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Journal of Biotechnology 140 (2009) 270–277

Contents lists available at ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

ristinamycin-inducible gene regulation in mycobacteria

rancesca Forti ∗, Andrea Crosta, Daniela Ghisottiipartimento di Scienze Biomolecolari e Biotecnologie - Università degli Studi di Milano - Via Celoria 26 - 20133 Milano - Italy

r t i c l e i n f o

rticle history:eceived 2 December 2008eceived in revised form 26 January 2009ccepted 3 February 2009

eywords:

a b s t r a c t

In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This systemis based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and theinducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reportergene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated thatthe ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold inMycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was

nducible promoterycobacteria

ristinamycinonditional mutants

fully repressed in the absence of the inducer.Three mycobacterial genes were cloned under pptr–Pip control, both in sense and antisense direction;

both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reductionof the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditionalmutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer

owe

pristinamycin I. Thus it sh

. Introduction

About two million human deaths every year are caused byycobacterium tuberculosis infection, resulting in a severe burden

or mankind. No vaccine is able to fight efficiently the infection, thushe research for new drugs represents the only way for hoping in aetter future.

Inducible gene expression systems are powerful tools for study-ng gene function and validating drug targets in bacteria, allowingo over-express proteins in a controlled way, to generate knock-owns using antisense RNAs, and to construct conditionally lethalutants where the expression of an essential gene can be con-

rolled. So far, only a few regulated expression systems are availableo control gene expression in mycobacteria (Mahenthiralingam etl., 1993; Parish and Stoker, 1997; Lim et al., 2000; Ehrt et al., 2005;lokpoel et al., 2005; Carroll et al., 2005; Hernandez-Abanto etl., 2006; Guo et al., 2007; Gandotra et al., 2007). The acetamide-ontrolled expression system is based on the inducible acetamidaseene promoter of Mycobacterium smegmatis, controlled by two pos-tive and one negative regulators (Mahenthiralingam et al., 1993;arish and Stoker, 1997; Roberts et al., 2003). It has been used

or over-expression of mycobacterial genes, antisense expressionnd for conditional expression of essential genes in M. smegma-is and M. tuberculosis (Triccas et al., 1998; Dziadek et al., 2003;reendyke et al., 2002; Gomez and Bishai, 2000). However this

∗ Corresponding author. Tel.: +39 02 50315021; fax: +39 02 50315044.E-mail address: francesca.forti@unimi.it (F. Forti).

168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2009.02.001

d to be an effective inducible system in mycobacteria.© 2009 Elsevier B.V. All rights reserved.

system demonstrated to be relatively unstable (Brown and Parish,2006), not tightly switched off (Parish and Stoker, 1997; Parish etal., 2001; Roberts et al., 2003), and is not suitable for use in vivo.

Other inducible systems that have been used in mycobacte-ria are based on the use of anhydrotetracycline as inducer, andon tetracycline regulatory systems derived from Escherichia coliTn10 (Ehrt et al., 2005; Carroll et al., 2005; Guo et al., 2007),Corynebacterium glutamicum (Blokpoel et al., 2005), and Strepto-myces coelicolor (Hernandez-Abanto et al., 2006). These systemsallowed gene regulation in M. smegmatis and M. tuberculosis andhave proved to work in macrophage infection too (Blokpoel et al.,2005; Ehrt et al., 2005). Two kinds of M. tuberculosis conditionalmutants have also been constructed: one whose growth dependson the presence of tetracycline (Carroll et al., 2005; Gandotra et al.,2007), the other which showed the mutant phenotype only whentetracycline is added (Gandotra et al., 2007).

