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 <i>Microbacterium testaceum, Paenibacillus validus</i>, and
<i>Clavibacter michiganensis</i> subsp. <i>michiganensis</i> have strong
carotenoid bands in the regions 1155–1157 cm<sup>–1</sup> and 1516–1522
cm<sup>–1</sup> that differentiate them from other tested Gram-negative bacteria. In
the Raman spectrum of Gram-positive bacteria <i>Bacillus megaterium</i> excited with
785 nm laser, the Raman bands at 1157 and 1521 cm<sup>–1</sup> 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 (<i>v</i><sub>1</sub>) 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 <i>Xanthomonas axonopodis</i> pv.
<i>dieffenbachiae</i>, and it is a unique Raman marker for the bacteria. The
representative bands for xanthomonadin were the C–C stretching
(<i>v</i><sub>2</sub>) vibrations of the polyene chain at 1135–1136 cm<sup>–1</sup>
and the in-phase C=C (<i>v</i><sub>1</sub>) vibrations of the polyene chain at
1529–1531 cm<sup>–1</sup>, which were distinct from the carotenoid bands of other
tested bacteria. The tyrosine peak in the region 1170–1175 cm<sup>–1</sup> 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<sup>–1</sup> is a distinguishable feature of <i>Enterobacter cloacae</i>. The
Gram-negative <i>Agrobacterium rhizogenes</i> and <i>Ralstonia solanacearum</i> 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.
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