Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level

Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance.

acid 29,30 . In the β -lactam-producing Actinomycetes, LAT has been shown to catalyze the first steps of β -lactam antibiotic biosynthesis pathway 31 . Rv3290c, encoding a Lysine ε -aminotransferase (LAT) in M. tuberculosis, was upregulated over 40-fold in nutrient-starved persistence models 32 . In this study, we initially reported the involvement of LAT in mycobacterium persister formation.

Materials and Methods
Antibiotics. Ampicillin, kanamycin, hygromycin, norfloxacin were bought from Sangon Biotech Co., and their stock solutions were freshly prepared, filter-sterilized, and used at indicated concentrations.
Bacterial culture and starvation conditions. The bacterial strains and plasmid used in this study are shown in Table 1. E.coli strains were grown on LB broth agar or in LB broth, Mycobacterium smegmatis mc 2 155 was grown in 7H9 liquid medium (Difco) supplemented with 0.05% w/v Tween 80, 0.5%glycerol and 0.5%glucose or were grown on 7H10 agar supplemented with 1% glycerol and 0.5% glucose. The starvation culture condition as described 32,33 with minor modifications. In brief, exponential phase cultures were pelleted and washed twice with 1 × PBS before being resuspended in 1 × PBS, transferred to standing flasks or microwell and incubated at 37 °C, 110 rpm. For viability determination during starvation, bacteria were cultured in 50 ml volumes in 250 ml bottles (Shuniu), and the number of cfu/ml was determined by plating serial dilutions onto 7H10 agar from triplicate cultures at several time points (0 h, 24 h, and 72 h).
Knockout mutant construction and complementation. The lat gene of M. smegmatismc 2 155 was disrupted using specialized transduction previously described 34 . PCR and sequencing of lat Msm was used to confirm the deletion. For knockout mutant complementation, the M. tuberculosis H37Rv Rv3290c coding region was amplified by polymerase chain reaction using the primers: 5′ -CGCGGATCCGCGTCCTGCTATCATAGCGTCATG-3′ , bearing a BamH I restriction site followed by the start codon of Rv3290c; and 5′ -GGAATTCCATATGGAATTCCGGCTGCCTTACGTCACCAC-3′ , consisting of the final three C-terminal amino acids of Rv3290c and a TAA termination codon followed by a Nde I restriction site. The gel-purified polymerase chain reaction product was digested with BamH I and Nde I, yielding a 1395-base pair BamH I/Nde I fragment. The fragments were ligated into the plasmid pALACE digested by BamH I and NdeI, to produce the plasmid pALACE-lat Mtb . Sequencing of pALACE-lat Mtb confirmed the correctness of the constructed fragment. Competent cells of lat Msm mutant were prepared as described 34 , and pALACE containing lat Mtb gene was used to transform lat Msm mutant competent cells. This was followed by electroporation 35 into the mutant as previously described 34 . Transformed cells were streaked on 7H10 plates containing 100 μ g/ ml ampicillin and 50 μ g/ml hygromycin. Briefly, 50 mg dried sample was put into a 15 * 150 mm testtube, and then 6 ml of 6 M HCl were added into the testtube containing bacterial cells. The upper part in the testtube was removed and the testtube was sealed after 10 min vacuumization. The treated testtube was hydrolyzed for 22 hours in a 110 °C ± 1 °C oven. The testtube was taken out and cooled to room temperature, mixed and filtered.1 ml of filtrate was put into a 50 ml beaker, and waterbathing evaporated at 60 °C, 2-fold diluted by adding 0.02 M HCl, the sample was filtered by 0.22 um membrane, and loaded into a Hitachi L-8800 amino acid analyzer. The analysis cycle is 53 min, using two columns during the analysis process: (1) Separation column: (4.6 mm × 60 mm) Eluent flow rate is 0.4 ml/min, the column temperature was 70 °C, column pressure was 11.627 MPa.
MIC assay and drug treatment of cultures. The MICs of antibiotics were determined by using serial two-fold dilution of the antibiotics in 7H9 medium as previously described in reference 37 . The initial cell densities were 10 8 cfu/ml of exponential culture, and the samples were incubated for 3 days at 37 °C.

