Chemicals, enzymes and DNA
Hygromycin B was purchased from Calbiochem. All other chemicals were purchased from Merck, Roche or Sigma at the highest purity available. Enzymes for DNA restriction and modification were purchased from New England Biolabs. Isolation and modification of DNA was performed as described (
Ausubel et al., 1990). Oligonucleotides were obtained from Integrated DNA Technologies (
Table S1).
Bacterial strains and growth conditions
All bacterial strains used in this study are listed in . Mycobacterial strains were grown at 37°C in Middlebrook 7H9 liquid medium (Difco Laboratories) supplemented with 0.2% glycerol, 0.025% Tyloxapol or on Middlebrook 7H10 agar (Difco Laboratories) supplemented with 0.2% glycerol, unless indicated otherwise.
Escherichia coli DH5α was used for all cloning experiments and was routinely grown in LB medium at 37°C.
M. tuberculosis strains were grown in Dubos broth or Hartmans de Bont (HdB) minimal medium (
Smeulders et al., 1999) or on 7H10 agar plates supplemented with 0.2% glycerol and 10% Middlebrook OADC enrichment (BBL) at 37°C. In HdB medium, (NH
4)
2SO
4 was replaced by Na
2SO
4 and 15 mM asparagine was used as a nitrogen source. Antibiotics were used when required at the following concentrations: hygromycin (200 μg ml
−1 for
E. coli; 50 μg ml
−1 for mycobacteria) and kanamycin (50 μg ml
−1 for
E. coli; 30 μg ml
−1 for mycobacteria).
Construction of ompATb expression vectors for mycobacteria and E. coli
To construct the
ompATb operon (
ompATb-rv0900-rv0901) expression plasmid pML763 driven by the native promoter of
ompATb, the
ompATb-rv0900-rv0901 operon and the adjacent upstream 1.0 kb promoter region was amplified from
M. tuberculosis genomic DNA using the primer pair 1091/1149 and ligated into pML003 (
Song et al., 2008), which was digested by
XbaI and
SwaI. The
psmyc-ompATb operon expression plasmid pML1450 was constructed by removal of the
ompATb-rv0900-rv0901 operon via digestion of pML763 using
HpaI and
HindIII, and ligation into similarly digested pML003. The N-terminally truncated
ompATb73-326 expression plasmid was constructed by PCR amplification of
ompATb73-326 from pML752 with the primer pair 2277/2184. A second PCR amplification of the product using the primer pairs 2276/2184 allowed for addition of a shine-delgarno sequence, which was followed by digestion with
PacI and
HindIII, and ligation into similarly digested pML003.
To construct an integrative expression vector for
ompATb, the plasmid pCV125 (
Steyn et al., 2003) containing the integrase gene and attachment site (
attP) of the mycobacteriophage L5 and a kanamycin resistant gene was used. The
pimyc-ompATb fragment was removed from pML588 by
XbaI and
ClaI digestion, and ligated into similarly digested pCV125. The resulting plasmid was named pML759 () and integrated into the L5
attB site of
M. tuberculosis ML163 (Δ
ompATb::loxP). The complemented strain was named ML168 (Δ
ompATb::loxP,
attB::pML759). All plasmid constructions were verified by restriction enzyme digestion and sequencing and are listed in , while all strains are listed in .
To construct a C-terminally his-tagged
ompATb overexpression vector for
E. coli, the
ompATb gene was amplified from pML588 by PCR using the primers 960 and 962 (
Song et al., 2008). This fragment was digested with
NcoI and
SwaI and ligated into similarly digested pET28b+ (Novagen), resulting in pML591.
Overexpression of ompATb in E. coli
For protein expression and purification, a 1 L culture was grown to OD600 of 0.6-1.0, and induced with 0.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 2 h at 37°C. Bacteria were harvested by centrifugation, resuspended in 20 ml lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) and sonicated on ice for 15 min. Then, the cell suspension was centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was removed, filtrated through a 0.22 μm filter, and loaded onto a Ni2+ charged resin column (HIS-Select™ Spin Columns, Sigma) according to the manufacturer's protocol. Bound proteins were washed (50 mM Tris-HCl, 100 mM NaCl, 5 mM imidazole, pH 8.0) and then eluted from the column using elution buffer (50 mM Tris-HCl, 100 mM NaCl, 250 mM imidazole, pH 8.0).
