Elastic and Inelastic Light Scattering from Single Bacterial Spores in an Optical Trap Allows the Monitoring of Spore Germination Dynamics

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Abstract
Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual Bacillus subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade spores’ peptidoglycan cortexes. Conclusions from these measurements include (1) CaDPA release from individual wild-type germinating spores was biphasic; in a first heterogeneous slow phase, Tlag, CaDPA levels decreased ∼15%, and in the second phase ending at Trelease, remaining CaDPA was released rapidly; (2) in l-alanine germination of wild-type spores and spores lacking SleB (a) the ESLI rose ∼2-fold shortly before Tlag at T1, (b) following Tlag, the ESLI again rose ∼2-fold at T2 when CaDPA levels had decreased ∼50%, and (c) the ESLI reached its maximum value at ∼Trelease and then decreased; (3) in CaDPA germination of wild-type spores, (a) Tlag increased and the first increase in ESLI occurred well before Tlag, consistent with different pathways for CaDPA and l-alanine germination, (b) at Trelease, the ESLI again reached its maximum value; (4) in l-alanine germination of spores lacking both CLEs and unable to degrade their cortex, the time ΔTrelease (Trelease − Tlag) for excretion of ≥75% of CaDPA was ∼15-fold higher than that for wild-type or sleB spores; and (5) spores lacking only CwlJ exhibited a similar but not identical ESLI pattern during l-alanine germination to that seen with cwlJ sleB spores and the high value for ΔTrelease.

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