Here we report the development of a new gene regulationsystem for controlling gene expression in fast and slow growingmycobacteria. The system is based on the inducer streptograminpristinamycin I (Barrière et al., 1994), on the pristinamycin-responsive protein Pip of S. coelicolor (Folcher et al., 2001), and onthe ptr promoter of the multidrug resistance gene ptr of Strepto-myces pristinaespiralis (Blanc et al., 1995; Salah-Bey and Thompson,1995). Pip is a pristinamycin sensitive repressor, able to bind to three

operator sites upstream of ptr, overlapping the promoter region(Fig. 1). Upon addition of pristinamycin I, Pip is released and tran-scription can initiate (Salah-Bey and Thompson, 1995; Fusseneggeret al., 2000; Frey et al., 2001). Such a system has been successfullyused both in plants and mammalians (Fussenegger et al., 2000; Frey

F. Forti et al. / Journal of Biotechnology 140 (2009) 270–277 271

F , −35, the +1 and ATG codon are underlined. The regions bound by the Pip repressor areG

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Fig. 2. Plasmids pMY696, pMY718, pMY719, and pMY769. The plasmids are deriva-tives of the integrative pSM128 (Timm et al., 1994). In pMY696 the ptr promoterregion of S. pristinaespiralis was cloned upstream of the reporter lacZ. In pMY718and pMY719, the pip gene of S. coelicolor was cloned under the control of eitherpfurA102 or pfurA104. In the unique HindIII site of pMYS769 was cloned the codingor antisense regions of pknB, glnA1 and fadD32, under the control of ptr promoter.

Table 1Regions cloned in plasmids.

Plasmid Gene/antisense Coordinates of the cloned regiona

pMYT770 fadD32 +4 to +1928pMYT771 Antisense glnAI −22 to +525pMYT772 glnAI +4 to +1452pMYT773 Antisense pknB −11 to +592pMYT774 Antisense fadD32 −12 to +528pMYT775 pknB +4 to +1894pMYT795 5′ end fadD32 −16 to +930

′

Fb

ig. 1. Sequence of the S. pristinaespiralis ptr promoter cloned in pMY696. The −10TACnnnGTAC and are highlighted in grey.

t al., 2001), where it showed a low baseline expression and a highnduction rate.

In this work we developed a pristinamycin I inducible systemPip-ON) that allows the efficient regulation of gene expression bothn M. smegmatis and in M. tuberculosis. This system also allowed theonstruction of two conditional mutants in the fadD32 and pknB M.uberculosis genes.

. Materials and methods

.1. Bacterial strains, growth media and transformationonditions

M. smegmatis mc2155 (Snapper et al., 1990) was grown in LDedium (Sabbattini et al., 1995) containing 0.2% (vol/vol) glycerol

nd 0.05% (vol/vol) Tween 80 and supplemented when necessaryith spectinomycin (100 �g/ml). M. tuberculosis H37Rv (labora-

ory stock) was grown in Middlebrook 7H9 broth or 7H10 agaredium supplemented with 0.05% Tween 80, 0.2% glycerol and

0% ADN (Albumin, Dextrose, NaCl). When necessary streptomycinas added at 20 �g/ml, hygromycin at 50 �g/ml. Pristinamycin I

Sanofi-Aventis, Paris, France) was dissolved in DMSO (50 mg/ml)nd diluted to different concentrations. E. coli DH10B (Grant et al.,990) was used as host strain for cloning and plasmid propaga-ion. The preparation of electrocompetent cells and conduction oflectroporation were performed following standard procedures.

.2. Plasmids

pMF150 was bought from Cistronics Cell Technology GmbH andsed as template for pip gene amplification; pMYT127 is an integra-ive vector, derivative of pSM128 (Timm et al., 1994), carrying theacZ reporter gene; pMY696 is a derivative of pMYT127, in whichhe ptr promoter region of S. pristinaespiralis was cloned upstreamf the reporter lacZ (Figs. 1 and 2). In pMY718 and pMY719, the pipene of S. coelicolor was cloned under transcriptional and transla-ional control of either pfurA102 or pfurA104 (Figs. 2 and 3). pMY769s a derivative of pSM128 in which the lacZ gene was substituted byhe pip gene controlled by the pfurA102 promoter. In the uniqueindIII site of pMY769 were cloned the coding or antisense regionsf pknB, fadD32 and glnA1, controlled by the ptr promoter, obtain-ng plasmids for over-expression of the genes (pMYT775, pMYT770

nd pMYT772), and for over-expression of the antisense RNAspMYT773, pMYT774 and pMYT771). The fragments cloned in theix plasmids are indicated in Table 1.

