Articles

PhoY2 of Mycobacteria Is Required for Metabolic Homeostasis and Stress Response

Chuan Wang, Yi Mao, Jia Yu, Lin Zhu, Ming Li, Decheng Wang, Dandan Dong, Jun Liu, Qian Gao
DOI: 10.1128/JB.01556-12

ABSTRACT

The ability of pathogenic mycobacteria to adapt to diverse environments is essential for their success as pathogens. Here we describe a transposon-inactivated phoY2 mutant of Mycobacterium marinum. PhoY2 of mycobacteria is a functional homologue of PhoU in Escherichia coli and an important component of the Pho regulon. We found that PhoY2 is required for maintaining intracellular inorganic phosphate (Pi) homeostasis and balanced energy and redox states. Disruption of phoY2 resulted in elevated levels of intracellular poly-Pi and ATP and an elevated NAD+/NADH ratio, and the mutant strain exhibited increased sensitivity to environmental stress conditions, including nutrient deprivation as well as SDS and antibiotic treatments. Taken together, our results suggest that PhoY2 is required for maintaining metabolic homeostasis and adaptation to stress conditions, which may provide an explanation for the suggested role of PhoY2 in drug tolerance.

INTRODUCTION

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one-third of the world's population and causes approximately 2 million deaths annually (1). In the majority of individuals infected with M. tuberculosis, the bacteria establish a latent, asymptomatic infection that can persist for decades (2, 3). About 5 to 10% of latently infected individuals develop active disease in their lifetime, and host immunosuppression (e.g., HIV coinfection) markedly increases the risk of reactivation (4). The success of M. tuberculosis as a pathogen is partly due to its ability to adapt to and persist in diverse host microenvironments (5). These host environments include phagocytic vacuoles of macrophages, hypoxic and nutrient-limited environments within granulomas, and oxygen-rich alveolar air spaces. Adaptation to such diverse conditions requires controlled regulation of the expression of key genes that allow the bacillus to alter its physiology in response to changes in the environment. Studies of the M. tuberculosis response to several stress conditions, including hypoxia, nutrient deprivation, nitric oxide treatment, and growth in acidic media, have been described previously (6–11), and genes induced by certain stress conditions such as the DosR regulon (12–14) and EHR genes (15) in response to hypoxia have been identified. The ability of M. tuberculosis to survive and persist under these stress conditions is thought to be essential for establishing long-term infection (2, 5).

Phosphate is an essential nutrient for cell functions and life. Bacteria employ a sophisticated system, encoded by the Pho regulon, to manage inorganic phosphate (Pi) acquisition and metabolism (16, 17). A key component of the Pho regulon is the ABC-type phosphate specific transporter (Pst) system consisting of PstSCAB. PstS is a periplasmic protein that binds Pi with high affinity, PstC and PstA are components of the cytoplasmic membrane transporters for Pi translocation into the cytosol, and PstB is an ATPase that provides energy for the transporter. The Pho regulon is controlled by the PhoR/PhoB two-component regulatory system. Under Pi limitation, PhoR is autophosphorylated and transfers a phosphoryl group to PhoB, which, in turn, activates target genes, including the Pho regulon. When Pi is in excess, the activation is interrupted by PhoR acting as a phosphatase on phosphor-PhoB. PhoU, a peripheral membrane protein, is also required for dephosphorylation of PhoB and is essential for repression of the Pho regulon under Pi-rich conditions (18). The precise mechanism of PhoU action is unknown, but it may interact with PhoR/PhoB proteins and interfere with their functions (19, 20).

There are two PhoU homologues in M. tuberculosis, PhoY1 (Rv3301c) and PhoY2 (Rv0821c), which share 40% and 44% homology to Escherichia coli PhoU (21). Previous studies showed that disruption of phoY2 but not phoY1 in M. tuberculosis resulted in increased susceptibility to antibiotics and reduced bacterial burden in mice (21). The reduced drug tolerance phenotype of the phoY2 mutant is similar to that of the phoU mutant of E. coli (22). Therefore, it was suggested that PhoY2 is the functional homolog of PhoU (22). However, the underlying molecular mechanisms for the observed phenotypes associated with phoY2 mutation in mycobacteria remain unknown. In this study, we characterized a transposon-inactivated phoY2 mutant of Mycobacterium marinum. We found that the disruption of phoY2 resulted in elevated levels of intracellular poly-Pi and ATP in M. marinum. The expression of phoY2 was induced by environmental stress conditions, and the phoY2::Tn mutant exhibited increased sensitivity to SDS, antibiotics, and excessive levels of Pi. We also investigated the phenotypes of the phoY2::Tn mutant under hypoxia-induced dormancy. Taken together, our results indicate that PhoY2 is required for maintaining metabolic homeostasis and adaptation to environmental stress conditions.

