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

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, T-lag, CaDPA levels decreased similar to 15%, and in the second phase ending at T-release remaining CaDPA was released rapidly; (2) in L-alanine germination of wild-type spores and spores lacking SleB (a) the ESLI rose similar to 2-fold shortly before T-lag at T-1, (b) following T-lag, the ESLI again rose similar to 2-fold at T-2 when CaDPA levels had decreased similar to 50%, and (c) the ESLI reached its maximum value at similar to T-release and then decreased; (3) in CaDPA germination of wildtype spores, (a) T-lag increased and the first increase in ESLI occurred well before T-lag, consistent with different pathways for CaDPA and L-alanine germination, (b) at 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 Delta T-release (T-realease-T-lag) for excretion of >= 75% of CaDPA was similar to 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 dulling L-alanine germination to that seen with cwLJ sleB spores and the high value for Delta T-release.