In pMYT795 and pMYT797 (Fig. 7) the 5′ ends of fadD32 andknB (Table 1) were cloned in the NcoI and SphI sites of pAZI9479

ig. 3. Sequence of the M. tuberculosis wild type furA promoter regions. The first six basesold. The furA102 and furA104 promoter sequences carry the 6-bp substitutions indicated

pMYT797 5 end pknB −16 to +870

a The coordinates are arbitrary, with coordinate +1 corresponding to the firstnucleotide of each gene.

plasmid, kindly provided by AstraZeneca Bangalore, under the ptrpromoter.

2.3. Antibodies

Polyclonal rabbit antibodies anti-M. tuberculosis GlnA1 andPknB were kindly provided by Marcus Horwitz and Brigitte Saint-Joanis, respectively. Polyclonal rat antibodies against M. tuberculosisFadD32 were received from Ida Rosenkrands.

in italic are a restriction site for HindIII. The −35 and −10 consensus regions are inbelow the sequence.

272 F. Forti et al. / Journal of Biotechnology 140 (2009) 270–277

Table 2�-Galactosidase activity expressed by M. smegmatis mc2155 and M. tuberculosis H37Rv carrying the indicated plasmidsa.

Strain Description �-Galactosidase activity in Miller Units ± SD

mc2155(pMYT127) – 2.00 ± 0.00mc2155(pMY696) pptr-lacZ 4193.33 ± 206.63mc2155(pMY718) pptr-lacZ + pfurA102-pip 5.00 ± 3.56mc2155(pMY719) pptr-lacZ + pfurA104-pip 2.00 ± 0.82H37Rv(pMYT127) – 6.27 ± 1.93HH ip

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37Rv(pMY696) pptr-lacZ37Rv(pMY718) pptr-lacZ + pfurA102-p

a �-Galactosidase activity was measured in extracts obtained as described in Seviation.

.4. Preparation of crude extracts of M. tuberculosis and Westernlot

Crude extracts of M. tuberculosis were prepared from 40 ml loghase cultures (OD600 = 0.8). The cells were pelleted, washed in0 mM Tris–HCl pH 8, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT,0 mM KCl, and resuspended in 600 �l of 20 mM Tris–HCl pH 8,0 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 50 mM KCl, 1 mM PMSF,0% glycerol. The cells were lysed by vortexing for 5 min in the pres-nce of zirconia–silica beads. The cell lysate was spun for 10 min at5,000 × g and the supernatant (about 2 mg/ml) was used as therude extract.

About 15 �g of crude extracts were run on a 12% SDS–PAGEel, the proteins transferred on a nitrocellulose membrane, andhe blots were probed with polyclonal rabbit anti-M. tuberculo-is GlnA1 and anti-PknB antibodies, and polyclonal rat anti-M.uberculosis FadD32 antibody. The membranes were subsequentlyncubated with horseradish peroxidase-linked secondary antibod-es (Amersham), a chemiluminescent substrate (ECL Detectioneagents Amersham), and the proteins were visualized by exposureo X-ray film.

.5. RNA extraction from M. tuberculosis

40 ml of M. tuberculosis cultures carrying the different plas-ids (OD600 = 0.2–0.3) were pelleted and frozen in liquid nitrogen.

ells were then resuspended in 100 �l of Tris–EDTA buffer; 75 �lf RNA lysis buffer (4 M guanidinium thiocyanate, 0.01 M TrisH 7.5, 0.97% �-mercaptoethanol) were added and the suspen-ion was vortexed for 5′ in the presence of zirconia–silica beads.he RNA was then purified using the SV Total RNA Isolation Sys-em according to the manufacturer’s protocol (Promega). After ahenol–chloroform extraction the RNA was treated with DNasePromega), phenol–chloroform extracted and ethanol precipitated.

