Mycobacterium tuberculosis Expresses ftsE Gene Through Multiple Transcripts
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- Roy, S., Vijay, S., Arumugam, M. et al. Curr Microbiol (2011) 62: 1581. doi:10.1007/s00284-011-9897-1
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Bacterial FtsE gene codes for the ATP-binding protein, FtsE, which in complex with the transmembrane protein, FtsX, participates in diverse cellular processes. Therefore, regulated expression of FtsE and FtsX might be critical to the human pathogen, Mycobacterium tuberculosis, under stress conditions. Although ftsX gene of M. tuberculosis (MtftsX) is known to be transcribed from a promoter inside the upstream gene, ftsE, the transcriptional status of ftsE gene of M. tuberculosis (MtftsE) remains unknown. Therefore, the authors initiated transcriptional analyses of MtftsE, using total RNA from M. tuberculosis cells that were grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients. Primer extension experiments showed the presence of putative transcripts, T1, T2, T3, and T4. T1 originated from the intergenic region between the upstream gene, MRA_3135, and MtftsE. T2 and T3 were found initiated from within MRA_3135. T4 was transcribed from a region upstream of MRA_3135. RT-PCR confirmed co-transcription of MRA_3135 and MtftsE. The cloned putative promoter regions for T1, T2, and T3 elicited transcriptional activity in Mycobacterium smegmatis transformants. T1, T2, and T3, but no new transcript, were present in the M. tuberculosis cells that were grown under the stress conditions, which the pathogen is exposed to in granuloma in tuberculosis patients. It showed lack of modulation of MtftsE transcripts under the stress conditions tested, indicating that ftsE may not have a stress response-specific function in M. tuberculosis.
Bacterial ftsE gene codes for a potential ATP-binding protein, the presence of which has been found in Escherichia coli [6, 11, 13], Pseudomonas putida , Neisseria gonorrhoeae , Aeromonas hydrophila , Mycobacterium tuberculosis , Synechococcus elongatus PCC 7942 , and Bacillus subtilis . Several of these and other studies suggest that FtsE, in complex with the trans-membrane protein FtsX, participates directly or indirectly in diverse cellular processes. The FtsE-FtsX complex, in which FtsE has nucleotide binding domain, cannot yet be called as an importer or exporter type transporter protein since an associated substrate binding protein has not yet been identified . In E. coli, the diverse processes in which FtsE participate include cell division under normal [11, 13] and under low osmolarity conditions , translocation of potassium pump proteins , and prevention of endogenous DNA damage . Further, improper folding of FtsE, because of depletion of GroE, has been found to induce filamentous morphology in E. coli . FtsE has also been found to be required for opsonophagocytosis in Aeromonas hydrophila , membrane restructuring in M. tuberculosis , regulation of cellular differentiation in Bacillus subtilis , and DNA binding in Neisseria meningitidis . Considering its participation in such diverse-cellular functions, the demand for critical levels of FtsE and FtsX proteins could be of significance to any bacterial cell in general and to an intracellular human pathogen, like M. tuberculosis, in particular.
Regulation of expression of ftsE gene has been studied so far in E. coli , Pseudomonas putida PpG1 , and N. gonorrhoea . The authors had earlier shown that FtsE gene of M. tuberculosis (MtftsE) could only partially complement growth defect of E. coliftsE temperature-sensitive strain, MFT1181 [22, 43]. However, co-expression of MtftsE, along with the gene for the transmembrane protein, FtsX (MtftsX), efficiently complemented the growth defect of MFT1181, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function . Although FtsX gene of M. tuberculosis (MtftsX) has been shown to be transcribed from a promoter inside the upstream gene, MtftsE , transcriptional features of MtftsE remain unknown. Therefore, as the first step towards determination of the levels of MtFtsE in cellular processes, in this study, the authors initiated transcriptional analyses of MtftsE in M. tuberculosis cells grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients [19, 28, 39, 46]. MtftsE transcripts were identified, their putative promoters were mapped, the putative promoter regions were cloned and their activity was confirmed, detected co-transcription of MtftsE with its immediate upstream gene, and examined transcriptional status of MtftsE under the stress conditions.
