Mycobacterium tuberculosis complex is a genetically related group of Mycobacterium species that can cause tuberculosis. It characterizes for its ability to survive and grow within macrophages, in a stress environment provided with nutrient limitation and acidification^1-3. Its adaptation takes place through mechanisms that allow to detect its environment, regulating gene expression or repression during growth and reproduction⁴, favoring its persistence in the host⁵.

The cells can respond to external stimuli by initiating a process derived from the binding of an effector to a receptor joined with the membrane. This is a transduction mechanism in which stress factors can trigger a response through gene expression, preparing the bacteria during nutrient depletion^6,7. A fact that can be observed in vitro during the exponential phase and is decisive for adaptation to conditions present in the stationary phase and survival⁸. This involved that the cell accumulate protons from the environment to maintain pH homeostasis in the cytosol⁹ and polyphosphate to sustain energy¹⁰. In addition, glutamate also accumulates in M. tuberculosis and M. smegmatis¹¹ and asparagine¹², which is associated with induction dormancy of M. tuberculosis¹³.

The bacilli respond to stress conditions due to nutrient depletion, acid pH, and reactive oxygen and nitrogen species, caused by their growth in macrophages^7,8,14. In order to respond to these stimuli, mycobacteria secrete enzymes, such as ESAT-6 and CFP-10^2,15, which in the cells of the host, act in necrosis processes, such as lysis of alveolar epithelium and membranes of macrophages, induction of DNA fragmentation and permeability of mitochondrial membranes¹⁵. Among the metabolic functions of mycobacteria, they regulate the transfer of DNA¹⁶.

M. smegmatis mc²155, mutant of M. smegmatis wild-type strain, is 10 to 100 times more efficiently transformed with plasmid vectors, for use in the analysis of mycobacteria function¹⁷. Because of its fast growing, it has been proposed as model for understanding the changes that promote adaptation¹⁸ and persistence of pathogenic mycobacteria¹⁹. It shares 12 out of 19 virulence genes with M. tuberculosis²⁰, among them are esat-6 and cfp-10 genes, which encode corresponding proteins secreted by the Secretion Ejectosoma BOX²¹. Converse and Cox²² observed the effect of culture media in the ESX-1 secretion system, for which they suggest that this system can be sensitive to one or more culture media nutrients.

During the adaptation process, genes related to specific metabolic functions are expressed as a response to stress caused by nutrient depletion²³. The adaptation of cultured mycobacteria can be observed after the stationary phase, changing to fresh culture medium where they resume their activity; therefore, in the present study, the limiting nutrient conditions for the growth of M. smegmatis and its relationship with esat-6 gene expression was evaluated.

M. smegmatis mc²155, was grown at 37 °C in Middlebrook 7H11 agar. The initial aliquots of test culture and untreated control cultures were obtained by inoculum of a M. smegmatis colony in 10 ml of liquid Middlebrook 7H9 medium and was grown for 24 h until mid-log phase (0.8-1.0 DO at 600 nm).

The culture media used as control for comparing the metabolic behavior of bacilli and expression of esat-6 gene and Sauton’s medium without zinc, both supplemented with 0.5 and 6% of glycerol, respectively, and 0.05 % of Tween 80, for being media in which esat-6 gene expresses²². The initial inoculation was carried out using 100 μl of the initial culture in 225 ml flasks with 150 ml of liquid medium and were incubated at 37 °C and agitated at 250 rpm.

All assay culture media for nutrient limitation derived from HdB minimal medium; pH 7 was adjusted and 0.2 % of glycerol and 0.05 % of Tween 80 were added, except for depletion of C (HdB<C) to which glycerol was limited to 0.08 % (v/v). For the experiments in which nitrogen depletion was required (HdB<N), (NH₄)₂SO₄ was used at a concentration 100 times lower (0.15 mM) than the utilized for HdB minimal medium. For the experiments limited 100 times lower in Pi (HdB<Pi), K₂HPO₄ was added at a final concentration of 0.089 mM (0.0155 g L^-1) and NaH₂PO₄, at a concentration of 0.0708 mM (0.085 g L^-1), for replacing the loss of buffer capacity, 3-(Nmorpholino) propanesulfonic acid (MOPS) 50 mM was added²⁴. The growth and stationary phases of all cultures were monitored at 600 nm (OD600), until 144 h and final pH was recorded.

The predicted fragment incorporating esat-6 gene was obtained by amplification of M. smegmatis genomic DNA, using primers: F-5’ACAGGTATGGAATTTCGCCG-3’, R-5’-CAGGCAAACATTCCCGTGA-3’, which were designed using the Oligo TM Software DNA Star Program. For conducting the identification by enzymatic sequencing, the amplified product was purified by a protocol based on silica columns (QIAquick Gel Extraction Kit, QIAGEN®); the amplified fragment was observed in agarose gel at 1.2 % through a UV- light transilluminator and it was cut with a sterile scalpel, carrying out the aforementioned protocol. The quality and quantity of purified DNA was observed by agarose gel and this amplified sample was sent for sequencing. This process was carried out at the Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO) of the Centro de Investigación y Estudios Avanzados (CINVESTAV), Unidad Irapuato, Guanajuato, México, of the Instituto Politécnico Nacional (IPN), based on the ddNTPs method²⁵, using the 3730 XL DNA sequencer (Applied Biosystems, Foster City, CA) and the kit Big Dye Terminator 3.1 (Applied Biosystems, Foster City, CA).

