1 Department of Pathology and Infectious Diseases, Royal Veterinary College, London NW1 0TU, UK
2 Tuberculosis Research, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK
3 Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Correspondence
N. G. Stoker
nstoker{at}rvc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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E. coli culture conditions.
Strains were grown in Luria broth (LB) supplemented with antibiotics as needed or in M9 minimal media (Sambrook et al., 1989) containing 0·2 % glucose or 0·4 % glycerol. E. coli DH5
(Life Technologies) was used for all plasmid constructions.
Cloning of Rv1099c.
M. tuberculosis Rv1099c was cloned into expression vectors using the Tuberculist-predicted start codon (position 35 in Fig. 2). The Rv1099c coding sequence was amplified by PCR using primers Rv1099c-NdeI (GGAATTCCATATGGAGCTGGTCCGGGT) and Rv1099c-XhoI (TGACTCGAGGGCAATGGGTACACG). These primers introduced an NdeI site at the 5' end and an XhoI at the 3' end to allow the gene to be cloned in-frame into the expression vector pET-15b. Primers Rv1099c-PvuII (GGATCAGCTGATGGAGCTGGTCCGGGT) and Rv1099c-EcoRI (CGGAATTCGGGCAATGGGTACACG) were used to introduce a PvuII site at the 5' end and an EcoRI at the 3' end to allow the gene to be cloned in-frame into the expression vector pGEX-KG. The primers were each used at 300 nM final concentration. PCR was carried out using the Expand High Fidelity PCR system (Boehringer Mannheim) with M. tuberculosis DNA as the template and DMSO at 2 %. The temperature cycle used was: an initial 3 min at 94 °C to denature high-G+C DNA; 10 cycles of 45 s at 94 °C, 1 min at 63 °C and 1 min at 72 °C; 25 cycles of 45 s at 94 °C, 1 min at 63 °C and 1 min at 72 °C (this last increasing by 20 s per cycle); and finally an extension step of 7 min at 72 °C to complete primer extension. The PCR products were cleaved with the appropriate restriction enzymes and cloned into the vectors pET-15b or pGEX-KG. In this way, plasmids expressing the M. tuberculosis Rv1099c gene were generated, as both His-tag and GST-fusion constructs.
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To estimate the Km, the concentration of fructose 1,6-bisphosphate was varied from 2 mM (the concentration routinely used elsewhere; see Rashid et al., 2002) down to 3 µM. Fructose 1,6-bisphosphate was used at 20 µM in all experiments designed to test for potential inhibitors, including LiCl, that were included at the concentrations mentioned in Results. The potential inhibitors were mixed with extracts and incubated for 5 min before adding substrate to start the reaction.
Quantitative PCR.
RNA was prepared from an exponential (7-day) rolling culture of M. tuberculosis H37Rv (Betts et al., 2002), and cDNA synthesis was carried out using Superscript II (Invitrogen) according to the manufacturer's protocol. Real-time quantitative PCR (RTq-PCR) reactions were set up using the DyNAmo SYBR Green qPCR kit (MJ Research) and RTq-PCR was performed using the DNA Engine Opticon 2 System (Genetic Research Instrumentation). Reactions containing 1x DNA Master SYBR Green I mix, 1 µl cDNA product and 0·4 µM of each primer in 20 µl were set up on ice. Samples were heated to 95 °C for 10 min before cycling for 35 cycles of 95 °C for 30 s, 60 °C (Rv1099c) or 62 °C (sigA) for 20 s, and 72 °C for 20 s. Fluorescence was captured at the end of each cycle after heating to 80 °C to ensure the denaturation of primer-dimers. The experiment was repeated three times using cDNA from each of two independent RNA preparations.
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RESULTS |
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Rv1099c is the first gene in a cluster of three genes that we predict is an operon (see Fig. 3). The second gene, fum (30 bp downstream), encodes the only predicted fumarase in the genome, which is presumed to function in the citric acid cycle (Fig. 1
). Its location next to Rv1099c is conserved in all the sequenced mycobacterial genomes, and suggests a functional relationship between these two genes. Rv1097c, the start of which overlaps the end of fum in M. tuberculosis, is a putative Gly/Pro-rich membrane protein with unknown function that is not conserved in other species. A conserved hypothetical gene, Rv1100, is transcribed divergently from this putative operon, and its conserved synteny in other genomes is suggestive of a related function.
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This reassignment of the start of M. tuberculosis Rv1099c affects the predicted start for the divergently transcribed Rv1100, as it overlaps the newly assigned start codon for Rv1099c. While it is possible that both promoters lie within coding sequences, it is more likely that there is an intergenic gap. The corynebacterial Rv1100 homologues are located further upstream of glpX (200 bp), and have only one potential start site upstream of conserved regions (C. glutamicum: VAEK...). M. tuberculosis Rv1100 has a potential GTG start codon in a similar position in the aligned proteins; this aligns with the likely start in M. smegmatis (MTTQ...), and we propose that this is the genuine start codon. This would leave the intergenic gap between Rv1099c and Rv1100 at 57 bp. We suggest new translational starts for the M. leprae homologues using similar logic: glpX (ML1946) will start 81 bp upstream of the current assignment at base 2332629 (old start, MELV...; new start, MTAE...), and the Rv1100 orthologue ML1945 will start 84 bp of the current assignment at base 2332549 downstream (old start, MVND...; new start, VTFE...).
