Open Access

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

  • Zhuo Fang1Email author,
  • Mae Newton-Foot2, 3,
  • Samantha Leigh Sampson1 and
  • Nicolaas Claudius Gey van Pittius1
BMC Research Notes201710:426

https://doi.org/10.1186/s13104-017-2752-0

Received: 31 January 2017

Accepted: 23 August 2017

Published: 25 August 2017

Abstract

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.

Keywords

Iron ESX Tuberculosis Mycosin-3 Promoter

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 mycobactin-mediated (an iron chelator secreted by the mycobacteria) iron acquisition [35], and also affects heme acquisition [6]. Abolishing both pathways would be a promising anti-tuberculosis therapeutic strategy [7].

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].
Fig. 1

Genetic organization of the esx-3 gene cluster in Mycobacterium smegmatis. The positions of the promoters, pr1 and pr2, are indicated(Adapted from [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 MycP1 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 MycP1 and MycP5 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 MycP3 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.

Methods

Bacterial strains, culture media and plasmid DNA

Escherichia coli XL-1 blue (Stratagene, USA, Catalogue No. 200249) was used for manipulating and propagating recombinant plasmid DNA. Mycobacterium smegmatis mc2 155 (a gift from Rob Warren, South Africa) was used as the parent wildtype strain (WTms) from which the MycP3 deletion mutant (ΔMycP3ms) was derived, and as template to generate two complementation strains (ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms). E. coli was cultured using both Lysogeny Broth (LB) liquid media [1% (w/v) tryptone (Merck, USA, Catalogue No. 107213), 0.5% (w/v) yeast extract (Merck, USA, Catalog No. 113885), and 1% (w/v) sodium chloride (Sigma-Aldrich, USA, Catalogue No. S7653)] and solid media [LB liquid media supplemented with 1.5% (w/v) bacterial agar (Sigma-Aldrich, USA, Catalogue No. A5306)]. M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516). Fe-free 7H9 (omitting ferric ammonium citrate) and Fe-free Sauton’s medium (3.5 mM KH2PO4 (Sigma-Aldrich, USA, Catalogue No. NIST200B), 25 mM l-asparagine (Sigma-Aldrich, USA, Catalogue No. A0884), 10 mM citric acid (Sigma-Aldrich, USA, Catalogue No. 791725), 4 mM MgSO4·6H2O (Sigma-Aldrich, USA, Catalogue 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 MgSO4·6H2O) 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 MgSO4·6H2O (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.

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 ΔMycP3ms, and pMV306 [14] was used for MycP3 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 ΔMycP3ms 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 (PAg85-lacZ Phsp60-sacB) from pGOAL17 was inserted at the PacI restriction site p2Nil-UP-DOWN plasmid DNA and the final construct was electroporated into M. smegmatis mc2 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 ΔMycP3ms 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 ΔMycP3ms was about 200 bp (Additional file 1: Figure S1a).
Table 1

Primers used for ΔMycP3ms generation, MycP3 complementation and RT-qPCR assay

Experiment

Gene related

Primer name

Primer sequence

Restriction site (underlined)