MATERIALS AND METHODS

Bacterial strains and growth conditions.M. marinum M strain (ATCC BAA-535) was used as the wild-type (WT) strain for this study. The phoY2 mutant strain (phoY2::Tn) was generated by transposon mutagenesis as described previously (23, 24). M. marinum strains were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.2% glycerol, and 0.05% Tween 80 or on Middlebrook 7H10 agar supplemented with 10% OADC and 0.2% glycerol at 32°C. When necessary, the growth media were supplemented with antibiotics: kanamycin at 25 μg/ml or hygromycin at 75 μg/ml.

Cloning of phoY2 genes.The phoY2 (MMAR_4859) gene of M. marinum was PCR amplified using the forward primer 5′-CGCGGATCCAGGACTTACCACATGCGGACGGCCTACCAT-3′ and reverse primer 5′-CCCAAGCTTGATCGCCGGTCAGCGCGAGTGGGTTGGC-3′, which contain BamHI and HindIII sites, respectively (underlined). The amplified PCR product was cloned into the pSMT3L×EGFP vector (25), creating pMMphoY2, in which the open reading frame (ORF) of phoY2 was cloned downstream of the hsp60 promoter. The phoY2 (Rv0821c) gene of M. tuberculosis was cloned similarly using the forward primer 5′-CGCGGATCCAGGATTTAGCACATGCGGACCGCCTACCAT-3′ and reverse primer 5′-CCCAAGCTTGGAGCTTAAGTGGCCAAGCGGTTGGACCT-3′ to generate pRvphoY2. These constructs were electroporated into phoY2::Tn of M. marinum, and transformants were selected on 7H10 agar containing 75 μg/ml of hygromycin.

Measurement of intracellular poly-Pi.Intracellular poly-Pi was measured in cell suspensions by using a DAPI (4′,6-diamidino-2-phenylindole)-based fluorescence approach as described previously (26). M. marinum cells were grown in 7H9 broth to an optical density at 600 nm (OD600) of 0.5. Cultures were collected and resuspended in 7H9 broth containing various concentrations of Pi to a final OD600 of 0.5. After incubation at 32°C for 24 h, 10-ml cultures were harvested and the cell pellets were washed 3 times with 100 mM Tris-HCl, pH 7.4. DAPI at 10 μM was added to 2-ml cell suspensions (OD600 = 0.2) in 100 mM Tris-HCl, pH 7.4. After 5 min of agitation at room temperature in the dark, the DAPI fluorescence spectra (excitation, 415 nm; emission, 450 to 650 nm) were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian). The fluorescence of the DAPI–poly-Pi complex at 550 nm was used to measure intracellular poly-Pi concentrations because fluorescence emissions from free DAPI and DAPI-DNA are minimal at this wavelength (26).

Luciferase assay.To determine the expression profile of phoY2, the M. marinum WT strain carrying a PphoY2-luxAB reporter construct was generated. The 450-bp upstream sequence of the M. marinum phoY2 start codon was amplified using the forward primer 5′-GCTCTAGAGCCGCAAGAAGTCACCGTG-3′ and reverse primer 5′-CGGATCCGTGGTAAGCCTACGTTCTCG-3′, which contain a BamHI site and an Xbal restriction site (underlined). The amplified PCR product was cloned into the pSMT3L×EGFP vector by replacing the hsp60 promoter of luxAB, creating PphoY2-luxAB. An M. marinum WT strain caring this vector was cultured under various experimental conditions, and luciferase activity was determined by Glomax 20/20 luminometer (Promega).