.6. Quantitative RT-PCR

2 �g of RNA extracted as described above were reverse-ranscribed with Superscript II Reverse Transcriptase (Invitrogen)sing random primers. Double stranded DNA binding dye iQ SYBRreen Supermix (Bio-Rad) in an iCycler iQ real-time PCR detectionystem from Bio-Rad was used to quantify the number of targetDNA molecules in the different samples, using primers specificor each gene. Each reaction was run in triplicate and the melt-ng curves were constructed. The relative expression of mRNAs wasetermined using the �(�Ct) method (Ririe et al., 1997), with theigA mRNA as standard.

.7. Pristinamycin I minimal inhibitory concentration (MIC)eterminations

In M. smegmatis and M. tuberculosis the MICs of pristinamycin Iere determined by evaluating the colony forming units (cfu) of an

12146.50 ± 737.505.22 ± 3.29

2. The mean of two to four independent experiments is reported ± the standard

exponentially growing culture on increasing pristinamycin I con-centrations. In M. smegmatis with 500 �g/ml we observed a lowernumber of colonies (<50% of the control without pristinamycin I)of reduced size; with concentrations of 1000 �g/ml or higher nocolony could grow.

In M. tuberculosis with 60 �g/ml we found a decrease in the num-ber of colonies of reduced size; with a concentration of 100 �g/mlno colony could grow.

2.8. ˇ-Galactosidase activity

Independent cultures of M. tuberculosis H37Rv or M. smegma-tis mc2155 carrying the different plasmids were grown at 37 ◦Cto OD600 = 0.8. Cells were collected by centrifugation, resuspendedin 500 �l of TEDP (0.1 M Tris–HCl, 1 mM EDTA, 1 mM DTT and1 mM PMSF), and disrupted by vortexing for 5′ in the presence ofzirconia–silica beads. �-Galactosidase activity of the extracts wasmeasured as described in Miller (1972). The enzyme activity wasexpressed as nanomoles of o-nitrophenol-beta-galactopyranosideconverted to o-nitrophenol min−1 milligram protein−1. Each exper-iment was performed at least three times.

2.9. Generation of conditional mutants

pMYT795 and pMYT797 were electroporated into M. tuberculosisand homologous recombinants were selected on plates containinghygromycin 50 �g/ml and pristinamycin 2 �g/ml. The transfor-mants were analyzed by PCR to confirm the correct insertion ofthe plasmids in the chromosome.

3. Results and discussion

3.1. Construction of a new inducible promoter system inmycobacteria

We wanted to test in mycobacteria the pristinamycin induciblesystem already used in mammalian and plant cells (Fusseneggeret al., 2000; Frey et al., 2001). This system is based on thepromoter–operator site of S. pristinaespiralis ptr (Salah-Bey andThompson, 1995; Fussenegger et al., 2000) and on the S. coelicolorpristinamycin-responsive Pip repressor (Folcher et al., 2001). Wecloned the ptr promoter upstream of the lacZ reporter gene in theintegrative plasmid pMYT127, obtaining pMY696 (Fig. 2).

Subsequently, the S. coelicolor pip gene was amplified by PCRfrom pMF150 bought from Cistronics Cell Technology GmbH andcloned downstream of two constitutive mycobacterial promoters ofdifferent strength: the mutant pfurA102 and the stronger pfurA104

of M. tuberculosis. Both promoters carry mutations that eliminatethe negative control by FurA (Fig. 3; Sala et al., 2003). The pip genescontrolled by the two different promoters were cloned in plas-mids already carrying the lacZ gene controlled by the ptr promoter,obtaining pMY718 and pMY719 (Fig. 2).

