One-plasmid tunable coexpression for mycobacterial protein–protein interaction studies

Abstract

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA Δ⁹ desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.

Introduction

One of the outstanding challenges of proteomics research is the identification and preparation of interacting protein–protein partners. These interactions may include subunits of heteromeric quaternary structures, multicomponent enzymes such as oxygenases, desaturases, oxidases, or synthases, or more weakly interacting protein–protein pairs involved in the regulation of transcription, signaling, and other dynamic cellular processes. Many types of tools have been developed to investigate these protein–protein interactions, for example, coimmunoprecipitation (Co-IP), tandem affinity purification (TAP),1, 2 chemical crosslinking, and yeast two-hybrid screen.3, 4

Abbreviations: GFP, green fluorescent protein; DesA3, product of Mycobacterium tuberculosis H37Rv gene rv3229c, a stearoyl-CoA Δ⁹ desaturase; RFIP, red fluorescence inducible protein; Rv3230c, product of Mycobacterium tuberculosis H37Rv gene rv3230c, a DesA3:NADPH oxidoreductase.

Plasmid coexpression systems are also among the tools used for studying protein–protein interactions. For example, multiple Duet vectors (Novagen, Madison, WI) may be transformed into compatible host strains to coexpress up to eight target proteins, the pESC vectors (Stratagene, La Jolla, CA) can be used to coexpress two genes under control of the GAL1 and GAL10 yeast promoters, and the pIRES mammalian expression vector (Clontech, Mountain View, CA) allows high level expression of two genes of interest from the same bicistronic mRNA transcript. These vectors have been applied in various contexts for eukaryotic and bacterial studies.

The development of tools for study of mycobacterial proteins are of timely interest because tuberculosis, caused by the human pathogen Mycobacterium tuberculosis, is one of the world's deadliest diseases.5 Tuberculosis is resurgent due to the appearance of multiple-drug-resistant strains and the long time course required for drug treatment regimes.6–8 Presently, up to one-third of the world's population may be latently infected with M. tuberculosis and over two million people die from TB-related diseases each year.5, 6, 8 Methods and tools that can increase understanding of the M. tuberculosis proteome and the unique proteins and enzymes associated with pathogenicity have potential to facilitate the development of new drugs and vaccines. The TB Structure Initiative (http://www.doe-mbi.ucla.edu/TB/), dedicated to determining the structure of mycobacterial proteins, has given important insight into the proteome of M. tuberculosis.9

Several different types of systems have been developed for the study of mycobacterial protein–protein interactions. Some of these studies include the use of yeast or bacterial two-hybrid systems.10–12 Some coexpression systems have also been developed. For example, two compatible plasmid systems have been used to demonstrate coexpression of GFP and blue fluorescent protein and then used to coexpress M. tuberculosis extracelluar proteins in BCG or other mycobacterial hosts.13, 14 Alternatively, single plasmids have been used to express a single polycistronic transcript from one promoter.15, 16 Furthermore, a single plasmid that independently expresses GFP as a reporter and a second gene from two different heat shock promoters was used to monitoring the in vivo fate of recombinant proteins.17 In common, these previously developed vectors used constitutive or endogenous promoters for expression. Inducible gene expression systems have also been used to study gene function and validate drug targets for pathogenic mycobacteria. Acetamide-controlled expression systems have also been used to analyze conditional mutants and to express mycobacterial antigens in M. smegmatis.18, 19 More recently, tetracycline-inducible PtetO has been shown to allow controlled regulation of gene expression in M. smegmatis and M. tuberculosis either during grown in liquid culture or during infection of mammalian cells.20–22

In this study, we have combined expression cassettes using the PtetO inducible promoter22 and a constitutive promoter22, 23 into a single plasmid to provide a controlled, tunable coexpression vector. The operation of the coexpression system was validated using GFP and the newly identified red fluorescence protein (RFIP) from Geobacillus sterothermophilus strain G1.13. Results presented here establish RFIP as a useful expression reporter for mycobacterial studies. Furthermore, the vector was used to titrate expression of mycobacterial oxidoreductase Rv3230c relative to Rv3229c, an integral membrane acyl-CoA desaturase, leading to functional coexpression of the complex, thus establishing the utility of the coexpression system.