ATP measurement.ATP levels were determined by BacTiter-Glo microbial cell viability assay (Promega). Aliquots (0.5 ml) of bacterial cultures were collected, immediately inactivated at 90°C for 30 min, and stored at −80°C before use. Cell suspensions (25 μl) were mixed with an equal volume of the BacTiter-Glo reagent and incubated for 5 min in the dark. The emitted luminescence was detected by MicroLumatPlus/LB-96V microplate reader (Berthold) and was expressed as relative luminescence units. ATP ranging from 10 to 100 nM was also included in the experiments as the internal control and standard.

Assays of sensitivity to nutrient starvation, SDS, and antibiotics.To determine bacterial survival under nutrient starvation conditions, M. marinum strains were grown in 7H9 broth until stationary phase. Cultures were harvested, washed 3 times with phosphate-buffered saline (PBS) containing 0.05% Tween 80, resuspended in the same buffer to an OD600 of 0.01, and then incubated at 32°C. At various time points thereafter, cultures were collected and plated onto 7H10 agars to determine CFU.

For the SDS sensitivity assay, bacterial suspensions (5 μl; OD600 = 0.001) were spotted onto 7H10 agar with or without 0.01% SDS and incubated for 4 days. For antibiotic sensitivity assays, bacteria in 96-well plates were incubated with various drugs (erythromycin, rifampin, chloramphenicol, ethambutol, isoniazid, and streptomycin) at serial 2-fold dilutions. The MIC was defined as the minimal concentration at which no visible growth was observed after 7 days.

NAD+ and NADH measurement.The concentrations of NAD+ and NADH were determined as described previously (27). Bacterial cultures were harvested and resuspended in 0.25 ml of 0.2 M HCl for NAD+ extraction or 0.25 ml of 0.2 M NaOH for NADH extraction. After incubation at 55°C for 10 min, the cell suspensions were neutralized by adding 0.1 M NaOH (0.25 ml, for NAD+ extraction) or 0.1 M HCl (0.25 ml, for NADH extraction). After centrifugation, the supernatants were collected, transferred to a new tube, and used immediately. The concentrations of NAD+ and NADH were determined by measuring the rate of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction by the yeast type II alcohol dehydrogenase in the presence of phenazine ethosulfate (PES), which was measured at 570 nm using a Benchmark Plus microplate spectrophotometer (Bio-Rad).

Quantitative real-time PCR.Bacteria were harvested and resuspended in 2 ml of TRIzol (Life Technologies). Cells were disrupted by Mini-BeadBeater (Biospec), and the supernatants were collected for RNA isolation. DNA was removed by Ambion TURBO DNA-free DNase (Life Technologies). RNA (500 ng) was used for cDNA synthesis by using TaKaRa PrimeScript reverse transcriptase (RT) reagent kit (TaKaRa Bio Inc.). Quantitative real-time PCR was carried out by using a TaKaRa SYBR Premix Ex Taq kit in a 7500 real-time PCR system (Life Technologies).

Experiments under hypoxic conditions.The hypoxic cultures were established according to a published protocol (28). M. marinum cells grown in 7H9 broth to early exponential phase were washed and resuspended in 10 ml of 7H9 broth to a final OD600 of 0.2 and transferred to 15-ml tubes (Corning) in order to maintain an air/medium volume ratio of 1:2. Methylene blue (1.5 μg/ml) was added as an indicator of oxygen consumption. Tubes were sealed and incubated at 32°C with slow shaking (40 ppm). At various time points thereafter, cultures were collected and concentrations of ATP, NAD+, and NADH were determined.

RESULTS

Analysis of the phoY2 locus of mycobacteria.We identified a phoY2 mutant (phoY2::Tn) of M. marinum, which exhibited small colonies on 7H11 agar plates, from a transposon-insertion library generated as described previously (23, 24). The transposon was inserted 12 bp downstream from the translation start site of the MMAR_4859 gene (Fig. 1A), which is homologous to the M. tuberculosis phoY2 gene, with 87% identity in nucleic acid sequence. This gene is also conserved in other mycobacterial species. In some bacterial species such as E. coli, PhoU is encoded with the Pst system in the polycistronic pstSCAB-phoU operon (Fig. 1B). However, the genetic organizations of the pstSCAB-phoU operon in mycobacteria are different and vary among species. The phoY2 of M. marinum is in the orientation opposite that of the Pst system (pstA and pstC), while in M. tuberculosis, phoY2 is localized at a different genetic region of the pst operon. The phoY2 gene in M. marinum is transcribed separately and is not part of an operon, as revealed by our reverse transcription-PCR analysis (see Fig. S1 in the supplemental material). This suggests that in mycobacteria, the expression of phoY2 is not coupled to the Pst system.

