Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro

Background The ESX secretion system, also known as the Type VII secretion system, is mostly found in mycobacteria and plays important roles in nutrient acquisition and host pathogenicity. One of the five ESXs, ESX-3, is associated with mycobactin-mediated iron acquisition. Although the functions of some of the membrane-associated components of the ESX systems have been described, the role of by mycosin-3 remains elusive. The esx-3 gene cluster encoding ESX-3 in both Mycobacterium tuberculosis and Mycobacterium smegmatis has two promoters, suggesting the presence of two transcriptional units. Previous studies indicated that the two promoters only showed a difference in response under acid stress (pH 4.2). This study aimed to study the effect of a mycosin-3 deletion on the physiology of M. smegmatis and to assess the promoter activities in wildtype, mycosin-3 mutant and complementation strains. Results The gene mycP 3 was deleted from wildtype M. smegmatis via homologous recombination. The mycP 3 gene was complemented in the deletion mutant using each of the two intrinsic promoters from the M. smegmatis esx-3 gene cluster. The four strains were compared in term of bacterial growth and intracellular iron content. The two promoter activities were assessed under iron-rich, iron-deprived and iron-rescued conditions by assessing the mycP 3 expression level. Although the mycP 3 gene deletion did not significantly impact bacterial growth or intracellular iron levels in comparison to the wild-type and complemented strains, the two esx-3 promoters were shown to respond inversely to iron deprivation and iron rescue. Conclusion This finding correlates with the previously published data that the first promoter upstream of msmeg0615, is upregulated under low iron levels but downregulated under high iron levels. In addition, the second promoter, upstream of msmeg0620, behaves in an inverse fashion to the first promoter implying that the genes downstream may have additional roles when the iron levels are high. Electronic supplementary material The online version of this article (doi:10.1186/s13104-017-2752-0) contains supplementary material, which is available to authorized users.


Background
Tuberculosis, whose etiological agent is Mycobacterium tuberculosis (Mtb), was one of the top ten causes of death worldwide in 2015 (1.4 million deaths) [1]. Such a tremendous medical burden is exacerbated by the emergence of multidrug-resistant TB (MDR-TB), thus new drug target is urgently needed for new anti-TB treatment development. An ideal drug target should be responsible for essential metabolic functions in Mtb and it should have no homology to human proteins to minimize drug toxicity to the host. The Type VII secretion systems, or ESXs (with five members ESX-1 to -5), are a signature group of protein secretion systems in mycobacteria. They have been extensively studied, especially ESX-1, -3 and -5, because they are responsible for bacterial survival and pathogenicity during Mtb infection [2]. Unlike ESX-1 and ESX-5, ESX-3 is most conserved in both pathogenic and environmental mycobacteria, and it is associated with Open Access mycobactin-mediated (an iron chelator secreted by the mycobacteria) iron acquisition [3][4][5], and also affects heme acquisition [6]. Abolishing both pathways would be a promising anti-tuberculosis therapeutic strategy [7].