Construction of an unmarked ompATb deletion mutant of M. tuberculosis
To construct a mutant of
M. tuberculosis lacking
ompATb, fragments of approximately 1,000 bp of DNA up- and downstream of
ompATb were amplified by PCR from chromosomal DNA with the oligonucleotide pairs 779/780 and 982/983, respectively. The restriction sites for
AscI and
SwaI were introduced into the upstream fragment while
PacI and
BfrBI sites were introduced into the downstream fragments. The individual sequences were digested and ligated into the similarly digested temperature sensitive replication vector pML563 (
Song et al., 2008) flanking the
loxP-hyg-loxP cassette, and the resulting plasmid was named pML564. The cloned fragments were sequenced to ensure the absence of PCR errors.
The plasmid pML564 was transformed into M. tuberculosis H37Rv and selected on 7H10/OADC/Hyg plates at 37°C. After three weeks, a single colony was picked and inoculated into 10 ml of 7H9/OADC/Hyg medium and incubated at 37°C on a shaker to an OD600 of 1.0. Dilutions from 1 × 103 to 1 × 106 were plated on 7H10/OADC/Hyg plates and incubated at 41°C. After three weeks, 8 colonies were picked and separately transferred into 30 ml of 7H9/OADC/Hyg medium and cultured at 41°C to prepare chromosomal DNA. All eight clones were correct single cross-over candidates (SCO) and confirmed by southern blot analysis. One SCO candidate was named M. tuberculosis ML157. The SCO ML157 was grown in 7H9/OADC/Hyg medium to an OD600 of 1.0. A series of 10-fold dilutions were plated on 7H10/OADC/Hyg medium supplement with 2% sucrose and incubated at 41°C. After four weeks, 8 single colonies were picked and cultured in 7H9/OADC/Hyg media at 41°C to prepare chromosomal DNA. All eight clones were correct double cross-over candidates (DCO) and confirmed by southern blot analysis. One DCO was named M. tuberculosis ML160 (ΔompATb::loxP-hyg-loxP). The Cre recombinase expression vector pCreSacB1 (a kind gift from Dr. Adrie Steyn, University of Alabama at Birmingham) was used to excise the loxP-flanked hygromycin cassette from the chromosome of M. tuberculosis ML160. The plasmid pCreSacB1 was transformed into M. tuberculosis ML160 and selected on 7H10/OADC/Kan plates. After three weeks, 8 single colonies were transferred into 10 ml of 7H9/OADC/Kan medium and cultured at 37°C to an OD600 of 1.0. Whole cell PCR analysis was performed to confirm that the loxP-hyg-loxP cassette was removed from the genome using the primer pair 976/961. Three out of these 8 colonies were confirmed as having lost the loxP-hyg-loxP cassette. Then, a series of 10-fold dilutions from a single colony having lost the loxP-hyg-loxP cassette was plated on 7H10/OADC plates containing 2% sucrose and incubated at 37°C to counter-select against pCreSacB1. After three weeks, 24 single colonies were streaked in parallel on 7H10-OADC, 7H10/OADC/Kan and 7H10/OADC/Hyg plates to confirm the loss of the hyg cassette and pCreSacB1. Twenty out of these 24 candidates failed to grow on plates containing kanamycin, confirming the loss of pCreSacB1 in these candidates. Colony PCR was performed using the primer pair 976/961 and one amplified fragment was submitted for sequencing. Sequencing results confirmed that this colony was an unmarked ΔompATb mutant and was subsequently named M. tuberculosis ML163 (ΔompATb::loxP). In this mutant, 689 bp within the 981 bp ompATb gene were replaced by 41 bp representing the loxP site.
A specific probe (1128 bp) for
ompATb was amplified by PCR from chromosomal DNA of
M. tuberculosis using the primer pair 781/782. The genomic DNAs of the deletion candidates were digested with
PstI, and Southern blot hybridization was performed as previously described (
Stephan et al., 2004).
Preparation of detergent extracts from M. smegmatis and M. tuberculosis and analysis by Western blot
10 ml cultures of M. smegmatis or M. tuberculosis expressing ompATb genes were grown to high density, normalized to an OD600 of 3.0, washed in TBS containing 1% SDS (pH 7.2), and concentrated in 1 ml TBS containing 1% SDS lysis buffer. Cells in lysis buffer were transferred to glass bead Lysing Matrix Tubes (MP Biomedicals) and disrupted using a FastPrep FP120 (BIO101/Savant) at 6,000 × g for 90 sec. Whole extracts were incubated at 40°C for 2 h at 800 rpm shaking in a Thermomixer R (Eppendorf). Cell debris was removed by centrifugation at 4°C for 10 min at 13,000 × g and the supernatant was used for subsequent Western blot analysis.
