Vibrational spectroscopic characterisation of fluorescent-protein-tagged and wild-type bacteria in surface-associated...

Fluorescent protein (FP) tagging is widely used to differentiate bacterial populations in mixed-species and surface-associated systems. However, FP expression can impose a metabolic burden and alter cellular physiology, raising concerns about whether FP-tagged strains reliably represent their wild-type counterparts. Understanding the biochemical consequences of FP expression is therefore essential when interpreting data from fluorescence-based studies. In this study, we employed Fourier-transform infrared (FTIR) microscopy and Raman confocal microscopy to evaluate the impact of FP tagging in Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa labelled with eGFP- and mCherry-fluorescent proteins, respectively. Principal component analysis (PCA) of FTIR spectra revealed clear species-level discrimination driven by differences in lipid, protein, polysaccharide, and phosphate content characteristic of Gram-positive and Gram-negative bacteria. Importantly, no detectable spectral differences were observed between wild-type and FP-expressing strains. Raman spectroscopy offered stronger species-specific contrast owing to resonance enhancement of carotenoids in S. aureus and cytochromes in P. aeruginosa. PCA applied to Raman spectra from GFP-labelled Staphylococcus aureus strain and its wild-type counterpart again showed no difference. Together, these results demonstrate that FP expression under the experimental conditions used in this study did not induce detectable changes in vibrational spectral datasets, thus enabling species-level differentiation to be retained in the presence of FP tagging. This paves the way forward for correlative fluorescence and vibrational mapping of bacterial populations at surfaces.