BMC Research Notes
The expression of the esx-3 gene cluster in M. tuberculosis and M. smegmatis is governed by two promoters, the first located upstream of the first gene of the cluster (msmeg_0615) and the second located upstream of the esx genes (msmeg_0620 and msmeg_0621) [8] (Fig. 1). The first promoter is controlled by the transcriptional regulator IdeR in an iron-dependent manner [9]. The regulator of the second promoter has not been identified. Previously, the activities of the two promoters in M. smegmatis were only shown to differ in response to acid stress (pH 4.2) and no difference was observed under iron-rich or iron-deprived conditions [8].
Compared to the other membrane protein components of the ESXs consisting of EccBCDE which constitute the core membrane structure [10], the roles played by mycosins remain elusive [2]. It was found that MycP 1 cleaves EspB upon secretion, possibly facilitating the maturation of ESX substrates [11]. The stability of both the ESX-1 and ESX-5 complex could be compromised if MycP 1 and MycP 5 respectively, were absent, suggesting that mycosins are crucial for the integrity and functioning of the ESX [12]. However, how they facilitate the substrate secretion for their respective ESX systems remained poorly understood. The functional study on MycP 3 is even more limited with no functional data published in the literature. In this report, M. smegmatis was used as a model organism in which mycP 3 was deleted to generate the deletion mutant, and the mycP 3 complementation strains were generated from the mutant by introducing mycP 3 downstream of each of the two esx-3 promoters. This study investigated the impact of the mycP 3 deletion on bacterial growth and intracellular iron content under different iron conditions, as well as the activities of the two promoters under these conditions. No. 746452), 5% (v/v) glycerol, and 0.05% (v/v) Tween-80) were also used to monitor bacterial growth under iron-limiting condition. The iron depleted 7H9 and Sauton's media were prepared by mixing iron-free 7H9 and Sauton's media (omitting MgSO 4 ·6H 2 O) with 10 g/L Chelex resin (Bio-Rad, USA, Catalogue No. 1422822), a chelating agent, for 48 h and then filter-sterilized and supplemented with sterile MgSO 4 ·6H 2 O (4 mM) before culturing M. smegmatis. Additionally, the iron deprivation rescuing of the M. smegmatis cultured in iron-deprived 7H9 or Sauton's media was achieved by supplementation of ferric ammonium citrate in the same concentration of normal 7H9 or Sauton's media.

Bacterial strains, culture media and plasmid DNA
The CloneJet1.2 vector (Thermofisher, USA, Catalogue No. K1231) was used for insert DNA amplification before cloning into the target vectors. The p2Nil suicide vector and the pGoal17 selection gene cassette [13] were used to generate ΔMycP 3ms , and pMV306 [14] was used for MycP 3 complementation (the three vectors were provided by Rob Warren as a gift).