Results

Construction of a single plasmid two-promoter coexpression system

Figure 1 shows the critical functional elements of the coexpression plasmid pTetCoex, including transcription terminators (solid black bars), a hygromycin resistance selection cassette (hygR), the mycobacterial (myc ori), and E. coli replication origins (ColE1), and the tetracycline repressor (TetR) controlled by the constitutive promoter Psmyc.

Figure 1. Plasmid vectors and multicloning sites (MCS) used in this work. (A) pTetCoex. PTetO is used to provide tetracycline-inducible expression of a gene (gene A) cloned into the PacI and SbfI sites; the strong constitutive promoter Psmyc is used to control expression of the tetracycline repressor (TetR). (B) multicloning sequence in pTetCoex.

The placement of genes into pTetCoex for coexpression consists of two parallel efforts. In one, gene A that will be controlled by the tetracycline-inducible PtetO promoter is ligated into the PacI and SbfI restriction sites of the multicloning site in pTetCoex. In the other effort, gene B is first cloned into one of three pMYC4 vectors that provide three different strengths of constitutive promoter22, 23 (Psmyc, Phsp, and Pimyc, respectively, and see below). The circular maps of the pMYC4 variants and nucleotide sequences of their respective multicloning sites are shown in Figure 2. The PacI and SbfI restriction sites are used for cloning into the Psmyc and Pimyc promoter constructs, while the NdeI and SbfI restriction sites are used for the Phsp promoter construct. A sequence-verified pMYC4 variant that contains gene B is then digested using AsiSI and SrfI, and the promoter-gene B insert is ligated into similarly digested pTetCoex containing gene A (Fig. 1).

Figure 2. Plasmid vectors and multicloning sites (MCS) used in this work. (A) Vector for cloning and expression of a second gene (gene B) under control of constitutive promoters Psmyc or Pimyc. Gene B is ligated into the multicloning site (MCS) between either PacI or NdeI and SbfI restriction sites of pMYC4-S, or -I. (B) multicloning sequences in pMYC4S and pMYC4I. (C) Vector for cloning and expression of a second gene (gene B) under control of constitutive promoters Phsp. Gene B is ligated into the multi-cloning site (MCS) between NdeI and SbfI restriction sites of pMYC4-H. (D) Multicloning sequence in pMYC4H. The combination of desired constitutive promoter and gene B is cloned into pTetCoex by use of the AsiSI and SrfI restriction sites. Target genes cloned into the pMCY4 vectors can also be transferred into the PtetO-controlled position of pTetCoex by use of the PacI and SbfI restriction sites.

PacI, SbfI, AsiSI, and SrfI have 8-nucleotide recognition sites, and thus a high percentage of open reading frames can be cloned using these restriction enzymes. For example, greater than 95% of all open reading frames in M. tuberculosis can be cloned by these four restriction enzymes.14, 24 Because of the presence of shared restriction sites in both pTetCoex and pMYC4, genes cloned into either of these vectors can be conveniently swapped into positions controlled by either PtetO or the constitutive promoters using simple digestion and ligation. This design facilitates the optimization of coexpression by permitting parallel investigation of each of the different possible combinations of genes and promoters.

Analysis of promoter strength using GFP as a reporter

Figure 3(A) shows the results of GFP expression using the three different constitutive promoters provided by the pMYC4 vectors. The level of GFP fluorescence detected was consistent with the previously reported strength of the promoters, in the order of Psmyc > Phsp > Pimyc.22 Thus the three constitutive promoters can be used to obtain three different levels of protein expression. Figure 3(A) also suggested that PtetO was a weak promoter relative to Psmyc or Phsp, in effect comparable to Pimyc. Therefore, a mechanism to increase the level of PtetO-inducible protein expression was desired to improve the dynamic range of the coexpression.

