Biochemical Characterization of Gram-Positive and Gram-Negative Plant-Associated Bacteria with Micro-Raman Spectroscopy

Abstract

Raman spectra of Gram-positive and Gram-negative plant bacteria have been measured with micro-Raman spectrometers equipped with 785 and 514.5 nm lasers. The Gram-positive bacteria Microbacterium testaceum, Paenibacillus validus, and Clavibacter michiganensis subsp. michiganensis have strong carotenoid bands in the regions 1155–1157 cm⁻¹ and 1516–1522 cm⁻¹ that differentiate them from other tested Gram-negative bacteria. In the Raman spectrum of Gram-positive bacteria Bacillus megaterium excited with 785 nm laser, the Raman bands at 1157 and 1521 cm⁻¹ are weak in intensity compared to other Gram-positive bacteria, and these bands did not show significant resonance Raman enhancement in the spectrum recorded with 514.5 nm laser excitation. The Gram-positive bacteria could be separated from each other based on the bands associated with the in-phase C=C (ν₁) vibrations of the polyene chain of carotenoids. None of the Gram-negative bacteria tested had carotenoid bands. The bacteria in the genus Xanthomonas have a carotenoid-like pigment, xanthomonadin, identified in Xanthomonas axonopodis pv. dieffenbachiae, and it is a unique Raman marker for the bacteria. The representative bands for xanthomonadin were the C–C stretching (ν₂) vibrations of the polyene chain at 1135–1136 cm⁻¹ and the in-phase C=C (ν₁) vibrations of the polyene chain at 1529–1531 cm⁻¹, which were distinct from the carotenoid bands of other tested bacteria. The tyrosine peak in the region 1170–1175 cm⁻¹ was the only other marker present in Gram-negative bacteria that was absent in all tested Gram-positives. A strong-intensity exopolysaccharide-associated marker at 1551 cm⁻¹ is a distinguishable feature of Enterobacter cloacae. The Gram-negative Agrobacterium rhizogenes and Ralstonia solanacearum were differentiated from each other and other tested bacteria on the basis of presence or absence and relative intensities of peaks. The principal components analysis (PCA) of the spectra excited with 785 nm laser differentiated the various strains of bacteria based on the unique pigments these bacteria do or do not possess. Raman spectroscopy of diverse plant bacteria that are pathogenic and non-pathogenic to plants, and isolated from plants and soil, indicates the possibilities of using the method in understanding plant–bacterial interactions at the cellular level.