Construction of M. smegmatis mycP 3 gene knockout strain and corresponding complemented strains
Homologous DNA recombination was used to generate the ΔMycP 3ms strain (unmarked in-frame deletion) as previously described [13]. One thousand basepair (bp) fragments upstream (UP) and downstream (DOWN) of mycP 3ms gene (MSMEG_0624) were amplified using Phusion ® DNA polymerase (ThermoFisher, USA, Catalogue No. F532S) with two pairs of primers ( Table 1). The thermo-cycling conditions for producing these two PCR products were as follows: initial denaturation step at 95 °C for 30 s; 40 cycles of amplification at 95 °C for 5 s followed by 30 s at 60 °C and 1 min at 72 °C; final elongation step at 72 °C for 7 min. The UP and DOWN PCR fragments were blunt-end ligated into pJet1.2 vector individually according to the manufacturer's instructions. The UP and DOWN DNA inserts were restriction digested out of pJet1.2 by HindIII/XhoI and XhoI/BamHI restriction enzyme pairs respectively. The DNA inserts were simultaneously ligated into p2Nil, previously digested with HindIII and BamHI, via three-way cloning (three pieces of DNA joining together) using T4 DNA ligase (Promega, USA, Catalogue No. M1801), resulting in recombinant p2Nil-UP-DOWN plasmid DNA. The selection gene cassette (P Ag85 -lacZ P hsp60 -sacB) from pGOAL17 was inserted at the PacI restriction site p2Nil-UP-DOWN plasmid DNA and the final construct was electroporated into M. smegmatis mc 2 155 cells. Blue single-crossover colonies were selected on LB agar supplemented with 50 μg/mL kanamycin (Sigma-Aldrich, USA, Catalogue No. 17151) and 0.2% X-gal (Sigma-Aldrich, USA, Catalogue No. 11680293001). The colonies were picked and passaged in LB media in the absence of kanamycin to induce a second crossover event. Double crossover colonies were selected on LB agar supplemented with 5% sucrose (Sigma-Aldrich, USA, Catalogue No. E001888) and X-gal. White colonies were further screened by colony PCR using screening primers (Table 1) to distinguish between WT and ΔMycP 3ms strains. The colony PCR thermo-cycling conditions were as follows: initial denaturation step at 95 °C for 30 s; 40 cycles of amplification at 95 °C for 5 s followed by 30 s at 58 °C and 1 min at 72 °C; final elongation step at 72 °C for 7 min. The WT PCR product was approximately 1600 bp while that of the ΔMycP 3ms was about 200 bp (Additional file 1: Figure S1a).
The M. smegmatis esx-3 gene cluster contains two promoters, namely pr1 and pr2 which are upstream of the MSMEG_0615 and the MSMEG_0620 genes, respectively ( Fig. 1) [8]. Both promoters were used to make complementation constructs expressing MycP 3 as previously described [15]. The PCR thermo-cycling conditions for making the pr1, pr2 and mycP 3 respectively are as follows: initial denaturation step at 95 °C for 5 min, 40 cycles of amplification at 95 °C for 5 s followed by 30 s at 59 °C (pr1), 60 °C (pr2), and 62 °C (mycP 3 ) and 1 min of elongation step at 72 °C; final elongation step at 72 °C for 7 min. The four pairs of primers for making the two complementation constructs in integrative pMV306 plasmid DNA are given in Table 1. The reverse primer sequences for amplifying the two promoters are partially complementary to the sense primers of mycP 3 to facilitate single-joint PCR [16] connecting pr1/pr2 and MycP 3ms . The thermo-cycling condition for single-joint PCR is as follows: initial denaturation step at 95 °C for 5 min, an annealing step at 55 °C for 15 min and the last elongation step at 72 °C for 3 min. The final joined PCR products were amplified using the following thermo-cycling condition: initial denaturation step at 95 °C for5 min, 40 cycles of 95 °C for 5 s followed by 62 °C for 30 s and elongation step at 72 °C for 2 min, and final elongation step at 72 °C for 7 min. The final PCR products, named pr1-mycP 3ms and pr2-mycP 3ms were ligated into the pMV306 vector respectively using T4 DNA ligase. The recombinant pMV306-pr1-mycP 3 and pMV306-pr2-mycP 3 plasmids were electroporated into the M. smegmatis ΔMycP 3ms mutant strain to generate two MycP 3 complementation strains, ΔMycP 3ms ::pr1mycP 3ms and ΔMycP 3ms ::pr2mycP 3ms . The genetic integrity of the WT, ΔMycP 3ms and two complementation strains were confirmed by colony PCR (See Additional file 1: Figure S1a). The thermo-cycling condition for the colony PCR was the same as that of the WT and KO strains except the annealing temperature was at 62 °C. The eccE 3 gene is directly downstream of the mycP 3 gene with a tetra-nucleotide overlap. The expression level of eccE 3 gene was assessed via RT-qPCR to ensure there was no polar effect from mycP 3 deletion (See Additional file 1: Figure S1b).

Bacterial growth under iron-rich and iron-deprived conditions
M. smegmatis WT, ΔMycP 3ms , ΔMycP 3ms ::pr1MycP 3ms and ΔMycP 3ms ::pr2MycP 3ms strains were cultured in 7H9 broth, iron-free 7H9 broth and Sauton's media, iron-chelated 7H9 and Sauton's media from a starting OD 600nm of 0.01. They were incubated at 37 °C with a rotating rate of 200 rpm for 48 h during which the OD 600nm reading was taken every 3 h. Complete iron depletion of the culture was reached by sub-culuring the bacteria three times in iron-free 7H9 or Sauton's media and then finally into the iron chelated 7H9 or Sauton's media. The growth curves were performed in biological triplicate.

Intracellular iron quantitation
Intracellular iron quantitation was performed as previously described [17]. Fifty millilitres of bacterial cultures at mid-log phase (OD 600nm of 0.7-0.  sigA was selected as the reference gene due to its constitutive expression [18]. The expression of all genes of interest was normalized against that of sigA in the same RNA sample, which was done by dividing the number of cDNA copy number of mycP 3 by that of sigA.

ESX-3 promoter activity in response to iron levels
The promoter activity of the ESX-3 promoters in response to iron levels was assayed using RT-qPCR of the gene expression levels of mycP 3 in WT, ΔMycP3 ms , ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms strains under normal 7H9, iron deprived 7H9 and iron rescued 7H9 media. ΔMycP3 ms strain acted as the negative control.