Figure 3. A cell-mass normalized fluorescence obtained from GFP and RFIP expression driven by different promoters. (A) the GFP fluorescence observed from pMYC4 vectors expressing GFP under control of promoters PtetO, Psmyc, Phsp, and Pimyc was measured with excitation at 485 nm and emission at 515 nm. PtetO expression was induced by addition of 200 ng/mL of anhydrotetracycline. Psmyc, Phsp, and Pimcy was constitutive. Error bars are standard deviation from three different expression trials. (B) Use of RFIP as a reporter in Mycobacterium smegmatis. RFIP was expressed using three different constitutive promoters (Psmyc, Phsp, and Pimyc) and analyzed with excitation at 357 nm and emission at 605 nm. The cell-mass normalized relative expression levels observed for RFIP with these different constitutive promoters corresponded to the pattern observed with GFP [see Fig. 3(A)].

Stabilization of expression from PtetO by 3′ mRNA sequence

During the development of pTetCoex, we observed that ∼3- to 4-fold higher expression of GFP could be obtained when the intermediate plasmids contained the kan gene in the 3′ position from gfp. Figure 4 illustrates this difference in expression. As compared to gfp alone [Fig. 4(A)], when the full length kan was present [Fig. 4(B); Table I, 816 bp], the inducible expression of GFP was tunable over a range up to that provided by the strong constitutive promoters. Additional studies showed that this stabilization was not dependent on the presence of a ribosome-binding site for kan [Fig. 4(C)]. Instead, the stabilization was correlated with the length of the nucleotide sequence of the 3′ gene [Fig. 4(E,F)].

Table I. Genes Used to Replace kan as a 3′ Stabilizer of Gene Expression

Gene identifiera	Organism	Annotation^b	Nucleotide length (bp)
a Standard abbreviation or gene ontology number.
Kan	Streptomyces kanamyceticus ISP5500	Kanamycin nucleotidyltransferase	816
Trx	Escherichia coli	Thioredoxin	327
GO.34382	Mus musculus	Unknown protein	291
GO.13193	Arabidopsis thaliana	Unknown protein	453
GO.2361	Arabidopsis thaliana	Putative protein	456
GO.6042	Arabidopsis thaliana	Unknown protein	312
GO.35683	Mus musculus	Cysteine dioxygenase 1	603
GO.81370	Cyanidioschyzon merolae 10D	Unknown heme-binding protein	453
GO.605	Arabidopsis thaliana	Unknown protein	648

Figure 4. Effect of 3′ sequence on the cell-mass normalized level of GFP fluorescence observed from bi-cistronice expression of genes under control of the PtetO promoter. Influence of different downstream kan constructs on GFP expression. The starting constructs were gfp alone (A), and a bicistron of gfp and kan (B). The kan expression cassette was modified by removal of the ribosome binding site (C), shortening of the inter-cistron region (D), and truncation of either 441 bp nucleotides (E) or 696 bp nucleotides (F) from the 3′ end of kan (816 bp).

Because expression from kan might impart undesired drug resistance or incompatibility with other kan-selectable vectors, several other genes (including trx and seven other candidates from the UW Center for Eukaryotic Structural Genomics, Madison, WI) were tested for their ability to replace kan and still enhance expression of the upstream gene. These genes are all relatively small (Table I, ∼300 to ∼900 bp). As also shown in Figure 5, all of these different small genes enhanced GFP expression, and several constructs gave enhancement comparable to that observed with kan in the 3′ position. For the remainder of this work, GO.83170 was used as the enhancement insert.

Figure 5. Influence of different downstream genes on GFP expression. The abbreviation kan represents a plasmid construct with kanamycin nucleotidyl transferase gene placed in position x of panel A; other genes placed in the position of x are described in Table I.