ΔMycP3ms Generation

mycP 3

UP forward

5′-AAGCTTTCCCACGCACATCG-3′

HindIII

UP reverse

5′-CTCG AGATCACCTGTCGAGCACG-3′

XhoI

DOWN forward

5′-CTCGAGATGACCGCCCGGATAGC-3

XhoI

DOWN reverse

5′-GGATCCCCGGTCTCGGTGAC-3′

BamHI

ΔMycP3ms Construct verification

mycP 3

mycP 3 screening forward

5′-GCTCAACCCGAAGATC GCCTC-3′

N/A

mycP 3 screening reverse

5′-AGGAACATGCCTTTCCACCAGG-3′

N/A

MycP3 Complementa-tion

mycP 3

pr1 forward

5′-CCATGGGACGCTGAACGAGTGTTTAC-3′

NcoI

pr1 reverse

5′-GACGCCCAGACTCTTGTGGATCACATCGCGGTCGACCCGGGGCG-3′

N/A

mycP 3 -pr1 forward

5′-CGCCCCGGGTCGACCGCGATGTGATCCACAAGAGTCTGGGCGTC-3′

N/A

mycP 3 reverse

5′-AAGCTTTCATGTGGTCTTGTCCTTCC-3′

HindIII

pr2 forward

5′-CCATGGACGTGGGACGGCGACGA GAATC-3′

NcoI

pr2 reverse

5′-GACGCCCAGACTCTTGTGGATCACGACTGTTTCC TTTCGAAGGTGGTG-3′

N/A

mycP 3 -pr2 forward

5′-CACCACCTTCGAAAGGAAAC AGTCGTGATCCACAAGAGTCTGGGCGTC-3′

N/A

RT-qPCR assay

sigA

sigA forward

5′-GGGCGTGATGTCCATCTGCT-3′

N/A

sigA reverse

5′-GTATCCCGGTGCATGGTC-3′

mycP 3

mycP 3 forward

5′-GGATCATCGCGTTCGTGGGTAC-3′

mycP 3 reverse

5′-GTCTTGTCCTTCCGACGGTAGG-3′

eccE 3

eccE 3 forward

5′-GAGCCGTTGTTGACGGTTTG-3′

eccE 3 reverse

5′-GTTCGGTCGACAACGGGTTC-3′

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 MycP3 as previously described [15]. The PCR thermo-cycling conditions for making the pr1, pr2 and mycP3 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 mycP3 to facilitate single-joint PCR [16] connecting pr1/pr2 and MycP3ms. 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-mycP3ms and pr2-mycP3ms 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 ΔMycP3ms mutant strain to generate two MycP3 complementation strains, ΔMycP3ms::pr1mycP3ms and ΔMycP3ms::pr2mycP3ms. The genetic integrity of the WT, ΔMycP3ms 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, ΔMycP3ms, ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms strains were cultured in 7H9 broth, iron-free 7H9 broth and Sauton’s media, iron-chelated 7H9 and Sauton’s media from a starting OD600nm of 0.01. They were incubated at 37 °C with a rotating rate of 200 rpm for 48 h during which the OD600nm 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 (OD600nm of 0.7–0.9) was harvested by centrifugation. The cell pellet was washed twice with cold Tris–HCl buffer [5 mM Tris (Sigma-Aldrich, USA, Catalogue No. T3253), pH 7.6, 0.005% (v/v) Tween 80] and then mixed with equal volume of 0.1 mm diameter glass beads (Biospec, USA, Catalogue No. 11079101) and resuspended in 500 μL 50 mM NaOH (Sigma-Aldrich, USA, Catalogue No. S8045). The mixture was ribolyzed using a FastPrep®-24 ribolyzer (MP Biomedicals, USA) at 6.0 m/s for 30 s. This was repeated three times with 30 s incubation on ice between each repeat. The whole cell lysate was cleared by centrifugation at 12,000×g at 4 °C for 30 min. One hundred microliters of the whole cell lysate was used for the iron quantification assay and another 25 μL was used for protein quantitation using the RC-DC protein assay (Bio-rad, USA, Catalogue No. 5000121). For iron quantitation, the whole cell lysate was transferred into one well of a 96-well microtiter plate and mixed with 100 μL of 10 mM HCl (Sigma-Aldrich, USA, Catalogue No. H3162) and then 100 μL iron-releasing reagent [a freshly made solution of equal volumes of 1.4 M HCl and 4.5%, KMnO4 (Sigma-Aldrich, USA, Catalogue No. 1.09121) in distilled water]. This mixture was incubated at 60 °C for 2 h, cooled to room temperature, and 30 μL of iron-detecting agent [6.5 mM ferrozine (Sigma-Aldrich, USA, Catalogue No. 160601), 6.5 mM neocuproine (Sigma-Aldrich, USA, Catalogue. N1501), 2.5 M ammonium acetate (Sigma-Aldrich, USA, Catalogue No. A1542), and 1 M ascorbic acid (Sigma-Aldrich, USA, Catalogue No. A7506)] was added to the well and incubated for 30 min at room temperature. The absorbance was read at 550 nm on a photospectrometer. Ferric chloride (Sigma-Aldrich, USA, Catalogue No. F2877) was used as iron standards at the concentration of 10–80 μM in 50 mM NaOH. The iron concentration was normalized against the protein content, which was done by dividing the iron concentration by the protein concentration, resulting in the unit of nmol (of iron) per mg (of protein). The experiment was performed in triplicate.

RT-qPCR

Fifteen millilitres of each M. smegmatis strain was harvested at mid-log phase in normal 7H9, Fe-free 7H9 or Fe-rescued 7H9 by centrifugation. The supernatant was discarded and the pellet was resuspended in 1 mL FastRNA® Blue solution (MP Biomedicals, USA, Catalogue No. 6025-050) and ribolyzed as described above. The whole cell lysate was cleared by centrifugation at 12,000×g at 4 °C for 30 min, and 700 μL of the supernatant was transferred into a new 1.5 mL tube and thoroughly mixed with 300 μL chloroform (Sigma-Aldrich, USA, Catalogue No. C7559). The mixture was centrifuged at 12,000×g at 4 °C for 10 min. The top aqueous layer was transferred to a new 1.5 mL tube and mixed with 500 μL pre-chilled 100% ethanol (Sigma-Aldrich, USA, Catalogue No. 900642). The mixture was transferred onto the RNA purification column from NucleoSpin® RNA isolation kit (Macherey–Nagel, Germany, Catalogue No. 740955) and further total RNA purification was done according to manufacturer’s instructions. The quality of the total RNA was assayed using a Bioanalyzer (Agilent Technologies, USA) at the Central Analytical Facility (Stellenbosch University, South Africa).