Proteins were separated in a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane using a standard protocol (
Ausubel et al., 1987). OmpATb was detected with a rabbit antiserum against OmpATb ((
Senaratne et al., 1998); gift from Dr. Philip Draper) while RNA polymerase was detected with the mouse anti-
E. coli RNAP mAb 8RB13 (Neoclone). A horseradish peroxidase-conjugated goat-anti-rabbit antibody (Sigma) or goat-anti-mouse antibody (Sigma) served as the secondary antibody for OmpATb and RNAP, respectively. The blot was developed using luminol substrates (ECL, Pierce) and detected in an EpiChemi
3 Darkroom (UVP BioImaging system). Purified recombinant rOmpATb
His from
E. coli served as a standard reference.
Growth of M. smegmatis strains on agar plates
Cultures of M. smegmatis SMR5/pMS2, ML16/pMN016, ML16/pMS2, ML16/pML003 or ML16/pML1450 were grown to an OD600 of about 1.0, filtered through a 5 μm filter, and dilutions were plated on minimal HdB medium supplemented with 0.2% glycerol. After 4 days of incubation at 37°C, pictures of colonies were taken under white light at 10 × magnification using a Stemi 2000-C stereomicroscope (Zeiss) and an AxioCam MRc camera (Zeiss).
Uptake experiments
Glucose, glycerol and serine uptake measurements were carried out as described before for glucose (
Stephan et al., 2005) with minor modifications. The cells were grown on Middlebrook 7H10 plates containing 0.1% Tween 80 at 37°C. The cells were scraped off the plates, suspended in Middlebrook 7H9 medium and filtered through a 5.0 μm filter (Sartorius). The cell suspension was used to inoculate 100 ml of Middlebrook 7H9 medium for
M. smegmatis or Dubos medium for
M. tuberculosis containing 1 mM of either glucose, glycerol, serine, or asparagine. The cells were harvested at an OD
600 of 0.6 by centrifugation (3750 ×
g at 4°C for 10 min), washed twice in uptake buffer (50 mM Tris-HCl pH 6.9, 15 mM KCl, 10 mM (NH
4)
2SO
4, 0.05% Tween 80) and resuspended in the same buffer. Radio-labelled [
14C]glucose (GE healthcare) and non-labelled glucose, [
14C]glycerol (GE healthcare) and non-labelled glycerol, [
14C]serine (GE healthcare) and non-labelled serine, or [
3H]asparagine (Moravek Biochemicals and Radiochemicals) and non-labelled asparagine, were mixed and added to cell suspensions to obtain final concentrations of 20 μM accordingly. The mixtures were incubated at 37°C and 1 ml samples were removed at the indicated times. The cells were collected on a 0.45 μm Spin-X centrifuge tube filter (Costar) by mixing with an equal volume of 10% buffered formalin phosphate (Fisher) containing 0.1 M LiCl, and counted in a liquid scintillation counter (Beckman). The uptake rate was expressed as nmol/mg cells (dry weight).
Whole cell enzyme-linked immunosorbent assay (ELISA)
To examine the cellular localization of OmpATb, an enzyme-linked immunosorbent assay (ELISA) using whole cells (
Huff et al., 2009) was employed with modifications. 10 ml cultures of
M. smegmatis expressing
ompATb were diluted to an OD
600 of 1.0, harvested by centrifugation, and washed in 10 ml TBST (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM MgCl
2, 0.025% tyloxapol). Cells were concentrated in 1 ml TBST and 200 μl were transferred in 4 replicates to a U-bottom 96-well microtiter plate (Becton Dickinson). The plate was centrifuged at 3200 ×
g for 5 min at 4°C and cells were blocked with 2.5% skim milk (Difco) in TBST for 1 hour at 37°C. Cells were then washed twice with TBST and incubated for 1 hour at 37°C with an anti-OmpATb antiserum (
Raynaud et al., 2002). Following three washes with 200 μl TBST (3200 ×
g, 5 min, 4°C), cells were then incubated with an anti-rabbit IgG secondary antibody conjugated to alkaline phosphatase and incubated for 1 hour at 37°C. Cells were then washed three times in TBST (3200 ×
g, 5 min, 4°C) and resuspended in 100 μl substrate buffer (0.1 M glycine, 1 mM ZnCl
2, 1 mM MgCl
2, 1% 4-nitrophenyl phosphate (
pNPP)). After incubation for 2 hours at 37°C, the reaction was stopped with the addition of 100 μl 2 M NaOH. The plate was centrifuged and 100 μl of the supernatant was transferred to a flat-bottom 96-well microtiter plate (Nunc). Phosphatase activity was quantified by reading the absorption at 405 nm using a microplate reader (Synergy HT, Bio-TEK Instrument Inc, USA).
pH-dependent growth of M. tuberculosis at pH 7.2 and pH 5.5
M. tuberculosis ML163 (Δ
ompATb::loxP) was transformed with either the integrative
ompATb expression vector pML759 (
pimyc-ompATb), replicative vector pML588 (
pimyc-ompATb) or pML763 (
pnative-ompATb operon). The pML759 plasmid was integrated into the genome after plating on 7H10/OADC/Kan plates and confirmed by Western blot. This resulting strain was named
M. tuberculosis ML168 (Δ
ompATb::loxP;
attB::ompATb).