A new reporter protein for Mycobacterium smegmatis

Because the mycobacterial cell wall is relatively impermeable and difficult to disrupt, stable reporter proteins that can be detected with high sensitivity and without the requirements of external substrates or cellular disruption are desirable. Among commonly used reporter systems (GFP, firefly luciferase, β-galactosidase, chloramphenicol acetyltransferase, and alkaline phosphatase), only GFP or its variants were previously known to match these criteria. A less often used reporter, uroporphyrinogen III methyltransferases (E. coli cysG25 or Propionibacterium freudenreichii cobA26) also meets these criteria. Expression of either of these proteins results in the intracellular accumulation of sirohydrochlorin (factor II) and a trimethylated product termed trimethylpyrrocorphin, which emit strong red fluorescence upon illumination with UV light.27 CobA had been used as a reporter in E. coli and yeast, while the coexpression of CobA and GFP had been achieved in mammalian cells.28

In this work, we tested the use of a putative uroporphyrin-III C-methyltransferase gene rfip from Geobacillus sterothermophilus strain G1.13 as a second reporter for coexpression in M. smegmatis. The rfip gene (a 780 bp open reading frame, encoding red fluorescence inducible protein, RFIP) shares 49% and 46% identity with CysG25 and cobA.26 Like the homologous genes, overexpression of rfip in E. coli results in the intracellular accumulation of sirohydrochlorin and trimethylpyrrocorphin, which subsequently emit red fluorescence upon illumination with UV light.27 Figure 3(B) shows that expression of rfip in M. smegmatis also yields a strong fluorescence signal.

Figure 3(B) shows that the level of red fluorescence detected from the three different pMYC4 constructs containing rfip corresponded well with the relative strengths of these three promoters, and also paralleled the fluorescence response given by gfp with these three promoters [Fig. 3(A)]. These results indicated that expression of RFIP could be monitored independently and quantitatively in coexpression tests as a second reporter gene along with GFP.

Tunable coexpression

To demonstrate tunable gene coexpression in M. smegmatis, pTetCoex was prepared to contain both gfp and rfip as reporter genes. Figure 6 summarizes this construct and the experimental results. The tetracycline-inducible promoter PtetO was used to control the bi-cistronic expression of GFP along with the small gene GO.81370 as the downstream stabilizer, while the intermediate strength constitutive promoter Phsp was used to express RFIP as a single cistron. Figure 6(B) shows that level of fluorescence obtained from each protein in this coexpression system was comparable to that in the single gene expression vector controlled by the same promoter (i.e., inducible by PtetO for GFP, and constitutive by Phsp for RFIP), suggesting that the two expression cassettes in the coexpression vector functioned independently. This is consistent with the intentional placement of transcription terminators between the expression cassettes. As shown in Figure 6(C,D), the PtetO-driven expression of GFP could be controlled either by induction time at a fixed inducer concentration or by increasing the inducer concentration. Either of these methods was capable of producing changes in the ratio of GFP and RFIP. Moreover, similar titratable expression of GFP could be achieved when the Phsp promoter was replaced by Psmyc or Pimyc, yielding either higher or lower levels of RFIP, respectively, and thus expanding the range of protein: protein ratios achievable.

Figure 6. Demonstration of tunable expression of GFP during coexpression with RFIP using pTetCoex. (A) Construct map showing the bi-cistron of GFP and downstream stabilizer gene GO.81370 (identified in Table I) under control of the inducible PtetO promoter; RFIP is under control of the constitutive Phsp promoter. (B) Comparison of fluorescence levels observed with GFP and RFIP expressed either separately or in tandem from coexpression construct A. The dark gray columns represent uninduced samples and the light gray columns represent samples induced by 200 ng/mL of anhydrotetracycline for 6 h. (C) Time-dependent increase in the level of GFP expression in the presence of 200 ng/mL of anhydrotetracycline relative to constitutive expression of RFIP. (D) Anhydrotetracycline-dependent expression of GFP observed 6 h after induction relative to constitutive expression of RFIP. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Tunable coexpression of M. tuberculosis DesA3 complex

M. tuberculosis DesA3 consists of an NADPH oxidoreductase Rv3230c and a membrane-bound, iron-containing desaturase that reacts with stearoyl-CoA to produce oleoyl-CoA.7, 29 Prior studies of this enzyme complex showed that uncontrolled expression of oxidoreductase Rv3230 was apparently toxic (Y. Chang and B.G. Fox, unpublished results), and indeed, attempts to express Rv3230 gene using the constitutive promoter Phsp failed to yield M. smegmatis transformants. In contrast, desaturase Rv3229c could be expressed from constitutive promoter Phsp without any apparent effects on cell viability. With this information, obtained from use of the pMYC4 vectors, a pTetCoex construct shown in Figure 7(A) was assembled to have Rv3230c under inducible control of PtetO and Rv3229c under constitutive control of Phsp.