Five micrograms of total RNA was treated with Turbo DNase (ThermoFisher, USA, Catalogue No. AM2238) according to the manufacturer’s instructions. One microgram of Turbo DNase-treated total RNA was used for cDNA synthesis using the PrimeScript™ 1st strand cDNA synthesis kit (TaKaRa, USA, Catalogue No. DRR037A) with the appropriate reverse primers (Table 1) as per manufacturer’s instructions. Quantitative PCR was conducted using SYBR® Premix Ex Taq™ mastermix (TaKaRa, USA, Catalogue No. RR82WR) on a Bio-Rad CFX96™ Real-Time PCR Detection System (Bio-Rad, USA) with the following cycling conditions: initial denaturation at 95 °C for 30 s; 39 cycles of amplification at 95 °C for 5 s followed by 30 s at 60 °C. The subsequent melt curve started with a denaturation step at 95 °C for 10 s and then a melting step from 65 °C to 95 °C with 5 s staying at each 0.5 °C interval. 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, ΔMycP3ms, ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms strains under normal 7H9, iron deprived 7H9 and iron rescued 7H9 media. ΔMycP3ms strain acted as the negative control.

Statistical analysis

Differences of intracellular iron concentrations and gene expression levels of mycP 3 between WTms, ΔMycP3ms 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.

Results

Mycobacterium smegmatis ΔMycP3 mutant showed similar growth as the WT under low iron conditions

MycP3 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, ΔMycP3 mutant and the two complementation strains, ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms 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 ΔMycP3 mutant appears to have a slightly lower growth rate and OD600 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 OD600nm 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).
Fig. 2

Growth curves of the WT, ΔMycP3 mutant and the two complementation strains, ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms under Fe-free 7H9 (a), Fe-free Sauton’s media (b). The growth curves were done in triplicate, error bars show standard deviation

Fig. 3

Growth curves of the WT, ΔMycP3 mutant and the two complementation strains, ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms under Fe-depleted 7H9 (a), and Fe-depleted Sauton’s media (b). The growth curves were done in triplicate, error bars show standard deviation

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 ΔMycP3ms 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.
Fig. 4

The comparison of the intracellular iron levels in WTms, ΔMycP3ms, ΔMycP3ms::pr1MycP3ms and ΔMycP3ms::pr2MycP3ms 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 WTms

Functional analysis of the ESX-3 promoters

We used both promoters from the esx-3 gene cluster to construct the MycP3 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 WTms 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).
Fig. 5

Gene expression analysis of mycP 3 in WTms, ΔMycP3ms, ΔMycP3ms::Pr1MycP3, and ΔMycP3ms::Pr2MycP3 strains under normal 7H9 (iron rich), Fe-free 7H9, and Fe-rescued 7H9 media. The results were normalized against the RNA copy number of sigA. The p values obtained using two-way ANOVA statistical analysis (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001, non-significant comparison is not shown)

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]. MycP3 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 MycP3 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 WTms and ΔMycP3ms 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 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-mycP3 couple was independent of the esx-3 cluster in the M. smegmatis genome. The transcription of mycP 3 from the promoter (pr1) in the ΔMycP3ms::pr1MycP3ms strain responds to different iron levels in the same fashion as WTms (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 WTms. In the integrative complementation vector, the mycP3 gene was positioned directly downstream of the promoter, rather than being 9 genes downstream as in the WTms 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 WTms. 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 EspG3 (MSMEG_0622), which may be required in other metabolic pathways or even nutrient acquisition [24].

Conclusion

Our study confirms that MycP3 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 in the bacteria which is of great interest for further investigation.

Abbreviations

WT: 

wildtype

ESX: 

ESAT-6 (6 kDa early secretory antigenic target) protein family secretion systems

Declarations

Acknowledgements

Not applicable.

Authors’ contributions

ZF and NCGvP conceived the study; SLS, MN and NCGvP supervised the study; ZF, MN and NCGvP designed experiments; ZF performed experiments and analysed data; All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its Additional file 1].

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This project was not funded by any specific Grant from Funding agencies in the public, commercial, or not-for-profit sectors.

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Authors’ Affiliations

(1)
DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, US/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Stellenbosch
(2)
Division of Medical Microbiology, Department of Pathology, Faculty of Medicine and Health Sciences, University of Stellenbosch
(3)
National Health Laboratory Services, Tygerberg Hospital

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