Codon-optimized DsRed fluorescent protein for use in Mycobacterium tuberculosis.

OBJECTIVE: We have previously codon-optimized a number of red fluorescent proteins for use in Mycobacterium tuberculosis (mCherry, tdTomato, Turbo-635). We aimed to expand this repertoire to include DsRed, another widely used and flexible red fluorescent protein. RESULTS: We generated expression constructs with a full length DsRed under the control of one of three strong, constitutive promoters (Phsp60, PrpsA or PG13) for use in mycobacteria. We confirmed that full length DsRed (225 amino acids) was expressed and fluoresced brightly. In contrast to mCherry, truncated versions of DsRed lacking several amino acids at the N-terminus were not functional. Thus, we have expanded the repertoire of optimized fluorescent proteins for mycobacteria

Codon-optimized DsRed fluorescent protein for use in Mycobacterium tuberculosis.

OBJECTIVE: We have previously codon-optimized a number of red fluorescent proteins for use in Mycobacterium tuberculosis (mCherry, tdTomato, Turbo-635). We aimed to expand this repertoire to include DsRed, another widely used and flexible red fluorescent protein. RESULTS: We generated expression constructs with a full length DsRed under the control of one of three strong, constitutive promoters (Phsp60, PrpsA or PG13) for use in mycobacteria. We confirmed that full length DsRed (225 amino acids) was expressed and fluoresced brightly. In contrast to mCherry, truncated versions of DsRed lacking several amino acids at the N-terminus were not functional. Thus, we have expanded the repertoire of optimized fluorescent proteins for mycobacteria

Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the PmtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly

Detection of fluorescent reporter in <i>M. tuberculosis</i> H37Rv.

<p>FP expression plasmids were transformed into <i>M. tuberculosis</i> and colonies isolated on hygromycin-containing plates. (A) Expression of several fluorescent reporters gave rise to coloured colonies. (B) Microscopy of fluorescent mCherry reporter driven by either P_hsp60 or P_smyc. Scale bar is equal to 10 µm.</p

Far-red fluorescent reporters are functional in <i>M. smegmatis</i> mc²155.

<p>Fluorescent reporters were expressed from P_hps60 or P_smyc in <i>M. smegmatis</i> and assayed in liquid culture. Cultures were diluted (from left to right) to an OD₅₈₀ of 0.25, 0.10, 0.05 and 0.01, representing cell densities of 7.8×10⁷, 2.3×10⁷, 9.2×10⁶ and 1.8×10⁶ per mL respectively. Fluorescence was quantified at the following wavelengths: mCherry 587/610 nm, mKat 588/635 nm, mPlum 590/649 nm, tdTomato 554/581 nm, Turbo-635 588/635 nm. Data are the mean and standard deviation from three independent transformants. A value of 1015 corresponds to saturation of the machine. The reporter, promoter and vector backbone are indicated. WT = wild-type oriM; ΔHC – high copy number derivative oriM.</p

Microscopy of <i>M. smegmatis</i> mc²155 strains expressing mCherry.

<p><i>M. smegmatis</i> transformants were grown in liquid for microscopy. Cells were visualised by differential interference contrast and fluorescence microscopy. Panel A - transformants carrying P_hsp60-mCherry. Panel B - transformants carrying P_smyc-mCherry. 1 - Differential interference contrast; 2 - Texas Red filter; 3 - overlaid image. Scale bar is equal to 10 µm.</p

FP activity under hypoxic conditions.

<p><i>M. smegmatis</i> (A to C) transformants expressing FPs from P_smyc were cultured aerobically (black lines) or under limiting oxygen (grey lines) conditions. Fluorescence intensity (solid squares) and OD₅₈₀ (open squares) were measured. A - <i>M. smegmatis</i> expressing mCherry. B -<i>M. smegmatis</i> expressing tdTomato. C - <i>M. smegmatis</i> expressing Turbo-635. D - <i>M. tuberculosis</i> expressing mCherry under hypoxic conditions. <i>M. tuberculosis</i> transformants expressing FPs from P_smyc were cultured under limiting oxygen conditions. Fluorescence intensity (open squares) and OD₅₈₀ (solid squares) were measured.</p

Far-red fluorescent reporters are functional in <i>M. marinum</i> strain M.

<p>Fluorescent reporters were expressed from P_smyc in <i>M. marinum</i> and assayed in liquid culture grown either in the dark or in the light. Cultures were diluted (from left to right) to an OD₅₈₀ of 0.25, 0.10, 0.05 and 0.01, representing cell densities of 9.8×10⁵, 4.2×10⁵, 1.9×10⁵ and 6.5×10⁴ CFU/ml respectively. Fluorescence was quantified at the following wavelengths: mCherry 587/610 nm, Turbo-635 588/635 nm. Data are the mean and standard deviation from three independent transformants. A value of 1015 corresponds to saturation of the machine. The reporter and promoter backbone are indicated.</p

Correlation between cell viability and fluorescence in <i>M. tuberculosis</i> during antibiotic treatment.

<p>Transformants expressing FPs from P_smyc were grown in liquid and treated with the bactericidal antibiotic kanamycin (grey line) or the static antibiotic chloramphenicol (black line). Fluorescence intensity (solid squares) and OD₅₈₀ (open squares) were measured. A. mCherry, B. tdTomato and C. Turbo-635.</p

Detection of fluorescent reporters in <i>E. coli</i> DH5α.

<p><i>E. coli</i> transformants were cultured and diluted (from left to right) to an OD₅₈₀ of 0.25, 0.10, 0.05 and 0.01, representing cell densities of 5.5×10⁶, 1.5×10⁶, 7.7×10⁵ and 1.1.×10⁵ per mL respectively. Fluorescence was quantified at the following wavelengths: mCherry 587/610 nm, mKat 588/635 nm, mPlum 590/649 nm, tdTomato 554/581 nm, Turbo-635 588/635 nm. Data are the averages and standard deviations from three independent transformants. A value of 1015 corresponds to saturation of the machine. The reporter, promoter and vector backbone are indicated. WT = wild-type oriM; ΔHC – high copy number derivative oriM.</p