M. tuberculosis H37Rv wt, ML163, ML168 and the strains complemented with replicative vectors were inoculated into 10 ml 7H9 Middlebrook medium and grown to an OD
600 of 1.0. Cells were then harvested, washed twice with sterilized Millipore water, and transferred into 200 ml Hartmans de Bont (HdB) minimal medium (
Smeulders et al., 1999) (pH 5.5, without OADC) supplemented with 15 mM asparagine as the sole nitrogen source. The initial OD
600 for all cultures was 0.01. Samples of 3 ml were taken every day from each culture for 20 days and filtrated through 0.22 μm filters before measuring the pH using a pH meter (Symphony, VWR) and the ammonia concentration as described below. Samples of 1 ml were taken from the cultures every day and mixed with equal volume of formalin for 30 min. The OD
600 of these samples was measured using a spectrophotometer (SmartSpec™ Plus, Biorad).
Chemical analysis of the culture medium of M. tuberculosis
To identify chemical differences in the supernatants, wt
M. tuberculosis and the Δ
ompATb mutant ML163 were grown in Dubos medium for 14 days. Samples of 5 ml were removed every day from each culture and filtrated through two polyvinylidene fluoride-membrane (PVDF) membrane filters (0.22 μm, Millipore) to remove live cells and kept at −20°C before analysis. These samples were filtered again through Nylon Luer-Lock membrane filters (0.22 μm, Roth) and then injected into a gas chromatograph with a mass selective detector (HP 5971A MSD). Ammonia was separated from other compounds using a capillary column containing a cross-linked polyethyleneglycol matrix (HP Innowax) and identified by comparison with analytical GC/MS data available from a mass spectral data base (
NIST, 2008).
To quantitatively determine ammonia concentrations, we used a spectroscopic method based on the specific formation of the yellow/brownish adduct HgO•Hg(NH
2)I upon addition of Nessler's reagent to an ammonia-containing solution. Formation of the adduct in the supernatants of
M. tuberculosis cultures was determined directly by measuring the absorbance at 520 nm (
London et al., 1974). The analytical procedure was done as described earlier (
Yuen & Polland, 1954). A linear calibration curve was obtained using bis-ammonium salt of citric acid.
As a control experiment to determine whether the compound which reacted with Nessler's reagent was volatile, the supernatants of M. tuberculosis were adjusted to pH >12 using NaOH and distilled by high-vacuum or increased temperature using a Schlenk apparatus in combination with a trap cooled with liquid nitrogen. Then, the ammonia content of the distillate was determined photometrically using Nessler's reagent as described above.
Enzymatic determination of ammonia in culture supernatants of M. tuberculosis
To determine the ammonia concentration in culture supernatants, wt M. tuberculosis, the ΔompATb mutant ML163, and the ML163 complemented strains with pML588 or pML763 were grown in HdB medium for 30 days. Samples of 3 ml were removed every day from each culture and filtrated through two PVDF membrane filters (0.22 μm, Millipore) to remove cells. These samples were frozen at −20°C before analysis. The ammonia concentration in these samples was determined by using an enzymatic method based on the synthesis of glutamate from 2-oxoglutarate and ammonia by glutamate dehydrogenase. The oxidation of nicotinamide-adenine dinucleotide (NADH) during this reaction is stoichiometric to the amount of ammonia and is measured by the loss of absorbance of NADH at 340 nm. The method was used as described in the manufacturer's protocol (Roche).
pH-dependent expression of ompATb in M. tuberculosis
Two cultures of wt
M. tuberculosis H37Rv were grown in Dubos medium at pH 7.2 and 5.5 at 37°C. The pH of the medium was adjusted to pH 7.2 or 5.5 using 1 M NaOH or 1 M HCl before autoclaving. Samples of 20 ml were harvested at an OD
600 of approximately 3.0 by centrifugation and resuspended in 2 ml PEN buffer (80 mM Na
2HPO
4, 20 mM NaH
2PO
4, 100 mM NaCl, 0.1 mM EDTA, pH 7.2) containing 1% SDS, and mixed with 1 ml glass beads (Q-Bio Gene). The cells were disrupted using the Fastprep FP120 beater (BIO101) twice at 6,000 ×
g for a total of 90 seconds, and incubated for 2 h at 40°C while shaking. The cell debris was removed by centrifugation at 16,000 ×
g for 20 min and the detergent extracts were analyzed by gel electrophoresis. The proteins were then blotted on a polyvinylidene fluoride-membrane, and detected by a rabbit anti-OmpATb antiserum as described previously (
Song et al., 2008).
