The Gram-positive anaerobic bacterium Clostridioides difficile (C. dificile) has emerged as one of the biggest public health threats in the past two decades, in part because of the high rate of antibiotic failure when treating C. difficile infections. The precise molecular mechanisms causing the extreme antibiotic resistance of C. difficile are unknown. Molecular analysis is urgently needed as C. difficile’s resilience against antibiotics becomes more evident. The Photostable, Improved Light, Oxygen or Voltage sensing domain (phiLOV) is a green fluorescent oxygen-independent reporter used in live-cell microscopy in obligate anaerobes such as C. difficile. Reporters using phiLOV have been used to identify extracellular stresses that activate transcription of stringent response genes rsh and relQ in C. difficile. However, the green autofluorescence of the C. difficile cell envelope makes visualization with phiLOV challenging and precludes single-cell analysis, because of the poor signal-to-noise ratio. The bilin-binding fluorescent protein IFP2.0 emits in the far-red range of visible light and has recently been used as a reporter in other anaerobic bacteria. The existing C. difficile rsh and relQ reporter plasmids will be restriction digested with SacI and BamHI to excise the phiLOV open reading frame. The open reading frame encoding IFP2.0 will be PCR amplified with primers that introduce SacI and BamHI cut sites and the PCR product will be digested. Then the IFP2.0 gene will be ligated into the reporter plasmids to create red fluorescent transcriptional reporters for the C. difficile rsh and relQ genes. These plasmids will be transformed into E. coli mating strains that will be used to introduce these plasmids into C. difficile by conjugative transfer. The resulting C. difficile reporter strains will be exposed to stringent response activating stresses during live cell microscopy and grown in biofilms on coverslips for microscopy. This will allow exploration of previously unanswerable questions, such as whether individual genes are expressed heterogeneously in a population of cells and whether gene expression is spatially regulated within living biofilms.

Red anaerobic reporters for single-cell visualization of transcription in live anaerobic Clostridioides difficile

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