Statistical analysis
Differences of intracellular iron concentrations and gene expression levels of mycP 3 between WT ms , ΔMycP3 ms and two complementation strains under different iron concentrations were evaluated by Two-way ANOVA using GraphPad Prism 5 software. The comparison was considered significant when p value is smaller than 0.05.

Mycobacterium smegmatis ΔMycP 3 mutant showed similar growth as the WT under low iron conditions
MycP 3 is an important component of the ESX-3 protein secretion system although its detailed function has not been revealed. The growth profiles of Mycobacterium smegmatis WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms were assessed under low iron and iron-deprived conditions in 7H9 and Sauton's media to see whether the knockout of mycP 3 gene would have a negative impact on the bacterial growth. However, no major difference in the exponential growth rate and the starting point of exponential growth phase was observed between the strains in these media, although ΔMycP 3 mutant appears to have a slightly lower growth rate and OD 600 reading at plateau than the WT and two complementation strains in these media except for iron-depleted Sauton's medium (Figs. 2, 3). The differences of endpoint bacterial loads of all strains were observed, however, it was possibly due to bacterial clumping making the OD 600nm reading inaccurate. Clumping of all six cultures started to become visible when the growth reached plateau. It persisted although a range of Tween-80 concentrations and sonication intensity were applied to the culture (Results not shown).

The mycP 3 gene does not impact on bacterial intracellular iron level
Deletion of the mycP 3 gene did not significantly affect the growth of ΔMycP 3ms under low iron conditions. But this does not rule out an impact on iron homeostasis, we therefore investigated whether mycP 3 influenced bacterial iron acquisition by measuring intracellular iron levels. No significant differences between the strains were detected under three culturing conditions (Fig. 4). Intracellular iron levels dropped dramatically for all four strains after they were sub-cultured three times in Fe-free 7H9 and finally in Fe-depleted 7H9, showing an approximately 75% reduction. In contrast, intracellular iron level rose to a significantly higher level (approximately twofold) when iron was added to the Fe-depleted 7H9 media in the same concentration as conventional 7H9 medium.

Functional analysis of the ESX-3 promoters
We used both promoters from the esx-3 gene cluster to construct the MycP 3 complementation strains to see how the activities of the two promoters in the ESX-3 gene cluster in M. smemgatis differ in different iron levels. The promoters were incorporated into the two complementation strains separately and the strains did not show significant differences in either bacterial growth or mycobacterial intracellular iron levels under different iron concentrations (Fig. 4). We then assessed the promoter activity by determining the mycP 3 gene expression levels in the four strains under different iron conditions (Fig. 5). In iron rich conditions, mycP 3 under control of the first promoter was expressed at similar levels as observed in WT ms while mycP 3 expression is highly elevated under control of the second promoter. This expression profile was inverted in iron-deprived media, and restored when iron was added to the iron-deprived media (Fig. 5).

