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Microbiol Res. Author manuscript; available in PMC 2010 January 1.
Published in final edited form as: Microbiol Res. 2009 ; 164(2): 228232. doi:10.1016/j.micres.2007.06.003.

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The lipF promoter of Mycobacterium tuberculosis is upregulated specifically by acidic pH but not by other stress conditions
Laetitia Richter1,2 and Beatrice Saviola1,* 1 Basic Medical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, 309 E. Second St. Pomona CA 91766 2 Department of Biological Sciences California State Polytechnic University Pomona 3801 West Temple Ave, Pomona CA 91768

Abstract
The lipF gene of Mycobacterium tuberculosis has been implicated in pathogenesis and its promoter has been shown to be upregulated by acidic stress. To further define the acidic pH that upregulates the lipF promoter from M. tuberculosis and to establish that it is specifically upregulated by acid stress and not by other environmental stresses, promoter expression levels were measured under a variety of conditions. The conditions measured were pH, temperature, oxidative stress and hypoxic stress.

Keywords Acidic stress; lipF; lipase; Mycobacterium tuberculosis

Introduction
The lipF gene of Mycobacterium tuberculosis encodes an esterase and has been shown to be important in pathogenesis (Zhang et al., 2005; Camacho et al, 1999). A transposon insertion between the promoter region for lipF and the gene resulted in a bacterium significantly reduced in its ability to grow in a mouse lung (Camacho et al., 1999). The same mutant grew well in nutrient broth medium in vitro and an additional transposon insertion into the gene showed it to be non-essential (Lamichane et al., 2003; Sassetti et al., 2003). We had previously identified a 479 base pair (bp) region of DNA upstream of the lipF gene that is transcriptionally upregulated by exposure to growth media at pH 4.5 (Saviola et al., 2003). In support of the lipF genes involvement in a response to acidic stress, disruption of a homologue of the gene in M. smegmatis led to a decreased ability to replicate at lower pHs (OBrien at al., 1996; Rao et al., 2001; Tran et al., 2005). As there may be additional pHs that upregulate the lipF promoter, we sought to define the maximum pH that can induce promoter expression. In addition, to determine if the lipF promoter is responsive to many stresses, or if it is responsive specifically to acidic stress we sought alternative stress conditions that may result in upregulation of the lipF promoter. We

*To whom correspondence should be addressed. Tel: +1 909 469-5373, Fax: +1 909 469-5698; Email: E-mail: bsaviola@westernu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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examined lipFs inducibility due to heat shock, cold stress, oxidative stress, hypoxic stress, and alkaline stress.

