The pNIT series of E. coli-mycobacteria shuttle plasmids contain the artificial regulon depicted in . The expression cassette consists of the nitR gene, encoding the regulatory protein, under the control of the inducible nitA promoter. A separate cistron contains a second nitA promoter followed by a multiple cloning site (MCS). The native ribosome binding site of the nitA gene is preserved in the MCS allowing any gene to be cloned in frame using the NdeI site that overlaps the nitA start codon. This arrangement of regulatory elements was designed to maximize the inducibility of the target gene by creating a positive-feedback loop in which the expression of the regulatory protein is simultaneously induced. pNIT-2 contains the same arrangement of regulatory elements and differs largely in the cloning sites available in the MCS. The sequence of pNIT-1 has been deposited in GenBank (accession # FJ173069).
Figure 1 (A) Design of pNIT-1. The expression cassette contains a multiple cloning site (MCS) into which a target gene is inserted (here shown as the reporters, gfp or xylE) and the gene encoding the regulatory protein (nitR) under the control of the NitR-targeted (more ...)
To determine if pNIT plasmids could direct gene expression in mycobacteria, green fluorescent protein (gfp
) and catechol-2,3-dioxygenase (xylE
) genes were used as reporters. Initially, we used the nitrile analog, ε-caprolactam as an inducer, because this inexpensive and non-toxic compound had been shown to induce nitA
expression in both Rhodococcus
spp. expressing NitR.11,13
In M. smegmatis
transformed with pNIT-2::xylE
, ε-caprolactam addition caused a dose-dependent increase in reporter gene activity (). Optimal induction was achieved with 28 mM ε-caprolactam, which caused a >100-fold increase in reporter gene expression.
Figure 2 pNIT plasmids direct expression of exogenous reporter genes in mycobacteria. (A, B) Expression from pNIT plasmids depends on the dose of inducer. M. smegmatis transformed with pNIT-2::xylE (A) or pNIT-1::gfp (B) were treated with the indicated concentration (more ...)
However, very little reporter gene expression was observed when this compound was added to M. tuberculosis transformed with pNIT2::xylE (not shown). We suspected that this was due to the relatively impenetrable hydrophobic cell wall of the slow-growing pathogenic mycobacterial species. To surmount this barrier, we tested a number of more hydrophobic nitriles. Of the compounds tested, isovaleronitrile (IVN) was the most effective, inducing maximal expression at ~5 µM in both M. smegmatis and M. tuberculosis ( and data not shown). Greater than 100-fold induction was routinely achieved in both mycobacterial species (). In M. smegmatis, similar kinetics of GFP induction are observed for both compounds (data not shown). At the concentrations used in this study, neither ε-caprolactam nor IVN had a detectable effect on the growth rates of M. smegmatis or M. tuberculosis (not shown).
We then compared pNIT with two existing methods for regulating gene expression in mycobacteria. After 24 h of induction, pNIT-2 was found to direct equivalent expression of XylE as the previously described acetamidase-inducible promoter in “pACET” (described in Section 3). However, induction from pNIT-2 was considerably more rapid (). A GFP reporter was used to compare pNIT-1 with a commonly used TetR-based expression plasmid, pUV15TetO.1
Upon induction, both plasmids produced fluorescence with approximately the same kinetics (not shown), but pNIT-1::GFP was found to produce ~3-fold higher fluorescence. For some applications, the most critical parameter is not the maximal induction level, but the absence of basal (uninduced) transcription. While we assume that no expression system can be completely repressed, neither the xylE
reporters used in this study proved sensitive enough to reliably detect this low level of protein production. Thus, while we conclude that the basal transcription from these promoters is reasonably low, these experiments do not allow the direct comparison of these systems.
Figure 3 Comparison of pNIT with other mycobacterial expression systems. (A) M. smegmatis transformed with pNIT-2::xylE or pACET::xylE were induced (with ε-caprolactam or acetamide, respectively), and XylE activity was assayed at the indicated time points. (more ...)
While the overall induction achieved by pUV15TetO and pNIT-1 appeared roughly similar at the population level, the circuitry controlling these regulons varied significantly. Transcription from the pUV15TetO promoter is controlled by a repressor expressed at constitutively high-levels. Often, regulons designed in this way produce titratable expression in individual cells. In contrast, the pNIT regulatory circuit includes positive feedback component in which NitR induces its own expression. This raised the possibility that the pNIT-encoded circuit may behave like other positive feedback loops and exist only in two distinct states, fully induced or fully repressed. To investigate this question at the single cell level we analyzed GFP production by FACS after induction at a half-maximal inducer concentration. As shown in , pNIT-based expression showed a distinct bistable nature, whereas pUV15TetO expression was titratable in individual cells.
An ideal expression system for studying mycobacterial pathogenesis would be effective during infection, but this introduces several challenges. Due to the intracellular lifestyle of this pathogen, the inducer must permeate eukaryotic membranes. In addition, M. tuberculosis is growing slowly, if at all, during important stages of infection, and it is generally difficult to produce robust induction of gene expression in such slowly-replicating organisms.
To assess the usefulness of mycobacterial expression systems under these more demanding conditions, we assessed the induction of a GFP reporter by pNIT-1 and pUV15TetO during different growth phases in M. smegmatis (). While both systems were less inducible in stationary phase than in exponentially growing cultures, GFP production could be detected in every condition. The higher fluorescence attainable with pNIT1::GFP was evident in all growth phases, indicating that pNIT may be an attractive option for controlling gene expression under a variety of conditions.
Figure 4 pNIT induction can be achieved in stationary phase and during intracellular growth. (A) M. smegmatis transformed with pNIT-1::gfp or pUV15tetO::gfp were grown to late stationary phase (OD600 >4) and either induced directly (arrow) or diluted to (more ...)
We suspected that the small and relatively hydrophobic inducer of NitR, IVN, was likely to diffuse through mammalian membranes. Therefore, we determined if it was possible to induce gene expression from pNIT-1::GFP during intracellular growth in macrophages. Indeed, the addition of 250 nM IVN to infected macrophages caused robust expression of the GFP reporter that could be detected by microscopy (). Confocal microscopy was then used to verify the intracellular location of these bacteria. The colocalization of GFP-expressing bacteria with host actin in multiple optical sections () verified that these bacteria were inside of the macrophages. The relatively low concentration of IVN used for gene induction did not appear to be toxic to or alter the morphology of the macrophage cell line used in these experiments. However the long-term effects of this concentration of IVN on mammalian cells or whole animals remains to be rigorously tested.
In summary, we have demonstrated that pNIT can direct nitrile-inducible gene expression in both saprophytic and pathogenic mycobacteria. This complements existing expression systems and may provide a number of advantages under certain circumstances, such as when high-level overexpression or induction in slowly-replicating cells is desirable. In addition, the availability of multiple compatible inducible systems should allow the independent regulation of multiple genes in the same cell.