Fig 1
Fig 1

(A) Genetic locus of phoY2 in the genomes of M. marinum and M. tuberculosis. Open reading frames with more than 78% identity at the amino acid level between M. marinum and M. tuberculosis are in dark gray. (B) Genetic organization of the pstSCAB-phoU operon in E. coli.

PhoY2 is required for the maintenance of intracellular Pi homeostasis.Since PhoY2 of M. tuberculosis was found to be a functional homolog of PhoU (22), which is known to be involved in the uptake of Pi in E. coli (29), we first examined if PhoY2 plays a role in M. marinum adaption to growth media with various Pi concentrations. In standard 7H9 broth and 7H10 agar media, which contain ≈25 mM Pi, the phoY2::Tn mutant of M. marinum showed a slight growth delay (Fig. 2A and B), and this growth defect was more pronounced at higher Pi concentrations (Fig. 2B). At 250 mM Pi, the mutant strain showed little growth, whereas the WT strain exhibited growth comparable to that at lower Pi concentrations. Importantly, the growth defect of the mutant was mostly complemented by the wild-type copy of phoY2 of M. marinum or M. tuberculosis (Fig. 2A and B), indicating that the disruption of phoY2 is responsible for the growth defect of the mutant at high Pi concentrations.

Fig 2
Fig 2

PhoY2 is required for the maintenance of intracellular Pi homeostasis. (A) Growth of M. marinum strains in 7H9 broth. The phoY2::Tn mutant exhibited a growth delay at early time points. (B) The phoY2::Tn mutant was defective in growth at high Pi concentrations. A total of 2.5 × 104 CFU of M. marinum was spotted on 7H10 agar containing various concentrations of Pi. Growth was visualized after incubation for 6 days. (C and D) Measurement of intracellular poly-Pi. M. marinum was cultured in 7H9 broth containing the indicated concentrations of Pi for 24 h. Poly-Pi contents were determined by measuring the fluorescence emission of the DAPI–poly-Pi complex at 550 nm. The spectra shown in panel C are representative of three independent experiments, and the data shown in panel D are from three independent experiments (means + SDs). AU, arbitrary fluorescence emission units. Significant differences between the phoY2::Tn mutant and the indicated strains were obtained by Student's t test and are indicated as follows: ***, P < 0.001; **, P < 0.01; and *, P < 0.05.

Within cells, inorganic phosphate is stored as a high-molecular-weight inorganic polyphosphate (poly-Pi), which is a linear polymer containing from a few to several hundred residues of orthophosphate that are linked by energy-rich phosphoanhydride bonds (30). We next determined the intracellular poly-Pi content by using a DAPI (4′,6-diamidino-2-phenylindole)-based fluorescence approach (26). Interestingly, the intracellular poly-Pi content of the phoY2::Tn mutant was nearly 3-fold higher than in WT cells in standard 7H9 broth (Fig. 2C and D), and this difference was enlarged with increasing external Pi concentrations. The poly-Pi content of WT cells remained essentially unchanged as the external concentration of Pi increased from 25 to 200 mM. In contrast, poly-Pi was accumulated in the phoY2::Tn mutant in a Pi-dose-dependent manner (Fig. 2D). At 200 mM Pi, the level of poly-Pi in the phoY2::Tn mutant was about 7-fold greater than that in the WT strain. The elevated level of poly-Pi in the mutant strain was fully reversed to WT levels by complementation with the phoY2 gene of M. marinum or M. tuberculosis (Fig. 2D). These results suggest that PhoY2 negatively controls the intracellular poly-Pi level under high external Pi concentration, which is consistent with the suggested role of PhoU in E. coli (18). Consistently, we found that the expression of phoY2 in WT M. marinum, as measured by the promoter luciferase assay (31), was increased at higher Pi concentrations. The activity of the phoY2 promoter was increased 2-fold at 100 mM Pi and 4-fold at 200 mM Pi after a 24-h treatment (Fig. 3A). Furthermore, the expression of Pst homologue genes, the phoS2-pstC2-pstA1 operon (MMAR_4580-MMAR_4579-MMAR_4578) and phoT (MMAR_4860; pstB homologue), in the phoY2::Tn mutant was increased, as determined by RT-PCR (Fig. 3B), suggesting that PhoY2 represses the expression of the pst system in M. marinum. Taken together, our results indicate that expression of phoY2 is induced at high Pi concentrations, which is necessary for maintaining a constant intracellular poly-Pi level by negatively controlling the uptake of external Pi.