F. Forti et al. / Journal of Biotechnology 140 (2009) 270–277 273

Table 3Induction of �-galactosidase activity by pristinamycin I in M. smegmatis mc2155a.

Plasmid Pristinamycin I (�g/ml) OD600 after induction �-Galactosidase activity in Miller Units Incrementb

pMY718 0 1.943 10 120 1.953 133 1340 1.963 296 3080 1.696 373 37

160 1.856 524 52

pMY719 0 1.818 2.8 120 1.902 4.3 1.540 1.866 5.2 1.980 1.210 7.7 2.7

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induction with 2 �g/ml of pristinamycin I.Overnight induction of H37Rv(pMY775), in which pknB was

cloned, was excessive and caused cell death. Thus, we reduced theinduction time to 6 h with 2 or 4 �g/ml of pristinamycin I. At leastsix bands were visualized: one band corresponds to the apparent

Table 4Induction of �-galactosidase activity by pristinamycin I in H37Rv(pMY718)a.

Pristinamycin I (�g/ml) �-Galactosidaseactivity in MillerUnits ± SDb

Incrementc

0 4.61 ± 2.87 10.015 933.00 ± 304.12 2020.15 1906.33 ± 124.67 4130.50 2057.33 ± 759.94 4461.5 1762.80 ± 561.09 3825 806.00 ± 231.06 174

a

160 1.926

a The strains were grown to OD600 = 0.2–0.3, pristinamycin I was added at the indb Ratio of the �-galactosidase activity after induction relative to the uninduced va

We first tested the system in M. smegmatis: strain mc2155 wasransformed with the plasmids, selecting for spectinomycin resis-ance, and strains in which the plasmids integrated at the attB sitef mycobacteriophage L5 (Hatfull and Sarkis, 1993) were obtained.

�-Galactosidase activity expressed by these strains was mea-ured as described in Section 2 (Table 2). The activity expressedrom pMY696 was high (more than 4000 Miller Units), meaninghat the ptr promoter has a strong activity in M. smegmatis, and thusuitable for performing the repression test in the presence of Pip.oth plasmids pMY718 and pMY719, expressing the Pip repressor,howed a very low �-galactosidase activity, near to the backgroundevel, indicating that Pip could repress pptr efficiently (about 840-old repression).

Then we tested the inducibility of our system by pristinamycin I.he MIC of pristinamycin I for mc2155 was about 500–1000 �g/mlsee Section 2), thus we used pristinamycin I concentrations in

range from 20 to 160 �g/ml (Table 3). In mc2155(pMY718) �-alactosidase activity increased in dependence of pristinamycin Ioncentrations (up to more than 50-fold increase in Miller Units),hereas in mc2155(pMY719) the response was clearly lower (up to

-fold increase). From these results we concluded that the repres-ion in pMY719 was too strong and decided to test only pMY718 in. tuberculosis.

We transformed H37Rv with pMY696 and pMY718 and mea-ured �-galactosidase activity of the strains obtained (Table 2).s in M. smegmatis, pptr showed to have a high activity (about2,000 M.U. for H37Rv[pMY696]) and to be effectively repressedy Pip (about 5 M.U. for H37Rv[pMY718], 2400-fold repression).e then tested the inducibility of the system by pristinamycin I.

he MIC of pristinamycin I for M. tuberculosis was 60–100 �g/mlsee Section 2), thus the inducer was added at doses of 0.015,.15, 0.5, 1.5 and 5 �g/ml to H37Rv(pMY718). In response toristinamycin I we could obtain a maximum induction of about00-fold with 0.15–0.5 �g/ml of pristinamycin I (Table 4). Adding�g/ml or higher concentrations of pristinamycin I, cell growthas partially inhibited (data not shown), and the activity decreased

Table 4).Thus, in M. tuberculosis the system appears to be tightly con-

rolled and efficiently induced at concentrations that are about00-fold below the MIC of pristinamycin I.