RNA Isolation and reverse transcription-PCR (RT-PCR). M. smegmatis cultured under starvation
conditions in 50 ml volumes in 250 ml bottles (Shuniu).Three 50 ml cultures were harvested by centrifugation after 24 h and 72 h of starvation. Control samples were prepared by washing log-phase cultures twice with PBS then resuspended in PBS, as described for the preparation of starved bacteria, and harvesting 50mlculture by centrifugation at time zero (t = 0). Pellets were pulverized in liquid nitrogen and homogenized in Trizol solution (Invitrogen) and RNA was isolated according to the manufacturer's instructions. The subsequent steps were performed according to the reference 38 .
Real-Time PCR. 1 μ g of total RNA was reversely transcribed to cDNA using a first strand cDNA synthesis kit (Roche) according to the manufacturer's instructions. Resultant cDNA was used for real-time PCR. Advanced SYBRGreen Supermix (BIO-RAD) were used to quantify cDNA in a 20 μ L reaction containing 10 μ M each primer, 10 μ l supermix (2X), 4 μ L cDNA. Primers used are listed in Table 1  To test this, we first measured the MIC of norfloxacin, rifampicin and isoniazid, the MIC value showed that the deletion of lat has no effect upon the antibiotic sensitivity for the M. smegmatis wild-type and complement strain (2 μ g·ml −1 for norfloxacin, 8 μ g·ml −1 for rifampicin and 4 μ g·ml −1 for isoniazid). The deletion of genes involved in persistence usually has no effect upon MIC 42,43 . To test the effect of lat deletion on the MIC, the strains were exposed to high concentration antibiotics. The survival rate of Δ lat Msm strain is lower than wild-type and complement strain under 20 μ g·ml −1 norfloxacin exposure (Fig. 4A).
Since LAT was significantly upregulated under nutrient starvation in M. tuberculosis. It is interesting to know whether the deletion of lat will compromise the survival of M. smegmatis under starvation. Therefore, we tested the viability of mutant strain in 1 × PBS buffer as described by Betts et al. 39 . As shown in Fig. 5, no difference  can be seen between wild-type and Δ lat Msm strain after 72 hours starvation. To explore whether the deletion of lat Msm only affect the persistence of mutant strain in high antibiotic concentrations, strains were subjected to antibiotic exposure and the ratio of persisters was determined. The 24 hours and 72 hours-starved cultures were exposed to norfloxacin for 48 h and the number of survived strains was assessed. We can see that 24 h and 72 h (Fig. 4B,C) starvation cultures of three strains exhibit higher level of norfloxacin persistence than 0 h time point. With the increase of starvation, wild type and complement strains persisted, but not Δ lat Msm strain (Fig. 4D). Complementation of the mutant with Rv3290c restored the phenotype of wild type strain. After 24 h and 72 h starvation, all strains showed a higher tolerance to norfloxacin than non-starvation cultures. This result indicated that deletion of lat Msm failed to affect the viability under nutrient starvation, but can impair the persistence of the mutant strain under norfloxacin treatment.
Inactivation of lat Msm decreased the intracellular amino acids content. 13 C-isotope profiling of the persisters of Staphylococcus aureus revealed an active amino acid anabolism in this subpopulation, including Ala, Asp, Glu, Ser, Gly and His 44 . Dysregulation of intracellular amino acids level has been shown to lower the survival capability within macrophage exemplified by M. tuberculosis pknG mutants, which encodes protein serine/threonine kinase involved in the regulation of amino acids level 45 . To examine whether the inactivation of MSMEG_1764 has an effect on the amount of intracellular amino acids, the intracellular levels of the amino acids were determined for the wild-type, mutant, and complemented strains. As shown in Fig. 5A, the concentration of glutamic acid, glycine, methionine and total amino acids content in the Δ MSMEG_1764 mutant strain was decreased, but without significant differences for the lysine content between the mutant strain and the wild-type strains. This might be due to the feedback repression of lysA (Diaminopimelate (DAP) -decarboxylase, the enzyme involved in the last step of lysine biosynthesis) by the excess of lysine. This shows that the rate of lysine biosynthesis was regulated by the intracellular lysine amount 46 . To test whether lysA control the lysine level in M. smegmatis, we measured the expression level of lysA in these strains. As expected, the transcription of lysA in Δ lat Msm strain was decreased (Fig. 5B). Genomic context analysis showed that there is a transcriptional regulator leucine-responsive regulatory protein (LrpA) upstream of lat (Fig. 2), a regulator capable of directly binding to the upstream region of lat 47 . To test whether the transcription of lrpA was also decreased, RT-PCR was applied. The result showed that lrpA transcription was also lowered in Δ lat strain (Fig. 5B).The results suggested   48 and bacterial persistence 49 , was found to accumulate rapidly under starvation. The relationship between relA and lrpA has previously been noted 50,51 .The expression of lrp is positively controlled by ppGpp, namely the lower ppGpp level means fewer Lrp 50 . Moreover, the expression of lrp was significantly reduced in the strains failed to produce ppGpp 50 . Rel Mtb , the M. tuberculosis bifunctional enzyme responsible for both (p)ppGpp synthesis and hydrolysis 52 , and has been shown to play an important role in the survival of bacteria during nutrient starvation condition 53 . To test whether the downregulation of lrpA resulted from the decreased expression of Rel in M. smegmatis, the transcription level of rel Msm , the rel Mtb homolog of M. smegmatis 54 , was measured at different intervals under starvation. No significant difference can be spotted between wild type and knockout strain at 0 h starvation (Fig. 6A). But rel Msm was markedly downregulated in Δ lat Msm strain after 24 h starvation (Fig. 6B). Complementation of M. smegmatis Δ lat MSm with the M. tuberculosis lat Rv3290c can partially restore the expression of rel Msm . This result suggested that knockout lat Msm has no effect upon the intracellular (p)ppGpp content under normal conditions, but can influence the synthesis and hydrolysis of (p)ppGpp under nutrient starvation.