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Materials and Methods

Strains and Media The Mycobacterium smegmatis strain MC2155 (ATCC) was used in all experiments. Mycobacteria were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% ADC (bovine serum albumin, dextrose and NaCl), 0.025% Tween 80 (polyoxyethylenesorbitan monooleate) and 0.2% glycerol. For exposure to acidic stress, 7H9 growth media was adjusted in pH by the addition of concentrated HCl, and then filter sterilized through a Nalgene disposable polyethersulfone filter with a 0.45 m pore size. For exposure to alkaline stress, 7H9 growth media was adjusted in pH by the addition of concentrated NaOH, and then filter sterilized through a Nalgene disposable filter as previously described. Promoter inductions with acidic media Mycobacterium smegmatis containing either, the plasmid pFPV27 with a promoterless gene for enhanced green fluorescent protein (gfp) from the jelly fish Aequorea Victoria (Cormack et al., 1996; Valdivia et al., 1996), or plipF with a 59 bp DNA region of the lipF promoter fused to gfp, was grown overnight to an approximate optical density of 0.7 at 600 nm (OD600). M. smegmatis was centrifuged and resuspended in 7H9 culture media (middlebrook) at pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5 and neutral pH 7.0. All promoter inductions were performed at 37C and were exposed for 3 hrs, and 20 hrs. All samples were vortexed with 4 mm glass beads to eliminate clumping and were diluted to the same optical density at 600 nm. The samples were measured on a TD-700 Turner designs fluorometer with a 486 nm excitation filter and a 510700 nm emission filter. All pH points were tested in triplicate. Adjusted fluorescence units were determined to be fluorescence units/O.D. units. To further narrow the maximum pH that induces the lipF promoter, M. smegmatis bearing pFPV27, or plipF was exposed to 7H9 media at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, and pH 7.0 for 20 hours and was measured on a TD-700 Turner designs fluorometer as previously described. Fluorescence was confirmed on a Nikon AFX-DX fluorescence microscope with a 450490 nm excitation filter and a 510 nm long pass emission filter (data not shown). Induction with other environmental conditions M. smegmatis containing pFPV27, plipF, or pBEN with a constitutive heat shock promoter from M. tuberculosis fused to gfp, was grown to OD600= 0.7 in 7H9 culture media pH 7.0. To test for temperature stress, 1 ml samples were pipetted into test tubes and exposed to a temperature of 4C in a Kenmore refrigerator or to a temperature of 42C in a VWR heat block for 3 hours. To test for oxidative stress, hydrogen peroxide was added to a final concentration of 5 mM in 7H9 culture media pH 7.0 for 3 hours and 20 hours. This hydrogen peroxide concentration was used as it had previously been determined to be minimally lethal to the mycobacteria (Springer et al., 2001). For hypoxic stress, mycobacterial aliquots were maintained in 5 ml culture tubes in 7H9 culture media pH 7.0 and placed into an anaerobic jar using the GasPak system (BBL) for 3 hours and for 20 hours. As a control, M. smegmatis was also exposed to culture media pH 4.5 for 3 hours. All samples were measured as previously described with a TD-700 Turner Designs fluorometer. Fluorescence was confirmed on a Nikon AFX-DX fluorescence microscope as previously described (data not shown). M. smegmatis bearing pFPV27, plipF, or pBEN was grown to an OD600= 0.7 in 7H9 media. Cells were spun down and resuspended in 7H9 media pH 4.5, 7.0, pH 8.0, pH 9.0 and pH 10.0 and measured after 3 hours on a TD-700 Turner Designs fluorometer as previously described.

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Fluorescence was confirmed with a Nikon AFX-DX fluorescence microscope as previously described (data not shown).