Fig 3
Fig 3

(A) Expression of phoY2 is induced at high concentrations of Pi. The M. marinum WT strain carrying PphoY2-luxAB was inoculated in 7H9 broth containing various concentrations of Pi. Aliquots (0.5 ml) were harvested at different time points and assayed for promoter activity. The results were normalized to RLU (relative light unit)/CFU by plating appropriate culture dilutions on 7H10 agar plates and enumerating. (B) The expression of the Pst system is induced in the phoY2::Tn mutant. Quantitative RT-PCR analysis of phoS2 (MMAR_4580) and phoT (MMAR_4860) in M. marinum was carried out. The expression of each gene was normalized to the level of sigA. Results are from three independent experiments (means + SDs). Significant differences between the phoY2::Tn mutant and the indicated strains were obtained by Student's t test and are indicated as follows: ***, P < 0.001; **, P < 0.01; and *, P < 0.05.

PhoY2 is required for maintaining ATP homeostasis.The high-energy bonds within poly-Pi make it an important energy source; poly-Pi can be converted to ATP by phosphotransferases or substitute for ATP (30). We therefore hypothesized that the elevated level of poly-Pi in phoY2::Tn may alter the energy status of the mutant strain. To test this, we measured the intracellular ATP levels at different Pi concentrations. As expected, the ATP level in the WT and complemented strains remained relatively constant as the external Pi concentration increased from 25 to 200 mM, demonstrating the ability of the bacteria to maintain an ATP homeostasis (Fig. 4). In contrast, the intracellular ATP level of the phoY2::Tn mutant is nearly 2-fold that in the WT or the complemented strains at 25 mM Pi and increased with increasing external Pi concentrations, reaching 5-fold that in the WT and complemented strains at 200 mM Pi (Fig. 4). These results suggest that PhoY2 is required for maintaining ATP homeostasis.

Fig 4
Fig 4

Measurement of intracellular ATP level of M. marinum. M. marinum was cultured in 7H9 containing various concentrations of Pi for 24 h. ATP was measured by using a Promega BacTiter-Glo microbial cell viability assay kit. The results were normalized to RLU/OD600. Results are from three independent experiments (means ± SDs). Significant differences between the phoY2::Tn mutant and the indicated strains were obtained by Student's t test and are indicated as follows: ***, P < 0.001; **, P < 0.01; and *, P < 0.05.

The phoY2::Tn mutant is sensitive to environmental stresses.Cells having an imbalanced level of poly-Pi were shown to exhibit a defective response to environmental stress conditions in several microorganisms, including E. coli (32). Consistently, we found that the phoY2::Tn mutant survived less well under nutrient starvation conditions and exhibited increased sensitivity to SDS and several antibiotics. After a 4-day incubation in PBS, there was 2-log reduction of the CFU of the phoY2::Tn mutant, whereas the viability of the WT and the complemented strains was not affected (Fig. 5A). Inclusion of 0.01% SDS in 7H10 agar plates strongly inhibited the growth of the mutant but had no effect on the growth of the WT and complemented strains (Fig. 5A). The phoY2::Tn mutant was also 2- to 4-fold more sensitive than the WT and complemented strains to five of the seven antibiotics tested, including rifampin, ciprofloxacin, gentamicin, streptomycin, and ethambutol (Table 1). We also measured the luciferase activity of the phoY2 promoter under various stress conditions and found that the expression of phoY2 was induced by exposure to SDS or rifampin or in PBS (Fig. 5B). Taken together, our data indicate that PhoY2 is involved in stress responses, presumably by maintaining an intracellular balanced poly-Pi.