Utilization of amino acids as sole nitrogen sources by M. tuberculosis
To test which amino acids are utilized as nitrogen sources by M. tuberculosis under acidic conditions, wild-type H37Rv was grown in 200 ml HdB media (pH5.5, with OADC) supplemented with or without asparagine, aspartic acid, glutamine, glutamic acid, serine, arginine, methionine, proline, alanine, histidine, glycine, phenylalanine, leucine, valine, threonine, tyrosine, tryptophan, cysteine, isoleucine or lysine in parallel. The amino acid was added as the sole nitrogen source in HdB media. The optical density, pH and ammonia concentration were measured every two days as described above. For ammonia measurements, only the cultures supplemented with asparagine, glutamine, aspartic acid and glutamic acid were measured because no visible growth was found in the cultures supplemented with the other 16 amino acids.
Transcription of the ompATb-rv0900-rv0901 operon
M. tuberculosis RNA extraction and cDNA preparation were performed as described previously (
Hillmann et al., 2007). The primer pairs 976/977, 959/782, and 959/983 (
Table S1) were used to amplify the
ompATb-rv0900-rv0901 fragments as indicated in
Fig. S3. RNA and chromosomal DNA as controls were used as PCR templates to amplify fragments of the
ompATb-rv0900-rv0901 operon.
Mice infection experiments
Balb/c mice (female, 4-5 weeks old) were infected by aerosols with M. tuberculosis H37Rv wt and the isogenic ompATb operon mutant ML163. The cells were resuspended in sterile phosphate-buffered saline (PBS) at an OD600 of 0.2. Ten ml cell suspensions were loaded into a nebulizer of an aerosol chamber containing 40 mice. This provided a 1000 CFU/ml inoculum in lungs for both strains. After 0, 2, 4, 8 and 12 weeks of infection, the mice were sacrificed. Lungs and spleens were homogenized in 0.8 ml PBS by bead-beating, diluted 100x and 0.1 ml suspensions were plated on 7H10/OADC plates. The colonies on agar plates were counted after 1 month of incubation at 37°C.
Female, 6 to 8 week-old, specific pathogen-free BALB/c mice were purchased from Charles River Laboratories (Sülzfeld, Germany) and maintained in individually ventilated cages (IVC, Ebeco, Castrop-Rauxel, Germany) under biosafety level III conditions. All animal experiments performed were in accordance with the German Animal Protection Law and were approved by the Animal Research Ethics Board of the Ministry of Environment, Nature Protection and Agriculture (Kiel, Germany).
For infection experiments using the previously published wt and
ompATb mutant strains, cultures of wt
M. tuberculosis (strain 1424) or the
ompATb deletion mutant CK69 (
Raynaud et al., 2002) were grown to mid-log phase, harvested, aliquoted, and frozen at −80°C. After thawing, viable cell counts were determined by plating serial dilutions on Middlebrook 7H10 agar supplemented with 10% bovine serum (Biowest, Nuaillé, France). Aerogenic infections with approximately 200 CFU of the wild type or the mutant strains were carried out in a Glas-Col aerosol infection device (Glas-Col, Terre-Haute, IN). Actual inoculum size was confirmed 24 h after infection by determining the bacterial burden in undiluted lung homogenates of three infected mice per strain. For intravenous infection, 0.2 ml of a bacterial suspension containing approximately 1x10
5 CFU in PBS were injected into a lateral tail vein. The inocula were confirmed 24 h post infection by determining the bacterial load in undiluted lung homogenates and in serial ten-fold dilutions of spleen and liver homogenates (three infected mice per strain). To follow the course of infection, the bacterial load in lungs and spleens was determined at days 28 and 65 after infection. Organs from five animals per time point and strain were aseptically removed, weighed and homogenized in distilled sterile water containing 0.05% Tween 80. Ten-fold serial dilutions of organ homogenates were plated on Middlebrook 7H10 agar supplemented with 10% bovine serum and incubated at 37°C for 21 days. Colonies on plates were enumerated and results are expressed as log
10 CFU per organ.
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