Moreover, compared to the previously constructed TET systemEhrt et al., 2005) our system showed a higher induction factor (400-old compared to160-fold) and a more efficient repression (2400-old compared to 400-fold repression).

.2. Use of the Pip-ON system for over-expression andownregulation of genes in M. tuberculosis

In order to test whether the system could work for over-xpression and down regulation of mycobacterial genes, we

14.1 5.0

concentration, and the cultures were incubated overnight.

decided to clone under ptr-Pip control three M. tuberculosis genesand their antisense RNAs:

- pknB: the receptor-like protein kinase PknB from M. tuberculosisis an essential gene that is involved in control of cell shape andcell division (Av-Gay et al., 1999; Fernandez et al., 2006).

- glnA1: glutamine synthetase, an enzyme of central importance innitrogen metabolism; the M. tuberculosis mutant is auxotrophicfor l-glutamine (Tullius et al., 2003; Harth et al., 2005).

- fadD32: the acyl-AMP ligase FadD32 is required for the synthesisof mycolic acids and essential for mycobacterial growth (Portevinet al., 2005).

The three genes were PCR amplified with appropriate oligonu-cleotides, and cloned under the control of the pristinamycininducible ptr promoter in pMY769 (Fig. 2) both in sense andantisense orientation. Six different plasmids were obtained, three(pMYT770, pMYT772 and pMYT775) for over-expression of thegene products (FadD32, GlnA1, and PknB, respectively), and threefor over-expression of antisense RNAs (pMYT771, pMYT773, andpMYT774 against glnA1, pknB, and fadD32, respectively).

3.2.1. Over-expression of proteinsM. tuberculosis strain H37Rv was transformed with the plasmids

pMYT770 (FadD32), pMYT772 (GlnA1), and pMYT775 (PknB), andthe resulting strains were analyzed by Western blot in the inducedand uninduced condition (Fig. 4). For FadD32 and GlnA1, the inten-sity of the signal increased about 2- and 5-fold after overnight

H37Rv carrying pMY718 was grown up to OD600 = 0.3, induced with differentconcentrations of pristinamycin I, then grown for 48 h with shaking.

b The mean of three independent experiments is reported ± the standard devia-tion.

c Ratio of the �-galactosidase activity after induction relative to the uninducedvalue.

274 F. Forti et al. / Journal of Biotechnology 140 (2009) 270–277

Fig. 4. Over-expression of GlnA1, PknB, and FadD32 by H37Rv(pMYT772), H37Rv(pMYT775) and H37Rv(pMYT770). Immunoblot analysis of the cell lysates (15 �g of totalp ed wi1 rculosa GlnA1s

MtMmaabonTa

oHos

FOa

roteins per lane). Crude extracts from cultures at OD600 = 0.8, uninduced or induc2% SDS–PAGE gel, and the blots were probed with polyclonal rabbit anti-M. tubentibodies; MWM, molecular weights marker, in kilodaltons. The relative amount ofoftware (Molecular Dynamics).

W of the monomeric protein, which migrates a little faster thanhe 55 kDa molecular weight marker. Three bands had an apparent

W higher than the PknB monomer, in particular the slower oneay correspond to a dimer of the protein. The further two bands

re very faint. Two other bands had a MW lower than the monomernd are probably degraded forms of the protein. Cumulating all theands, their intensities were around 3.9 (induction with 2 �g/mlf pristinamycin I) and 4.9-fold (induction with 4 �g/ml of pristi-amycin I) the intensity of the signal of the non-induced condition.hus over-expression of PknB leads to the formation of aggregatesnd possibly also to partial degradation of the protein.