Fig 5
Fig 5

The phoY2::Tn mutant is sensitive to environmental stress conditions. (A) Sensitivity of the phoY2::Tn mutant to SDS. Stationary-phase cultures of M. marinum strains were incubated in 7H9 broth with or without 0.01% SDS. Cell viability was determined by plating. Results (means ± SDs) are from three independent experiments. (B) Viability of stationary-phase cultures of M. marinum strains in PBS containing 0.05% Tween 80. Results (means ± SDs) are from three independent experiments. (C) Luciferase reporter assay for the activity of the phoY2 promoter. M. marinum was grown in 7H9 containing 0.01% SDS or 0.25 μg/ml of rifampin or in PBS containing 0.05% Tween 80. The results were normalized to RLU/CFU. Results (means± SDs) are from three independent experiments.

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Table 1

Sensitivity of M. marinum to antibiotics

The phoY2::Tn mutant enters a nonreplicating state earlier under hypoxia.Oxygen limitation within caseous granulomas is thought to be a major factor that influences the metabolism of M. tuberculosis, leading to nonreplicating dormancy (5). We thus examined whether the phoY2::Tn mutant with altered levels of poly-Pi and ATP exhibited a phenotype in an oxygen-limited environment by using an in vitro progressive-hypoxia model, the Wayne model (9, 28). M. marinum standing cultures prepared in 15-ml polypropylene tubes were sealed and incubated at 32°C with slow shaking (40 rpm) for a long time (up to 60 days). Methylene blue was added to the cultures as an indicator of oxygen depletion. The growth of M. marinum strains under these conditions arrested at around day 8, as assayed by OD600 reading or CFU counting (Fig. 6A and B). Interestingly, during the early phase of hypoxic growth (days 6 to 12), the phoY2::Tn mutant appeared to have a decreased growth compared to the WT and complemented strains, where the CFU of the mutant were about 2-fold lower than those of the WT and complemented strains (Fig. 6A and B). The difference became negligible at the middle stage of hypoxia (days 14 to 40) but was apparent again at the late stage (days 50 to 60). Overall, the mutant appears to replicate at a lower level than the WT or complemented strains under the hypoxic conditions, although the phenotype is modest.

Fig 6
Fig 6

The phoY2::Tn mutant enters a nonreplicating state earlier under hypoxia. M. marinum standing cultures were grown at 32°C in sealed tubes. The growth determined by OD600 (A) or CFU/ml (B) is shown. (C) Oxygen consumption as revealed by methylene blue decolorization in M. marinum hypoxia broth in the adaptive phase to dormancy. Results (means ± SDs) are from three independent experiments. Significant differences between the phoY2::Tn mutant and WT strain were obtained by Student's t test and are indicated as follows: ***, P < 0.001; **, P < 0.01; and *, P < 0.05.

We also observed that the decolorization of methylene blue in the phoY2::Tn culture occurred more rapidly than that in the WT and complemented strains (Fig. 6C), indicating a more rapid oxygen consumption. Previous studies showed that an early M. tuberculosis response to hypoxia included the coordinated upregulation of 47 genes under the control of two sensor kinases (DosS and DosT) and the response regulator (DosR), known as the DosR regulon (12–14). We found by quantitative RT-PCR analysis that the expression level of dosR (MMAR_1516) in the phoY2::Tn mutant nearly doubled compared to that in the WT and complemented strains at day 4 after treatment with hypoxia (see Fig. S2 in the supplemental material). The higher level of DosR in the mutant may cause it to enter into the nonreplicating stage earlier than the WT, resulting in lower CFU during the early stage of hypoxia (Fig. 6A and B).

To gain further insight into the metabolic states of the bacteria under hypoxic conditions, we measured the levels of ATP, NAD+, and NADH in cells of the WT and the phoY2::Tn mutant. As shown in Fig. 7A, the ATP levels of the WT and complemented strains increased in the first 12 days under hypoxia before reaching a steady level, which corresponds to the initial replication of these strains under these conditions (Fig. 6A and B). Similarly, the ATP level in phoY2::Tn also increased before reaching a steady level at day 8, corresponding to its growth at the early phase of hypoxia. However, the ATP level of the mutant is much higher than that in the WT or complemented strains, especially at the early phase of hypoxia, but the difference diminished at later time points.