Figure 7. Coexpression of the M. tuberculosis stearoyl-CoA Δ⁹ desaturase complex. (A) Construct map. (B) Detection of C-terminal c-myc tagged DesA3 (green) and N-terminal FLAG-tagged Rv3230c (red) coexpressed in M. smegmatis by use of two-color infrared fluorescence Western blotting. Lanes 1–5 show mycobacterial lysates that had constitutive expression of DesA3 from Phsp and inducible expression of Rv3230c in the presence of 200 ng/mL anhydrotetracycline for 0, 1, 2, 4, and 6 h. Lanes 6 and 7 are control lysates from M. smegmatis incubated for 6 h either without or with 200 ng/mL of anhydrotetracycline, respectively. Lanes 8–13 show mycobacterial lysates that had constitutive expression of DesA3 and inducible expression of Rv3230c for 6 h in the presence of 0, 25, 50, 100, 200, and 400 ng/mL anhydrotetracycline, respectively. (C) Stearoyl-CoA desaturation. L1 to L5, total lysate with constitutive expression of DesA3 from Phsp and inducible expression of Rv3230c in the presence of 200 ng/mL of anhydrotetracycline for 0, 1, 2, 4, and 6 h, respectively, corresponding to lanes 1–5 of panel B; lane L7, lysate from empty vector transformed cells treated with anhydrotetracycline for 6 h corresponding to lane 7 of panel B. The separation of the substrate (18:0), product (18:1), and a side-product by thin-layer chromatography are indicated. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

When compared with control lysate prepared from M. smegmatis transformed with empty vector under either non-inducing or inducing condition [Fig. 7(B), lanes 6 and 7], DesA3 (Rv3229c) was detected at a constant level by Western blotting [Fig. 7(B), lanes 1–5 and lanes 8–13], while Rv3230c was increased in level either by induction time [Fig. 7(B), lanes 1–5] or by inducer concentration [Fig. 7B, lanes 8–13]. These responses were similar to those established for coexpression of GFP and RFIP in the inducible and constitutive positions, respectively.

Figure 7(C) shows that as the level of Rv3230c was increased relative to DesA3, stearoyl-CoA desaturase activity was dramatically increased as a threshold of coexpression was achieved. Desaturation activity first became detectable after 4 h of induction time [lane C4, corresponding to the appearance of Rv3230c in lane 4 of Fig. 7(B)], while increased desaturation activity was present after 6 h of induction time [lane C5, corresponding to increased presence of Rv3230c in lane 5 of Fig. 7(B)]. The specific activity obtained in the sample of lane C5 was comparable to that achieved previously from optimized mixing of an E. coli lysate containing Rv3230c with a mycobacterial lysate containing Rv3229c. This result demonstrates tunable coexpression of a multicomponent enzyme complex. This increase in activity also corresponds to the saturable behavior previously described for reaction of Rv3230c with Rv3229c.29

Discussion

Mycobacterial coexpression systems

Several types of mycobacterial coexpression systems have been previously developed. For example, a two-plasmid system was designed by coexpression of GFP and blue fluorescent protein in M. smegmatis,14 and then used to coexpress M. tuberculosis extracelluar proteins in M. tuberculosis BCG.13 Expression from the two-plasmid system had to be modulated by control of the copy number of two plasmids, which is more difficult to achieve than control of the copy number of a single plasmid as used here. Furthermore, owing to the use of either constitutive promoters or natural promoters, this previous two-plasmid system did not provide a mechanism to finely control the relative expression levels of the coexpressed genes to either match their physiological ratio or to minimize toxicity arising from an inappropriate level of constitutive expression.

