (A) and (B) were obtained with a one-way ANOVA with Tukey’s multiple comparisons. N.S. not significant, * p < 0.05, ** p < 0.01. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
https://doi.org/10.1371/journal.pgen.1012202.s001
(TIF)
S2 Fig. Complementation with rv3839, but not rv3840, shifts the growth profile of ∆rv3839-rv3840 Mtb in NO+ low iron conditions back towards WT Mtb.
WT, Δrv3839-rv3840, rv3839-rv3840*, rv3839*, and rv3840* Mtb were grown in aerated conditions in 7H9, pH 7.0 and sub-cultured in either 7H9, pH 7.0 (A, control), or in iron-depleted minimal media with 100 µM 2’2’-dipyridyl and treated with 6 doses of 100 µM DETA NONOate (B, NO + low iron) over 30 hours (shaded area). Bacterial growth was tracked by OD600 every day for 24 days. Data are shown as means ± SEM from 4-8 experiments. p-values in (B) were obtained with unpaired t-tests with Welch’s correction, comparing rv3840* to ∆rv3839-rv3840 Mtb. All comparisons of rv3839* to rv3839-rv3840* Mtb were non-significant. * p < 0.05. The numerical data underlying the graphs shown in this figure are provided in S1 Data.
https://doi.org/10.1371/journal.pgen.1012202.s002
(TIF)
S1 Table. Comparison of effect of iron limitation on genes differentially expressed ≥1 log2-fold in NO conditions in WT Mtb (4 hour exposure).
https://doi.org/10.1371/journal.pgen.1012202.s003
(XLSX)
S2 Table. Comparison of effect of NO on genes differentially expressed ≥1 log2-fold in low iron conditions in WT Mtb (4 hour exposure).
https://doi.org/10.1371/journal.pgen.1012202.s004
(XLSX)
S3 Table. Genes differentially expressed ≥1 log2-fold in NO+ low iron conditions compared to control conditions in WT Mtb (4 hour exposure).
https://doi.org/10.1371/journal.pgen.1012202.s005
(XLSX)
S1 Data. Numerical data underlying the presented graphs.
Excel file with numerical data underlying graphed data presented.
https://doi.org/10.1371/journal.pgen.1012202.s006
(XLSX)
Acknowledgments
We thank Amit Reddi (Georgia Institute of Technology) for generously providing the HS1 intrabacterial heme reporter plasmid, and acknowledge support from the University of Massachusetts Amherst IALS Mass Spectrometry Core (Director, Stephen Eyles). We thank Yue Chen for help with RNAseq analysis, and all members of the Tan laboratory for helpful discussions.
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