Fig 7
Fig 7

The phoY2::Tn mutant exhibited altered energy and redox status under hypoxia. (A) ATP measurement during hypoxia treatment. Samples from hypoxia broth were harvested at various time points. ATP was measured by using a Promega BacTiter-Glo microbial cell viability assay kit. Results (means ± SDs) are from three independent experiments. (B and C) Measurement of NAD+ and NADH during hypoxia treatment. Nucleotides were extracted and measured by using an alcohol dehydrogenase-based NAD+/NADH cycling assay. (D) Calculated NAD+/NADH ratios from data in panels B and C. Results (means ± SDs) are from three independent experiments. Significant differences between the phoY2::Tn mutant and the WT strain were obtained by Student's t test and are indicated as follows: ***, P < 0.001; **, P < 0.01; and *, P < 0.05.

The NAD+ and NADH levels of bacilli under the hypoxic conditions were measured by a published protocol (27). Similar levels of NAD+ were detected in the mutant and WT or complemented strains (Fig. 7B). However, the NADH level in the phoY2::Tn mutant was consistently lower than that in WT and complementary strains (Fig. 7C). The decreased level of NADH resulted in an elevated NAD+/NADH ratio in the mutant (Fig. 7D), and the difference between the mutant and WT or complemented strains became smaller at later time points. All strains reached a stable NAD+/NAD ratio at around 1.9 ± 0.3 (dashed line in Fig. 7D), which was similar to the level observed in M. tuberculosis under similar hypoxic conditions (33).

DISCUSSION

In this study, we have characterized a phoY2::Tn mutant of M. marinum. Our results demonstrate that PhoY2 is required for maintaining intracellular Pi homeostasis and balanced energy and redox states. Disruption of phoY2 resulted in elevated levels of intracellular poly-Pi and ATP and an increased NAD+/NADH ratio, and the mutant strain exhibited increased sensitivity to environmental stress conditions, including nutrient deprivation as well as SDS and antibiotic treatments. Taken together, our results suggest that PhoY2 is required for maintaining metabolic homeostasis and adaptation to various stress conditions. Previously, it was found that mice infected with the phoY2 mutant of M. tuberculosis contained 10- to 30-fold-lower bacterial counts in lungs and spleens than did mice infected with the WT strain of M. tuberculosis (21), suggesting that PhoY2 is involved in M. tuberculosis virulence. We also performed zebrafish infection experiments with a low dose (∼100 CFU/fish) to assess the role of PhoY2 in M. marinum virulence. However, we found no difference in the survival of zebrafish infected with the phoY2 mutant of M. marinum compared to that of zebrafish infected with WT or complemented strains (data not shown); the different in vivo phenotypes of the phoY2 mutants of M. tuberculosis and M. marinum may be caused by the different host environments encountered by these bacteria. Our finding that poly-Pi was accumulated at high levels in the phoY2::Tn mutant is consistent with a previous study which showed that deletion of phoU in E. coli and Synechocystis resulted in high-level accumulations of intracellular poly-Pi (34). That study also found that the uptake of Pi in the phoU deletion mutants was increased, which is in agreement with the current view that PhoU negatively regulates Pi uptake under Pi-rich conditions (18). Consistently, we found that the expression of the phoS2-pstC2-pstA1 operon and phoT was increased in the phoY2::Tn mutant, which is in agreement with previous findings that the pstSCAB operon was upregulated in the phoU mutant of E. coli (27) and that PhoU controls the activity of the PstSCAB transporter (35). It was suggested that Pi uptake is a rate-limiting step for poly-Pi accumulation, since increased uptake of Pi by overexpression of the pst system resulted in increased accumulations of poly-Pi in E. coli cells (36, 37). Consistently, we found that the levels of poly-Pi in the phoY2::Tn mutant exhibit a dose-dependent increase in response to increased external Pi concentrations, presumably as a result of increased uptake of Pi. The increased poly-Pi synthesis would require higher levels of ATP, which would require increased respiration (O2 consumption), leading to lower NADH levels, which is supported by our experimental data (Fig. 4, 6, and 7). The increased oxygen consumption may also explain the earlier induction of dosR expression in the phoY2::Tn mutant (see Fig. S2 in the supplemental material).