Complementation of E. coli mutants
It is possible to test for FBPase activity by genetic complementation, as an E. coli fbp mutant is unable to grow on medium with glycerol as sole carbon source (Donahue et al., 2000). We therefore cloned Rv1099c into two expression vectors, so that it would be expressed either with a short N-terminal His-tag (pFM142), or fused to the C-terminal end of GST (pFM149). These constructs were introduced into E. coli strains JLD2402, which lacks both fbp and glpX, and JLD2404, which lacks only fbp. Antibiotic-resistant transformants were selected and then plated onto minimal agar plates containing glucose or glycerol as carbon source, and IPTG to induce expression of Rv1099c. All of these strains grew on glucose agar, whereas the control strains only grew on glycerol agar (results with JLD2402 and pFM149 are shown in Fig. 4
). Complementation was not expected from the pET15b-based clone pFM142, because of the lack of a host T7 polymerase gene, but was detected; our experience is that this can occur due to read-through from other promoters. These results confirm that Rv1099c has FBPase activity. For historical reasons, the clones were made using the start site predicted by Tuberculist. The fact we obtained activity indicates that if the protein does have an extended N-terminus as we predict, this region is not critical for function.
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Gene expression levels in M. tuberculosis
We carried out RTq-PCR experiments to determine the level of expression of Rv1099c mRNA in exponential cultures of M. tuberculosis in Middlebrook 7H9 medium containing OADC supplement and Tween 80. Expression levels were normalized to those of sigA mRNA, and calculated based on the RNA used for reverse transcription. We showed that in mid-exponential-phase growth in supplemented Middlebrook 7H9 medium (which contains both glucose and Tween 80 as carbon sources), the level of Rv1099c mRNA is 0·49 (95 % confidence interval 0·390·63) that of sigA.
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DISCUSSION |
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We show here that M. tuberculosis Rv1099c has FBPase activity with saturable kinetics, and a Km at 15 µM that is similar to the affinity of bacterial class II FBPases for fructose bisphosphate. For example, the C. glutamicum enzyme has a Km of 14 µM and the E. coli GlpX has a Km of 35 µM. Lithium inhibits by interfering with the Mg2+ binding site of sugar phosphatases (Chen & Roberts, 1998), so its effect on the M. tuberculosis FBPase activity is consistent with this. The lack of effect of any of the intermediates is surprising as it is expected that GlpX needs to be regulated, for instance to avoid futile cycling when phosphofructokinase is active. However, their effects need to be investigated further on purified, possibly cleaved fusion protein before drawing any firm conclusions about regulation.
We suggest that Rv1099c may be the major FBPase of M. tuberculosis; this is supported by the recent report where the GlpX homologue of C. glutamicum was characterized (Rittmann et al., 2003). The authors showed that it contained FBPase activity, and demonstrated that a mutant lacked any detectable FBPase activity. The corynebacterial glpX mutant was incapable of growth on gluconeogenic substrates, which substantiates the observation that no other FBPase is predicted by the genome sequence of this organism. The C. glutamicum gene was accordingly named fbp; we, however, propose that Rv1099c be called glpX in order to avoid the implication that it is orthologous to the E. coli fbp gene; the glpX name is also used in the COGs database.
Interestingly, all the mycobacterial class II FBPases shown in Fig. 2 have a near-perfect Prosite (http://www.expasy.ch/prosite) inositol monophosphate phosphatase IMP1 motif (PS00629), although they have no IMP2 motif (PS00630). This may suggest a closer homology of the mycobacterial class II FBPases to IMPases than other members of the family. No activity with inositol 1-phosphate could be attributed to Rv1099c under the conditions examined (data not shown).
We demonstrated that Rv1099c is expressed in the cell. mRNA levels approximately half of the level of sigA, the major sigma factor of the cell, were detected. The fact that it is expressed is also indicated by the identification of the protein in 2D-PAGE analysis (http://www.mpiib-berlin.mpg.de/2D-PAGE/EBP-PAGE/index.html).
Support for a key biological role for the M. tuberculosis glpX gene comes from independent concurrent research using a genome-wide transposon-based method (TraSH) for identifying genes that are required for growth in different conditions (Sassetti et al., 2001; Sassetti & Rubin, 2003
). The data suggest that the fum gene (Figs 1 and 3
) is an essential gene for axenic growth in the presence of both glycolytic (glucose) and gluconeogenic (oleic acid) carbon sources together (Middlebrook 7H9+OADC+Tween 80). Mutants deficient in Rv1100 (which is conserved syntenically with Rv1099c in mycobacteria and corynebacteria) grew slowly, but it appeared that mutants deficient in Rv1099c grew normally. However, a comparison between axenic and in vivo growth showed that mutants in Rv1099c were among the most severely attenuated in a mouse model. This was confirmed in separate experiments showing that an Rv1099c transposon mutant is highly attenuated in vivo (Sassetti & Rubin, 2003
). The extreme attenuation of the glpX mutant is consistent with the essential role of gluconeogenesis for conversion of lipid carbon into cell wall glycan (Fig. 1
). These data, together with the demonstration that a C. glutamicum fbp mutant has lost all detectable FBPase activity, suggest that glpX encodes the major FBPase in M. tuberculosis.
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ACKNOWLEDGEMENTS |
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Received 1 April 2004;
revised 17 June 2004;
accepted 12 July 2004.
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