Discussion
This study investigated the effect of the deletion of mycP 3 , an important component of the ESX-3 protein secretion system, on the physiology of Mycobacterium smegmatis. ESX-3 has been implicated in iron homeostasis via the mycobactin and heme iron acquisition systems as well as virulence through the secretion of the EsxG-EsxH and PE5-PPE4 protein pairs, and is essential for in vitro growth of M. tuberculosis [19,20]. It could be a source of potential drug targets for anti-TB drug development.
The deletion of mycP 3 alone did not affect the growth of M. smegmatis significantly or disrupt iron homeostasis, which correlates with the findings from Siegrist and colleagues [19]. However, deletion of ESX-3 makes the mycobacteria unable to grow under low iron conditions [4]. MycP 3 possibly does not affect the secretion of the ESX-3 substrates significantly [19]. The deletion of mycP 3 might influence mycobactin-mediated iron acquisition, but the iron acquisition overall was not disrupted because M. smegmatis possesses an alternative exochelin-mediated iron acquisition pathway. The exochelin biosynthesis and transporters are distinct from those of mycobactin [7] therefore exochelin-mediated iron acquisition may have compensated for any potential malfunction of mycobactin-mediated iron acquisition. Interestingly, a double mutant with mycP 3 deleted and exochelin pathway disabled does not affect the bacterial growth in iron deprived media [19], suggesting that MycP 3 is dispensable in the function of the ESX-3. Surprisingly, the intracellular iron levels of all of the studied M. smegmatis strains, when iron rescued, were about twofold higher than when cultured in normal 7H9 media. This might be explained by the hypothesis that cell envelope-associated mycobactins serve as temporary storage for iron ions [21]. We reason that the production of mycobactin was suppressed under iron rich conditions through regulation by IdeR [9,22] when first cultured in commercially available normal 7H9; but derepressed during iron deprivation to produce a large amount of mycobactin which was transported into the cell envelope. When iron was added to rescue the iron-starved bacterial culture, normal iron uptake was restored meanwhile the abundant cell envelope mycobactins were able to bind the available iron resulting in high cellular iron levels. In addition, the insignificant impact of the mycP 3 gene knockout on the in vitro physiology of M. smegmatis is supported by the comparative proteomics between the WT ms and ΔMycP 3ms under iron rich condition (Fang et al. unpublished results).
The M. smegmatis ESX-3 is expressed under the control of two promoters [8]. Previous studies did not find major differences in the activity of the promoters under various culturing conditions including iron-rich and iron-deprived, except during acid stress [8]. In this study, the two promoters responded to different iron levels in an inverse fashion. A possible reason for the discrepancy ΔMycP3ms::pr2MycP3ms ΔMycP3ms::pr1MycP3ms + Hyg ΔMycP3ms::pr2MycP3ms + Hyg Fig. 3 Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-depleted 7H9 (a), and Fe-depleted Sauton's media (b). The growth curves were done in triplicate, error bars show standard deviation between our and Maciag's data is possibly due to the different experiment setup as they used the expression of the gene directly downstream of the promoters (msmeg0615 and msmeg0620 respectively) as the reporters while we used mycP 3 gene expression as the reporter and the promoter-mycP 3 couple was independent of the esx-3 cluster in the M. smegmatis genome. The transcription of mycP 3 from the promoter (pr1) in the ΔMycP3 ms ::pr1MycP3 ms strain responds to different iron levels in the same fashion as WT ms (Fig. 5) implying that the transcription of the mycP 3 gene is controlled by the first promoter (pr1) even though the gene is downstream of the 2nd promoter (pr2) (Fig. 1). However, the transcription level of recombinant mycP 3 under the control of the first promoter is significantly higher than endogenous mycP 3 expression in WT ms . In the integrative complementation vector, the mycP 3 gene was positioned directly downstream of the promoter, rather than being 9 genes downstream as in the WT ms genome. Such an artificial genetic arrangement brings the gene closer to the transcription start site thereby increasing the rate of transcription, increasing the number of transcripts [23]. The second promoter is not regulated in the same manner as the first, suggesting its independence from IdeR regulation. It was proposed that the first promoter in the esx-3 gene cluster is responsible for the transcription of the entire operon while the second promoter only influences the transcription of the 6 genes downstream of it [8]. Even when iron-deprived, the expression of mycP 3 from the second promoter reached the same level as in WT ms . When iron was sufficient, the second promoter was up-regulated by an unknown mechanism, possibly to express the downstream ESX pair, MSMEG_0620 and MSMEG_0621, and secreted protein EspG 3 (MSMEG_0622), which may be required in other metabolic pathways or even nutrient acquisition [24].

Conclusion
Our study confirms that MycP 3 is dispensable to the bacterial growth or iron homeostasis in M. smegmatis as previously shown in other studies. The two promoters in esx-3 gene cluster respond inversely to iron-rich and iron-deprived conditions which was not observed previously implying that the two promoters are not redundant and the second promoter may regulate the production of the downstream genes for other metabolic activities Fig. 4 The comparison of the intracellular iron levels in WT ms , ΔMycP3 ms , ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms strains under 7H9, Fe-depleted 7H9 and Fe rescued 7H9 media. The error bars show standard error of the mean (n = 4). The p values obtained using two-way ANOVA statistical analysis between different culturing conditions for all four strains are smaller than 0.0001 (***), an example is shown for the WT ms in the bacteria which is of great interest for further investigation.