A single plasmid and single promoter have been used to produce a single polycistronic transcript in M. tuberculosis and M. smegmatis.15, 16 With this plasmid, the levels of the individual, coexpressed target genes could not be controlled by external signals, but were instead determined by the properties of the polycistronic mRNA. As shown here, the 3′ region of the polycistronic mRNA might influence protein expression in M. smegmatis due to mRNA instability (Fig. 4), and this would complicate coexpression if the terminal, unprotected cistron were degraded. In a different approach, a single plasmid that simultaneously produced GFP and interleukin-2 from two different constitutive heat shock promoters was developed to study the in vivo fate of recombinant proteins in Mycobacterium bovis BCG.17 Although differences in the relative stabilities and levels of expression of the different target mRNAs produced from the two different heat shock promoters was noted, independent control of the relative amounts of the two coexpressed mRNAs could not be achieved.

Inducible expression in mycobacteria

Acetamide-controlled expression systems have been used to analyze conditional mutations of proteins and to express mycobacterial antigens in M. smegmatis.18, 19 Although this system works well in defined liquid medium, the induction by acetamide or short aliphatic amides is not suitable for regulation of gene expression during an infection. Also, the instability of the acetamide-inducible expression vector in M. tuberculosis has been reported.30

More recently, PtetO promoter and Tet repressor-controlled systems for conditional gene expression were developed for M. smegmatis and M. tuberculosis.20–22 These tetracycline-inducible vectors provide the ability to titrate gene expression by variation of either tetracycline concentration or induction time. Furthermore, because tetracycline can penetrate the cellular membranes of both mycobacterial and eukaryotic cells, the PtetO and TetR systems offer the advantage of controlled gene expression for mycobacterial strains in liquid culture or during infection of mammalian cells.

RFIP as an expression reporter

This work established that the Gst strain G1.13 rfip gene could be successfully used as an expression reporter in M. smegmatis. The rfip gene shares 49% and 46% identity with the E. coli and P. freudenreichii homologs, respectively. Despite the 33°C higher growth temperature optimum of Geobacillus sterothermophilus strain G1.13 relative to these other organisms, RFIP yielded UV fluorescence in E. coli that was easily visible as red coloration under conventional fluorescent lamps.

Properties of pTetCoex

The single plasmid described here places one gene under titratable control of PtetO, an intermediate strength promoter.22 The level of target protein expression observed from PtetO was enhanced by ∼3- to 4-fold upon the addition of an unrelated 3′ gene sequence to yield a bicistronic mRNA. This 3′ addition apparently served to increase the lifetime of the target sequence, presumably due to the slowing down of a 3′-directed degradation of mRNA in M. smegmatis. The other gene targeted for coexpression could be produced at one of three levels using different constitutive promoters,22, 23 and the relative strength of these promoters was assessed using fluorescence observed from expressed GFP and RFIP. In an application of the optimized pTetCoex vector (Fig. 7), stearoyl-CoA desaturase activity was dramatically stimulated as the level of oxidoreductase Rv3230c was increased above a threshold amount relative to a constant level of desaturase Rv3229c.

The tunable expression of Rv3230c allowed a way to overcome the toxicity previously observed with expression of this gene. With the low basal expression observed from the uninduced PtetO promoter, cell growth was able to proceed without expression of the toxic gene, while constitutive accumulation of nontoxic Rv3229c was allowed. Upon addition of anhydrotetracycline, the induction led to formation of the functional multiprotein complex, and enzymatic activity was detected as the level of Rv3230c and Rv3229c exceeded an optimal threshold. This combination of expression systems provides a simply controlled system for the investigation of protein–protein interactions in mycobacteria. This tool will complement the widely used yeast and E. coli two hybrid systems by permitting studies of mycobacterial protein–protein interactions in their own host. Although not yet implemented, assembly of inducible and constitutive promoter systems suitable for use with other organisms is reasonable based on the arrangements described here.