For RNA extraction, M. smegmatis culture was adapted to the corresponding culture media until the beginning of the stationary phase (96 h). In this phase, 30 % of cells were cultured, washed with PBS and placed in 150 mL of fresh culture medium under the same nutritional conditions and they were incubated for 14 h. RNA was extracted using TRlzol (Invitrogen®); it was observed by electrophoresis in agarose gels (1.2 %) using MOPS X (200 mM of MOPS, 50 mM of sodium acetate and 10 mM of EDTA, pH 7), and 3.15 % of formaldehyde. Electrophoresis was ran in TAE buffer solution (40/1 mM pH 8.0).

Northern blot hybridization was performed starting from 8 to 12 ng of extracted RNA, capillary transferred onto nylon membranes, sodium citrate and sodium chloride 20X. DNA was fixed using ultraviolet light for 5 min. The probe was derived from fragments of esat-6 gene, amplified by PCR and labelled as biotinylated alkaline phosphatase (Gene Images Alkphos Direct Labelling and Detection System-GE Health Care®). Hybridization was performed all night and was revealed by autoradiography (Hyperfilm-MP autoradiography, Amersham Pharmacia Bioscience®)²⁶.

The growth and pH data were obtained from six repetitions and Student’s t- test was used to express the results of the mean, standard error of the mean and corresponding comparisons between all M. smegmatis cultures²⁷.

In the testing culture media, M. smegmatis was observed with lower growth in HdB<N, HdB<C (0.659 and 1.697) media, in comparison to HdB minimal culture and HdB<Pi (2.170 and 1.982) (P<0.05). In addition, M. smegmatis in HdB<Pi has similar growth in HdB minimal culture (P>0.05) (Figure 1). This is indicative of a continuous growth in spite of stress conditions due to a decrease 100 times Pi. With the exception of HdB<Pi culture, in the rest of the testing and control media used, M. smegmatis resume its growth while changing it to a fresh culture medium, after 144 h of culture.

Figure 1 M. smegmatis grown until 144 h in culture media used for experiments: HdB<N (0.659±0.12), Sauton (1.559±0.03), HdB<C (1.697±0.07), HdB<Pi (1.982±0.11) HdB minimal (2.170±0.12) and 7H9 (2.638±0.12). 

In HdB<Pi culture medium, a relationship between growth, loss of acid-alcohol resistance of M. smegmatis, and pH was observed. That is, by higher number of culture hours (144), pH was decreased (5.4 ± 0.16), and the mycobacteria showed incapacity to recuperate its metabolic activity when the culture was changed to a fresh medium. This was observed in cumulus cells stained with the characteristic color of Ziehl-Neelsen technique, which disappeared from 100 h, until all cells were stained blue-colored (Figures 2a and 2b).

Figure 2 M. smegmatis a) Loss of acid-alcohol resistance in HdB<Pi culture medium through 96 h. b) Same culture changed to fresh medium 1 h after, where recovery of capacity of response to acid-alcohol is observed. 

With the exception of HdB<Pi culture medium, mycobacteria in limited nutrient culture media have a slow growth until they reach the stationary phase. Additionally, bacteria grown in HdB<P gradually lose their resistance to acidalcohol from 96 h, not recovering it until 144 h of culture, when bacteria were changed to fresh medium. For which the 96 h were taken into consideration for changing M. smegmatis to fresh culture media, extract total RNA after 12 ± 1 h (Figure 3A) and perform Northern hybridization. This assay reveals that M. smegmatis cultured in HdB<C, HdB<N and HdB minimal medium, does not express esat-6 gene. However, the culture limited 100 times in Pi (Figure 3B, lane 7) did showed the expression, in a similar way as the culture media used as control (7H9 and Sauton, lanes 1 and 3, Figure 3B).

Figure 3 M. smegmatis A) Total RNA of the bacteria in culture media: 1) 7H9, 2) HdB minimal culture, 3) Sauton without zinc, 4) not controlled variable 5) HdB<N, 6) HdB<C, 7) HdB<Pi. B) Same lanes with the results of Northern hybridization for esat-6 gene. 

The probe elaborated from esat-6 fragment was sequenced and was referred to GeneBank number SeqID KR363260. The homology comparison consulted on the database corresponds to esat-6 gene and is located in site 87 387 to 87 604 pb of the complete genome sequencing of M. smegmatis strain MC²155 (ID: gb|CP000480.1).