As previously reported (Kang et al., 2005), the effect

f PknB over-expression reduced mycobacterial growth of37Rv(pMYT775) (Fig. 5), whereas no growth defect could bebserved for H37Rv(pMYT772) and H37Rv(pMYT770) (data nothown).

ig. 5. Growth curve of H37Rv(pMYT775) upon induction. The culture was grown toD600 about 0.1, divided in two and pristinamycin I added to one of the subculturest 2 �g/ml. Closed squares: uninduced culture; open squares: induced culture.

th the concentration of pristinamycin I indicated on top of the lanes, were run onis GlnA1, anti-M. tuberculosis PknB, and polyclonal rat anti-M. tuberculosis FadD32, PknB, and FadD32, expressed upon induction, are quantified by using ImageQuant

Our conclusion is that this system led to a controlled and rela-tively effective over-expression of the three proteins tested.

3.2.2. Over-expression of antisense RNAsM. tuberculosis strain H37Rv was transformed with the plasmids

pMYT771 (anti-glnA1), pMYT773 (anti-pknB), and pMYT774 (anti-fadD32) for over-expression of antisense RNAs, and the strains werethen analyzed by real-time RT-PCR, Western blot and for the effectsof pristinamycin I induction on bacterial growth.

By real-time RT-PCR (Table 5) the antisense RNAs showed to betranscribed by the different strains in consistent although differentamount. Moreover, we evaluated if the level of the target RNAs var-ied upon induction: we observed a slight decrease for the pknB andglnA1 transcripts, whereas for fadD32 RNA the reduction was morerelevant (0.2–0.3 of the non-induced).

Western analysis (Fig. 6) showed that upon pristinamycin Iinduction the amount of PknB, GlnA1, and FadD32 decreased about0.5, 0.7/0.8, and 0.7/0.8-fold, respectively. Thus, the over-expressionof antisense RNAs appeared to reduce in a certain extent the levelof target RNAs and proteins.

H37Rv(pMYT773) and H37Rv(pMYT774) that over-express anti-PknB and anti-FadD32, respectively, when plated on agar platescontaining 2 �g/ml pristinamycin I, decreased their generationtime: the colonies formed after 2–3 weeks were visibly smallerthan the control colonies plated in the absence of pristinamycinI, although they continued to grow. We tried to confirm this growthdefect in liquid cultures, but the growth curves of the induced anduninduced cultures did not show any significant difference (datanot shown). This only very limited growth defect is in agreementwith the fact that, upon induction, the amount of FadD32 and PknBis only slightly reduced.

3.3. Construction of two conditional mutant strains of M.tuberculosis

Then we tested whether our inducible system was suitable toobtain conditional mutants in M. tuberculosis. We chose to mutage-nize pknB, coding for a Ser/Thr protein kinase already demonstratedto be essential in M. tuberculosis (Fernandez et al., 2006), andfadD32, coding for an enzyme required for the production of mycolicacids, essential in M. smegmatis (Portevin et al., 2005). We con-

structed the suicide plasmids pMYT795 and pMY797, carrying the5′ end of fadD32 and pknB, respectively, under the control of theptr promoter (Table 1, Fig. 7). The plasmids carried as well the pipgene under the pfurA102 promoter and a hygromycin resistancecassette. These plasmids were introduced into the M. tuberculosis

F. Forti et al. / Journal of Biotechnology 140 (2009) 270–277 275

Table 5Relative amount of antisense RNA and target gene transcripta.

Relative amount of antisense RNAb Relative amount of target transcript

Uninduced Induced Uninduced Inducedc

pknB 1 177 1 0.62–0.80glnA1 1 475 1 0.71–0.81fadD32 1 21 1 0.22–0.27

a Cultures of plasmid carrying strains H37Rv(pMYT773), H37Rv(pMYT771), and H37Rv(pMYT774) were grown to OD600 = 0.3, divided in two, and one of the subculturesinduced with 2 �g/ml of pristinamycin I for 6 h. The RNAs were then extracted, reverse-transcribed and quantified as described in Section 2.

b The amount of RNA was evaluated by quantitative RT-PCR, as described in Section 2.c Results of two independent experiments.