Materials and Methods

Cloning methods and materials

Total genomic DNA of Mycobacterium tuberculosis H37Rv obtained from the TB Research Materials Facility (Prof. J. Belisle, Director, NIH NIAD NO1AI75320, Colorado State University) was used as a PCR template to clone the rv3229c gene (encoding stearoyl-CoA desaturase, DesA3) and rv3230c gene (encoding NADPH:stearoyl-CoA desaturase oxidoreductase, Rv3230c). The vectors pUV15TetORs,22 pUV15TetORm,22 and pVV1623 were used as templates for PCR cloning of promoter sequences. The thioredoxin gene (trx) was amplified by PCR from pET-32a (Novagen, Madison, WI). Seven small genes with the Gene Ontology numbers GO.34382, GO.13193, GO.2361, GO.6042, GO.35683, GO.81370, and GO.605 were obtained from the University of Wisconsin Center for Eukaryotic Structural Genomics and also amplified by PCR (Table I).

Pfu DNA polymerase was from Stratagene (La Jolla, CA). Oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, IA). The pSMART-HC Amp CloneSmart blunt cloning kit (Lucigen, Middleton, WI) was used for initial cloning in Escherichia coli strain E. cloni 10G and transformants were used for preparation of plasmid DNA for sequencing. Big Dye DNA sequencing (Applied Biosystems, Foster City, CA) was performed at the University of Wisconsin Biotechnology Center to verify the coding sequence of all expression plasmids generated by PCR.

Isolation of red fluorescent inducible protein

Geobacillus sterothermophilus strain G1.13 was isolated from a 70°C grass compost in Madison WI. The organism was cultivated in YTP medium containing (per liter) 2.0 g yeast extract, 2.0 g tryptone, 2.0 g sodium pyruvate, 1.0 g KCl, 1.0 g KNO₃, 1.0 g Na₂HPO₄·7H₂O, 0.1 g MgSO₄, 0.03 g CaCl₂, and 2.0 mL of centrifuged tomato juice. Cells were harvested by centrifugation and frozen. Genomic DNA was prepared using phenol-chloroform, the DNA was sheared to 2–4 kb, end-repaired and ligated into pEZSeq vector (Lucigen). The shotgun genomic library preparation was transformed into electrocompetent E. coli cells (Lucigen), and the resulting colonies were inspected for fluorescent color using a 360 nm long wavelength handheld UV lamp. Four red fluorescent colonies were picked and sequenced using Sanger chemistry and Applied Biosystems capillary electrophoresis. An ∼1.5 kb insert was sequenced on both strands from these isolates, and revealed a putative uroporphyrin-III C-methyltransferase gene sharing 95% identity with the Geobacillus sp. WCH70 gene (YP_002948593.1) and 83% identity with the Geobacillus sp. Y412MC6 (ZP_03557305.1) gene. These latter genomes were sequenced at the DOE Joint Genome Institute. The deduced DNA and amino acid sequence are shown in Supporting Information Figure 1.

Fluorescence quantification of coexpression

M. smegmatis mc²155 ATCC 700084 was transformed by electroporation, plated onto selective Middlebrook 7H10 agar (Becton Dickinson, Spark, MD) containing 50 μg/mL hygromycin (Sigma-Aldrich, St. Louis, MO), and incubated at 37°C until visible colonies formed (∼3 days). Colonies from each different transformation were used to inoculate 500 μL of Middlebrook 7H9 broth (Becton Dickinson, Spark, MD) supplemented with 0.2% (w/v) glycerol, 0.05% (w/v) Tween-80 (Sigma-Aldrich, St. Louis, MO), and 50 μg/mL of hygromycin. The liquid cultures were incubated on a shaker (250 rpm) at 37°C to reach saturation, typically ∼16 h. Each culture was then diluted 100-fold into 5 mL of medium and grown as before to late logarithmic growth phase. At that time, cultures transformed with an inducible plasmid construct were supplemented with anhydrotetracycline (Sigma-Aldrich, St. Louis, MO) to the desired final concentration (ranging from 0 to 400 ng/mL) and monitored for ∼6 h with continued shaking. During this time, the fluorescence obtained from the pUV15TetORs control plasmid reached a maximum value with the inducer concentration of 200 ng/mL. After 6 h, the optical density was measured, the cells were harvested by centrifugation at room temperature, washed once in phosphate-buffered saline (0.01 M phosphate buffer, pH 7.4, 2.7 mM KCl and 0.14 M NaCl) containing 0.05% Tween20 (Sigma-Aldrich, St. Louis, MO), and pelleted by centrifugation. The cells were resuspended in the same buffer to a density of 2 OD₆₀₀ units per 0.1 mL, and 0.1 mL samples of the cell culture were transferred into black 384-well plates (ISCBioExpress, Kaysville, UT). The fluorescence was measured using a SPECTRA MAX GEMINIXS microplate reader (Molecular Devices, Sunnyvale, CA). GFP was detected with excitation at 485 nm and emission at 515 nm. RFIP was detected with excitation at 357 nm and emission at 605 nm.