The mycobacterial capacity to accumulate energy sources, allows them to in vivo persist the infection phase²⁸. In this sense, in vitro mycobacteria achieve to adapt to nutrient depletion and survive for more than 650 d²⁴, due to energy accumulation of the stored compounds and degradation of unnecessary proteins and bacterial RNA⁶. This can cause an emergency situation, where mycobacteria respond to the environment through genes which regulate adequate changes to the conditions imposed. Thus, the model of the present study allows to observe esat-6 gene expression of M. smegmatis in HdB<Pi.

The adaptation of M. smegmatis for up to 144 h is evident in all culture media, with the exception of the mycobacteria grown in HdB<Pi (Figure 1). For which it poses instability of the bacteria to the culture, because of its continuous growth with respect to the HdB minimal medium (P>0.05) and its inactive metabolism after 144 h when changing the mycobacteria to a fresh culture, where the loss of acid-alcohol capacity is observed (Figure 2: a and b); similar results in M. tuberculosis were obtained by Rifat et al²⁹, when limiting Pi in 7H9 medium.

This suggests, under the proposed conditions, the existence of a state of emergency, in which M. smegmatis can have different forms of adaptation, where several signal transduction pathways are involved by which mycobacteria detect and give response to the environment. These pathways can be associated with alarmone, which triggers the stringent response. An indispensable mechanism for bacterial adaptation during the transition of the exponential phase to the stationary phase^30-32. In this regard, in E. coli, lack of amino acids, carbon or Pi, is followed by an increase in ppGpp levels and polyphosphate accumulation^33,34.

Under limiting conditions of Pi and a sustained growth very near to HdB minimal medium (Figure 1), M. smegmatis apparently finishes the external sources and the internal energy reserves. In order to avoid this wear down of energy reserves, the stringent response has been proposed, which is characterized by ppGpp regulation that increases cyclopropanation³⁵, inhibition and synthesis of fat acids and phospholipids³⁶. Although the proposal of the stringent response is associated with the possibility of stabilization of the metabolism for adaptation of mycobacteria to stress conditions, that is, under conditions of abundance, polyphosphates can be used as donors of ADP, GDP and other reactions in which the Rel enzyme is involved, acting in the synthesis and hydrolysis of ppGpp^5,37. However, when there are not enough Pi reserves and energy reserves are minimal, it is probable that the stringent response fails so that M. smegmatis adapts and its metabolic state does not let it respond immediately to favorable changes to renew its growth, as it happened in the present study.

An increase in ppGpp levels and polyphosphates induces the transcription of 150 genes involved in the slowdown of growth and metabolism³⁸. Rather, what has been found in this study with respect to Pi limitation in HdB, is that growth is maintained similar to HdB minimal medium (P<0.05), where Pi is not limited. Conversely, the esat-6 gene expression in HdB<Pi that occurred at mRNA level (Figure 3B), which is comparable to 7H9 and Sauton, although the protein encoded by this gene is only secreted in Sauton medium²², it is necessary to determine whether under HdB<Pi culture conditions, esat-6 protein is secreted. This protein has also been proposed as an ATPase³⁹, which modifies the properties of the phagosome compartment, where M. tuberculosis reside, as evidenced by aberrant retention or imperfect acquisition of a range of proteins of the host, including Rab GTPases^40,41 and vacuolar (H+)-ATPase⁴².

In M. smegmatis and M. tuberculosis, polyphosphates regulate ppGpp synthesis through transcriptional control of relA via mprAsigE-relA pathway^37,43. There are other systems of the two components that can contribute to the regulation of relA. Consequently, it is proposed as prospect to study the relationships between regulators of Pi-dependent gene expression, including SenX3-RegX3 and its relationship with genes that regulate the ESX-1 system and secretion of the proteins in M. smegmatis⁴⁴. Pang et al⁴⁵⁾ confirm that in M. tuberculosis, esat-6 is not regulated by MprAB under growth in standard 7H9. Therefore, it is important to understand whether esat-6 of M. smegmatis is regulated by this system under Pi-limiting conditions. The studies of Rifat et al²⁹, focused on Pi limitation, show that esat-6 does not express in M. tuberculosis, when they limited Pi in 7H9 medium with L-glutamate as source of organic nitrogen; however, in this model, Pi is limited and the nitrogen source is inorganic. Pang et al⁴⁵ also report that at least there is an indirect regulation of ESX-1 by PhoPR. Thereon, it is suggested to research the relationships between Pi limitation and PhoPR, under the proposed model in this research.

In conclusion, M. smegmatis adapts to HdB minimal medium, HdB<C and HdB<N, and resumes its metabolic activity in fresh medium, but it does not express esat-6 gene. However, in HdB<Pi medium, M. smegmatis shows a continuous exponential growth phase, statistically similar to HdB medium (P<0.05), manifesting a metabolic state in which it loses resistant-acid capacity in the stationary phase and expresses the esat-6 gene after changing the culture medium to 96 h into the corresponding fresh culture medium. Consequently, the model of research for genetic expression under nutrient stress conditions is proposed.