Poly-Pi is a known stress response molecule which accumulates in microorganisms in response to nutrient deprivation, high salt concentrations, or other environmental stress conditions (30). E. coli cells having an imbalance in their poly-Pi have been shown to elicit a defective response to oxidative, osmotic, and thermal stresses (32, 38–41). Although most of these findings have been obtained from studies with E. coli, results from our study and two recent studies (42, 43) suggest that a similar conclusion can be drawn for mycobacteria. We found that the phoY2::Tn strain, which accumulates large amounts of poly-Pi, exhibited increased sensitivity to nutrient starvation, high Pi concentrations, SDS, and antibiotics. Poly-Pi is synthesized by polyphosphate kinase (PPK), which catalyzes the polymerization of the γ-phosphate of ATP into a poly-Pi chain (44, 45). Another enzyme, the exopolyphosphatase PPX, is involved in degradation and metabolism of poly-Pi (46). A ppk1 deletion mutant of M. smegmatis deficient in poly-Pi synthesis was more sensitive to oxidative stress and survived less well under long-term hypoxia (42). Conversely, disruption of ppx in M. tuberculosis resulted in increased accumulation of poly-Pi and the mutant was defective in growth under hypoxia and persistent infection in guinea pigs (43). That study also found that the ppx deletion mutant of M. tuberculosis exhibited increased resistance to isoniazid but not rifampin, which is in contrast to our finding that the phoY2::Tn mutant of M. marinum was more sensitive to multiple antibiotics, including rifampin (Table 1). This difference may be explained by the fact that the level of poly-Pi accumulation in our phoY2::Tn mutant is much higher than in the WT compared to the level of poly-Pi in the ppx deletion mutant relative to its WT strain. Our results are in accordance with the study of the phoY2 deletion mutant of M. tuberculosis (21).

The role of poly-Pi in stress response can be explained by its link to the stringent response. E. coli mutants that lack the stringent response molecule, (p)ppGpp, failed to accumulate poly-Pi (30), suggesting a link between poly-Pi and (p)ppGpp. This is explained by the finding that (p)ppGpp is able to inhibit PPX activity, causing an increase of poly-Pi accumulation (46). The (p)ppGpp-mediated accumulation of poly-Pi is responsible for activation of recA and rpoS, thereby activating the SOS response and increasing the general stress resistance of the bacteria (46). Therefore, poly-Pi acts downstream of (p)ppGpp and is a central component of the stringent response system in E. coli. Interestingly, in mycobacteria, poly-Pi instead acts upstream of (p)ppGpp. Mycobacteria use one gene, rel, for the synthesis of (p)ppGpp (47). Induction of rel is dependent on sigma factor sigE, which, in turn, forms a positive-feedback loop with a two-component system, MprAB (48, 49). Polyphosphate can act as a phosphodonor to MprAB, thereby activating the transcription of mprAB, sigE, and rel (42, 43, 50). The finding that poly-Pi acts upstream of (p)ppGpp raises an interesting question as to how the level of poly-Pi is regulated in mycobacteria. Future studies are needed to address this question.

ACKNOWLEDGMENTS

We thank E. J. Rubin, Harvard University, for providing the MycoMarT7 mariner transposon phage and D. B. Young, Imperial College London, for plasmid pSMT3. We also thank Chen Niu, Shanghai Medical College, Fudan University, for critical reading of the manuscript.

This work was supported by the international cooperation project of the Ministry of Science and Technology (2010DFA34440) and National Natural Science Foundation of China Grant 8271790 (to Q.G.) and Canadian Institutes of Health Research (CIHR) Grants CCI-85667, MOP-15107, and MOP-106559 (to J.L.).

FOOTNOTES

    • Received 23 August 2012.
    • Accepted 28 October 2012.
    • Accepted manuscript posted online 2 November 2012.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01556-12.

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