Coexpression of Rv3230c and DesA3

The method used to coexpress Rv3230c and DesA3 was similar to that used for GFP and RFIP, with the only difference being that 7H9 broth was supplemented with 10% OADC enrichment (oleic acid/albumin/dextrose/catalase, Becton Dickinson, Spark, MD). Rv3230c was placed under control of the tetracycline-inducible promoter, while DesA3 was placed under control of the constitutive promoter.

Two-color infrared fluorescence western blotting

Two-color infrared fluorescence Western blotting was used to detect Rv3230c and DesA3 coexpressed in M. smegmatis. Cells normalized by optical density were harvested and immediately boiled in denaturing electrophoresis sample buffer. Proteins were separated by 4–20% SDS-PAGE, and transferred to nitrocellulose membranes. The blotted proteins were blocked in Odyssey Blocker (LI-COR Biosciences, Lincoln, NE). The coexpressed N-terminal FLAG-tagged Rv3230c and C-terminal c-myc tagged DesA3 were detected simultaneously in the same Western blot by using two different primary antibodies (mouse anti-FLAG antibody and rabbit anti-c-myc antibody, Sigma-Aldrich), followed by two different infrared dye labeled secondary antibodies targeting the primary antibodies (goat anti-rabbit IRDye 800 (red color) and goat anti-mouse IRDye 680 (green color), LI-COR Biosciences). Signals were detected and quantified using the Odyssey infrared imaging system (LI-COR Biosciences).

Stearoyl-CoA desaturation assay

Methods to prepare mycobacterial cell-free extracts and to assay the reconstituted desaturase activity were as previously reported29 with the modification that separate expression of Rv3230c and Rv3229c was no longer needed. Reaction mixture contained 20 μmol of potassium phosphate and 250 μmol of NaCl in a final volume of 0.2 mL, and aliquots (20–50 μL) of the total cell lysate were used. The reaction was initiated by addition of 0.4 μmol of NADPH, 6 nmol of stearoyl-CoA, 0.03 μCi of [1-¹⁴C]-stearoyl-CoA and 0.2 nmol of FAD in a total volume of 200 μL. The reaction was incubated at 37°C for 1 h and stopped by the addition of 200 μL of 2.5 M KOH in ethanol. The mixture was heated at 80°C for 1 h and acidified by the addition of 280 μL of formic acid. The saponified fatty acids were extracted with 700 μL of hexane, the 200 μL of the extract was evaporated and then separated into saturated and unsaturated acids on a 10% AgNO₃-impregnated thin-layer chromatography plate using chloroform:methanol:acetic acid:water (90:8:1:0.8) as the developing solvent. Radioactivity was counted using a Packard Instant Imager (Packard, Meriden, CT).

Acknowledgements

The authors thank Prof. Sabine Ehrt (Department of Microbiology and Immunology, Weill Medical College of Cornell University) for providing vectors used as starting materials in this study. We also thank the University of Wisconsin Center for Eukaryotic Structural Genomics (Madison, WI) for providing small gene targets and Dr. Russell L. Wrobel from this Center for helpful discussions.