Materials and Methods
Bacterial Strains, Media, Culture Conditions
Mycobacterium tuberculosis H37Ra and Mycobacterium smegmatis mc2155  cells were grown in Middlebrook 7H9 (Difco, USA) liquid medium supplemented with 0.2% glycerol, 0.05% Tween 80 and ADC (albumin, dextrose, catalase) enrichment or in Middlebrook 7H10 agar (Difco, USA) medium supplemented with OADC (oleic acid, albumin, dextrose, catalase) enrichment. For experiments under hypoxia, cells were grown in Dubos broth base (Difco, USA) supplemented with ADC. E. coli JM109 cells were grown in liquid or solid Luria–Bertani medium (Difco). Hygromycin (Sigma, USA) was used at 150 μg/ml for E. coli and 50 μg/ml for mycobacterium. Kanamycin (Sigma, USA) was used at 50 μg/ml. For culturing cells under various stress conditions in vitro, an exponentially growing M. tuberculosis culture of OD600 nm of 0.6 at 37°C was divided into a series of 20 ml of cultures. The cells were harvested, suspended in 10 ml of appropriate stress medium, and subjected to stress for 2 h at 37ºC in shaker, except for heat shock culture, which was kept at standing condition [19, 48]. The stress conditions were: (i) 10 mM H2O2 (oxidative stress), (ii) pH 5 (acid stress), (iii) 0.05% SDS (detergent stress), (iv) 50°C (high temperature heat shock), (v) 5 M NaCl final concentration (hyper-osmotic stress; ), and (vi) 0.2 μg/ml final concentration of mitomycin C (DNA damage induction; ). The bacterial cells, which were exposed to different stress conditions, were chilled in ice, harvested, washed in LETS buffer (100 mM LiCl, 10 mM EDTA, 10 mM Tris–HCl, pH 7.8, 1% SDS) , and used for further analysis. M. tuberculosis cells were cultured under hypoxic condition exactly as described . The 12th day non-replicating persistent phase 2 (NRP2) cells were harvested, washed in Tween-saline (0.8% NaCl and 0.05% Tween 80) and used for total RNA preparation. For nutrient-depleted stationary phase culture, M. tuberculosis cells were grown in a rotary shaker at 37°C to OD600 nm of 2.5 (15 days) and then kept in standing non-shaking condition at 37°C for 30 additional days for gradual depletion of nutrients, along with micro-aerophilic submerged growth. The cells from the culture, which did not show appreciable lysis of the cells, were harvested at the end of 30 days and used for total RNA preparation.
Cloning of MtftsE Putative Promoter Sequences
Primers used in the study
5′ ctagcccgttgatttcgcctgcccgctaatctcaccgctacac 3′
5′ gatcgtgtagcggtgagattagcgggcaggcgaaatcaacggg 3′
5′ ctagccggcacctaccccaaatccgagccaccgacccgttg 3′
5′ gatccaacgggtcggtggctcggatttggggtaggtgccgg 3′
5′ gctctagagtgaagctcagcaaccagaaacggcactgg 3′
5′ cgggatcccggccgtcggacccggcc 3′
5′ gtgaagctcagcaaccagaaacggca 3′
5′ gccgacgatttgtactgcttggtgacatg 3′
5′ gaccatcgcggaccataatgtcgac 3′
5′ gaacaacgcgacaaaccacc 3′
5′ gcggatccatggccaccacccttcccgttc 3′
5′ gcaccggccaactacgtg 3′
5′ gcggaattctagagcgatccatcccgtagacgccacgctgttcgtc 3′
5′ ccggcacctaccccaaatccgag 3′
5′ gcgggatccgatatcatgatcaccctggaccatgtcaccaagcagtacaaatcg 3′
Primer Extension Analysis, cDNA Synthesis, and Real Time PCR
Total RNA was isolated from M. tuberculosis H37Ra cells and M. smegmatis mc2155 transformant cells containing promoter constructs, grown to OD600 nm of 0.6 or exposed to different stress conditions. The 525 bp region, which spans complete open reading frame (ORF) of the upstream gene MRA_3135 (438 bp), the MRA_3135-MtftsE intergenic region (43 bp), and the first 44 nt of MtftsE ORF, was amplified from genomic DNA, with MtRa3135f and MtEPE1r primers (Table 1) and Pfu DNA polymerase (MBI Fermentas). It was used as the template for the cycle sequencing reactions in primer extension. The 5′ 32P-end-labelled reverse primer MtEPE1r, the 3′ end of which anneals at 15 nt downstream of the ‘A’ of the ATG of MtftsE gene, was used for primer extension and for cycle sequencing reactions, as described .