Discussion
Bactericidal antibiotics usually can sterilize most bacteria rapidly. However, a sub-population will survive and be reactivated upon the withdrawal of the antibiotics 55 . Genes involving in the persisters formation are intensively studied 56 both for fundamental insights and translational medicine ends.
Here we showed that lat gene MSMEG_1764, the homologue of Mtb lat Rv3290c is involved in the persister formation (specifically tolerance to norfloxacin) via mediating the intracellular amino acid contents and altering the expression of ppGpp synthase expression in M. smegmatis.
Inactivation of the homologue of Rv3290c in M. smegmatis leads to more sensitive to norfloxacin than the wild type strain (Fig. 4), but without change to the MIC (Data not shown). Generally, mutation of genes involved in persister formation should not alter the MIC, but only affect the persistence 42,43 . LAT was previously showed to be involved in the L-lysine metabolism 57 . The data here showed that the knockout of lat Msm rendered a decrease of intracellular total amino acid, but without discernable effect on the accumulation of L-lysine (Fig. 5A). LAT is just one of the several genes involved in M. tuberculosis glutamate metabolism 58 , given the diversity and redundancy of genes to control the robustness of this important amino acid homeostasis, it is not quite unexpected that the disruption of LAT only slightly decreased the glutamate content in M. smegmatis.
Leucine-responsive regulatory protein (Lrp) is a global transcriptional regulator widespread among prokaryotes, and modulates the expression of a variety of genes involved in metabolism during starvation, especially in the amino acid catabolism and anabolism 59,60 . The knockout of lrp in E.coli downregulated the expression of the amino acid metabolism-related genes 61 . In our study, we found the expression of lrpA was downregulated in lat Msm knockout strain (Fig. 5B), which can explain why the intracellular amino acid content was decreased in knockout mutants. Previous study showed that lrpA is profoundly up-regulated during nutrient starvation conditions characteristic of persistent/latent phase in M. tuberculosis 39 , as well as in M. smegmatis (Fig. 5B). The persistence of lrpA inactivated Mycobacterium fortuitum was attenuated in a murine infection model 62 . The expression of lrp is stimulated by (p)ppGpp and LRP can regulate the biosynthesis of amino acid 50 . Here we have shown that the deletion of lat Msm downregulated the expression of rel Msm (Fig. 6). M. tuberculosis failed to produce (p)ppGpp under starvation was defective in long-term survival both in vitro 53 and in vivo 15 . In our study, the downregulation of rel Msm is coincident with the diminished bacterial tolerance to antibiotics.
In conclusion, we have shown that LAT is involved in persister formation in mycobacteria. Under nutrition starvation, (p)ppGpp is produced by Rel Msm and accumulates within bacteria, then upregulates the transcription of LrpA gene, which in turn upregulates the protein level of LAT and response for amino acid metabolism. Intracellular amino acid level regulates the accumulation of (p)ppGpp, and (p)ppGpp controls the dormant persister formation (Fig. 7). The lat deleted mutant strain failed to replenish the amino acid pool, then downregulated the synthesis of (p)ppGpp. This is largely due to the positive regulation of the transcription of lrpA by (p)ppGpp. The determination of the M. tuberculosis H37Rv LAT crystal structure and the identification of its active sites 63 will promisingly facilitate the discovery of novel LAT inhibitors 64 . Our finding has shown that LAT is a new player in mycobacterial persistence provided a potential drug target for inhibitors against persisters.