Label-Free Raman Microspectroscopy for Identifying Prokaryotic Virocells

Raman microspectroscopy has been used to thoroughly assess growth dynamics and heterogeneity of prokaryotic cells, yet little is known about how the chemistry of individual cells changes during infection with virulent viruses, resulting in socalled virocells. Here, we investigate biochemical changes of bacterial and archaeal cells of three different species in laboratory cultures before and after addition of their respective viruses using single-cell Raman microspectroscopy. By applying multivariate statistics, we identified significant differences in the spectra of single cells with/without addition of virulent dsRNA phage (phi6) for Pseudomonas syringae. A general ratio of wavenumbers that contributed the greatest differences in the recorded spectra was defined as an indicator for virocells. Based on reference spectra, this difference is likely attributable to an increase in nucleic acid versus protein ratio of virocells. This method also proved successful for identification of Bacillus subrilis cells infected with the double-stranded DNA (dsDNA) phage phi29, displaying a decrease in respective ratio, but failed for archaeal virocells (Merhanosarcina mazei with the dsDNA methanosarcina spherical virus) due to autofluorescence. Multivariate and univariate analyses suggest that Raman spectral data of infected cells can also be used to explore the complex biology behind viral infections of bacteria. Using this method, we confirmed the previously described two-stage infection of P. syringae's phi6 and that infection of B. subtilis with phi29 results in a stress response within single cells. We conclude that Raman microspectroscopy is a promising tool for chemical identification of Gram-positive and Gram-negative virocells undergoing infection with virulent DNA or RNA viruses. IMPORTANCE Viruses are highly diverse biological entities shaping many ecosystems across Earth. However, understanding the infection of individual microbial cells and the related biochemical changes remains limited. Using Raman microspectroscopy in conjunction with univariate and multivariate statistics, we established a marker for identification of infected Gram-positive and Gram-negative bacteria. This nondestructive, label-free analytical method at single-cell resolution paves the way for future studies geared towards analyzing virus-host systems of prokaryotes to further understand the complex chemistry and function of virocells.

Automated summary

Raman microspectroscopy is a promising tool for chemical identification of Gram-positive and Gram-negative virocells undergoing infection with virulent DNA or RNA viruses. This nondestructive, label-free analytical method at single-cell resolution allows for the exploration of complex biology and function of virocells.