The reaction mixture for the synthesis of cDNA, for real time PCR of MthspX and Mt16S rRNA, consisted of 500 ng DNA-free total RNA, 2 μl of RNase H-minus M-MuLV Reverse Transcriptase buffer, and 2 μl each of the respective reverse primer (MthspX-RTr for MthspX and Mt-16SrRNA-RTr for Mt16S rRNA; each of 12.5 μM concentration; Table 1), with the volume made up to 14.6 μl. The reaction mixture was denatured at 65°C, snap-cooled on ice for 5 min, kept at the annealing temperature of respective primer for 5 min, and again snap-cooled. Subsequently, 2 μl of RNase H-minus M-MuLV Reverse Transcriptase buffer, 2 μl of 10 mM of dNTPs mixture, 20 U of RNase inhibitor, and 40 U of RNAse H-minus M-MuLV reverse transcriptase were added. The reaction mixture was incubated at 42°C for 60 min and heat inactivated at 70°C for 10 min.
The cDNA, thus synthesised, was used for real time PCR, with DyNamo SYBR Green qPCR kit (Finnzymes). The reaction mixture (20 μl) for real time PCR contained 10 μl of 2× master mix, 0.4 μl of ROX dye, 2 μl of forward primer (MthspXf for MthspX and Mt-16SrRNA-RTf for Mt16S rRNA; each at 12.5 μM concentration; Table 1), 2 μl of reverse primer (MthspX-RTr for MthspX, and Mt-16SrRNA-RTr for Mt16S rRNA; each at 12.5 μM concentration; Table 1), 1 μl of cDNA and water. Real Time PCR was performed for 40 cycles in ABI Prism and the data were analysed using 7000 SDS software (Applied Biosystems, USA). Real time RT-PCR Cτ values for 16S rRNA were used to normalise the real time RT-PCR Cτ values for MthspX.
Flow Cytometry Analysis
Flow cytometry analysis for the expression of MYCGFP2+ protein in the M. smegmatis mc2155 transformants, which were carrying different promoter constructs or vector control (pMN406-ΔPimyc), was carried out as described .
MRA_3135-MtftsE Co-Transcription Analysis
For co-transcription experiments, cDNAs were generated from 5 μg of DNA-free total RNA using 20 pmol each of MtftsE2 and MtEPE1r primers (Table 1), independently, in the presence of 20 U of Ribolock Ribonuclease Inhibitor [MBI Fermentas] and 200 U of RevertAid™ Premium Reverse Transcriptase [MBI Fermentas] at 42°C for 1 h. The enzyme was inactivated by incubation at 70°C for 10 min. The cDNA was used for PCR using the appropriate pair of primers (MtRa3135f and MtEPE1r to give 525 bp, MtET3f and MtEPE1r to give 127 bp, and MtftsE1 and MtftsE2 to give 690 bp; Table 1) that encompass different regions overlapping MRA_3135-MtftsE sequences. As RT-minus control, total RNA was used for PCR (30 cycles) for the same different regions overlapping MRA_3135-MtftsE sequences, with the same pair of primers. As the template control, PCR was carried out using primers alone without either total RNA or cDNA template.
Results and Discussion
Identification of Four MtftsE Primer Extension Products
Promoter sequences and transcription start site of MtftsE gene
Nt gap between −10 and −35 seq
Promoter consensus to
T1, T2, and T3 are True Transcripts
Co-transcription of MtftsE and MRA_3135
MtftsE Transcripts Under Stress Conditions
In host cells, M. tuberculosis experiences stress conditions, namely low pH, reactive nitrogen and oxygen species and other DNA damaging conditions, surface structure damaging conditions like toxic, free fatty acids and peptides/proteins, and above all, reduced oxygen pressure (hypoxia) and nutrient depletion [19, 47, 50]. In E. coli cells, EcFtsE has been implicated to have some role in cell division , a process that gets arrested in dormant M. tuberculosis cells in response to hypoxia [45, 46] and nutrient depletion [4, 15]. Therefore, several bacterial cellular processes, in which FtsE is involved [11, 13, 18, 24, 26, 36, 43], may be influenced by altered expression of ftsE under stress conditions. For instance, presence of specific ftsE transcripts has been demonstrated in N. gonorrhoeae, which were exposed to stress conditions, reflecting the environment of genitourinary tract, namely anaerobiosis, presence of isoleucine, urea, and pH 6 . Similarly, E. coliftsE has been found to be required for cell division under low osmolarity condition , although it was non-essential for cell division under optimal growth and high osmolarity conditions.