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crplc

apolpatt

Fds

ig. 6. Effects of over-expression of antisense RNA against GlnA1, PknB, and FadD32as performed as indicated in the legend of Fig. 4.

hromosome by a single homologous recombination event, givingise to two strains in which the only functional copy of fadD32, orknB, were expressed from pptr (Fig. 7). The two strains were ana-yzed by PCR to control the correct insertion of the plasmids in thehromosome.

The phenotypes of the mutants were analyzed in the presencend absence of pristinamycin I. In solid media they grew in theresence of 2 �g/ml of pristinamycin I, but no growth could bebserved without the inducer (data not shown). When grown iniquid media with or without pristinamycin (Fig. 8), after an initial

hase during which the optical density of the cultures increasedt the same rate, at 48–72 h after the removal of pristinamycin I,he uninduced cultures stopped growing and started clumping. Onhe contrary, the growth curves of the induced cultures were indis-

ig. 7. Construction of conditional mutants. The recombination vectors pMYT795 and pMownstream of pptr into a plasmid which does not replicate in mycobacteria. Single-crostrains had one complete functional copy of fadD32 or pknB downstream of pptr as well a

7Rv(pMYT771), H37Rv(pMYT773) and H37Rv(pMYT774). The immunoblot analysis

tinguishable from those of the wild type strain. Plating on solidmedia containing different amount of pristinamycin I, we foundthat the minimal concentration sustaining optimal mutants growthwas 0.2 �g/ml; with 0.05 �g/ml the growth rate was slightly sloweddown, but with 0.005 �g/ml or lower concentrations we could notobserve any growth. Thus, with our inducible system, we demon-strated that, as already published (Fernandez et al., 2006), pknB isessential for the growth of M. tuberculosis. About the mutant strainin which the fadD32 promoter has been substituted, we could con-clude that fadD32 and/or pks13 and accD4, which are the second

and the third gene, respectively, of the same putative operon, arenecessary for M. tuberculosis growth.

These data showed that our system is efficient not only inrepressing genes put under its control, but also in inducing them:

YT797 were constructed by cloning the 5′ region of fadD32 and pknB, respectively,sover recombinants were generated by homologous recombination. The resulting

s the 5′ ends of the genes only.

276 F. Forti et al. / Journal of Biotechnology 140 (2009) 270–277

F ant. Th D600 am 2 �g/

ipatfaTt

eahh

A

Rhpwb

R

A

B

B

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ig. 8. Growth phenotypes of conditional mutants. (A) fadD32 mutant. (B) pknB mutygromycin 50 �g/ml and pristinamycin 2 �g/ml, pelletted and resuspended at Outant; triangles: pknB mutant; closed symbols: medium containing pristinamycin

n the absence of the inducer the growth of the mutants stops com-letely, but in the presence of pristinamycin they could grow as wells the wild type strain. Thus it is a real effective tool to study essen-ial genes in mycobacteria. Another advantage of this system is theact that pristinamycin I is readily bioavailable for eukaryotic cellsnd does not show significant interference with host metabolism.his opens up the possibility to control the expression of mycobac-erial genes in vivo as well as in vitro.

This study demonstrates the use of a mammalian cell-validatedxpression system in mycobacteria; the reverse case, in which

mycobacterial repressor–operator system was transferred touman cells for the discovery of new antituberculosis compounds,as been recently reported (Weber et al., 2008).

cknowledgements

We thank Marcus A. Horwitz, Brigitte Saint-Joanis, and Idaosenkrands for kindly providing antibodies, and Sudha Ravis-ankar and Haripriya Ramu from AstraZeneca Bangalore for theAZI9479 suicide vector. This work was supported by EC-VI Frame-ork Contract no. LSHP-CT-2005-018923 to Giovanna Riccardi, and

y PRIN2006, no. 2006064583 002 by Daniela Ghisotti.

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