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Results and Discussion

The lipF promoter of M. tuberculosis has already been shown to be significantly upregulated by exposure to acidic media pH 4.5 (Saviola et al., 2003). We sought to test the maximum pH that can induce the lipF promoter of M. tuberculosis to determine if higher pHs can also upregulate promoter activity. As a pH below pH 4.5 is unlikely to be encountered in vivo, we tested pHs above 4.5 (Clemens and Horwitz, 1995; Deretic and Fratti; 1999; Schaible et al., 1998, Via et al., 1997; Xu et al., 1994; MacMicking et al., 2003). By exposing M. smegmatis bearing a plipF-gfp fusion to a number of increasingly less acidic pHs, we have determined that the maximum pH that induces the lipF promoter after 3 hours of exposure is pH 5.5 (fig 1a). In this same experiment, the maximum pH to induce the lipF promoter after overnight exposure is pH 6.0 (fig 1a). Thus higher pHs may result in transcriptional upregulation of the lipF promoter but require a longer period of exposure to produce a full effect. Neither pH 6.5 nor pH 7.0 induced the lipF promoter after 3 hours of exposure or after overnight exposure. pH 6.0 had previously been found to induce the expression of lipF in M. tuberculosis after 36 hours of exposure (Zhang et al., 2005). As there may be a pH intermediary between pH 6.0 and 6.5 that can induce the lipF promoter, we sought to further define acid regulation by measuring gfp expression after 20 hrs of exposure of M. smegmatis bearing pFPV27, or plipF at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, and pH 7.0. It was determined that induction of the lipF promoter occurred with exposure to acidic media at pH 6.0, 6.1, 6.2, 6.3, and to a lesser extent pH 6.4. There was no detectible upregulation after exposure to acidic media at pH 6.5 or 7.0 (fig 1b). Thus the maximum pH that can induce the lipF promoter is pH 6.4 after 20 hours of induction (fig 1b). To verify that acidic stress uniquely induces the lipF promoter we investigated a number of stress conditions including temperature stress, oxidative stress, and hypoxic stress. None of the conditions examined resulted in upregulation of the lipF promoter other than exposure to acidic culture media pH 4.5 (fig 2a). All conditions examined resulted in significant and measurable activity of the heat shock promoter induced production of gfp. To investigate upregulation of the lipF promoter due to exposure to alkaline stress, gfp expression was measured under a range of pH levels. All promoter inductions were performed at 37C and were exposed for 3 hrs. The lipF promoter was upregulated by acidic stress at pH 4.5, but not by alkaline stress conditions at pH 8.0, 9.0, or 10.0. The lipF promoter appears to be upregulated specifically by acid stress. Other stresses that could be encountered in vivo, such as oxidative stress, temperature stress, and low oxygen tension did not appear to upregulate this promoter. Likewise, upregulation of the promoter does not appear to be a consequence of a general departure from neutral pH but as a result of exposure to acidic stress, as exposure to acidic pH but not alkaline pH induces the lipF promoter. The lipF promoter is transcriptionlly upregulated quickly by exposure to low external pH, but more slowly upregulated by moderately acidic pH. External acidity could damage surface exposed cell wall components that may be lipid or protein. Low pH (pH 4.5) may induce damage rapidly, while moderate acidity (pH 6.0) may induce damage more slowly. Mycobacterial surface damage may trigger transcriptional upregulation of the lipF promoter resulting in rapid upregulation at low pH and slow upregulation at moderate pH. Alternatively, transcriptional upregulation may be triggered by a decrease in cytosolic pH. At low external pH the cytosol may become acidic rapidly, while with moderate external acidity the cytosolic
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pH may decrease slowly. It has been shown that pH decreases in the cytosol of mycobacteria when they are exposed to external acidic pH, despite an active mechanism by the bacteria to maintain a neutral pH (Rao et al., 2001). Thus, decreasing cytosolic pH may also be an intracellular signal to increase transcription of plipF. LipF has been shown to function as an esterase (Zhang et. al., 2005). This protein may act to modify the mycobacterial cell wall to render the microorganism more resistant to acidic stress. Altered lipid composition within the cell wall may result in a mycobacterium that is less susceptible to acidic damage or may be less penetrable to external acidity. Alternatively, LipF may function to cleave lipids which could provide metabolic energy to withstand acidic stress. This metabolic energy may be essential to export protons from the mycobacterial cytosol to the external environment, thereby raising the internal pH. Thus specific induction of lipF may prime mycobacteria to be more resistant to acidic stress and be more likely to survive in vivo.

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Acknowledgments
This work was funded by a National Institutes of Health grant 5R03AI054794-02, an American Lung Association Grant, a California Lung Association grant, and a Potts Memorial Foundation grant.

References
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Tran SL, Rao M, Simmers C, Gebhard S, Olsson K, Cook GM. Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide mchlorophenylhydrozone. Microbiology 2005;151:665672. [PubMed: 15758213] Valdivia RH, Hromockyi AE, Monack D, Ramakrishnan L, Falkow S. Applications for green fluorescent protein (GFP) in the host-pathogen interactions. Gene 1996;173:4752. [PubMed: 8707055] Via LE, Deretic D, Ulmer RJ, Hibler NS, Huber LA, Deretic V. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab 5 and rab7. J Biol Chem 1997;272:1332613331. [PubMed: 9148954] Xu S, Cooper A, Sturgill-Koszycki S, van Heyningen T, Chatterjee D, Orme I, Allen P, Russell D. Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium infected macrophages. J Immunol 1994;153:25682578. [PubMed: 8077667] Zhang M, Wang J, Li Z, Xie J, Yang Y, Zhong Y, Wang H. Expression and characterization of the carboxyl esterase Rv 3487c from Mycobacterium tuberculosis. Prot Exp Purific 2005;42:5966.

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Fig. 1.

Effect of varying pHs on the lipF promoter. M. smegmatis bearing pFPV27 or plipF was exposed to growth media at a. pH 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 for 3 hours or 20 hours (o/n), or at b. pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, or 7.0 for 20 hours (o/n). AFU is adjusted fluorescence units.

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Fig. 2.

Effect of environmental stresses on the lipF promoter. M. smegmatis bearing pFPV27, plipF, or pBEN was exposed separately to a. neutral pH, pH 4.5, low temperature, heat shock, oxidative stress for 3 hours or 20 hours, or to hypoxic conditions for 3 hours or 20 hours, b. or to pHs ranging from pH 7.0 to pH 10.0 for 3 hours.

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