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Phenotypic diversification is key to microbial adaptation. Currently, advanced technological approaches offer insights into cell-to-cell variation of bacterial populations at a spatiotemporal resolution. However, the underlying molecular causes or consequences often remain obscure. In this study, we developed a workflow combining fluorescence-activated cell sorting and RNA-sequencing, thereby allowing transcriptomic analysis of 10 6  bacterial cells. As a proof of concept, the workflow was applied to study prophage induction in a subpopulation of  Corynebacterium glutamicum . Remarkably, both the phage genes and flanking genomic regions of the CGP3 prophage revealed significantly increased coverage upon prophage induction – a phenomenon that to date has been obscured by bulk approaches. Genome sequencing of prophage-induced populations suggested regional replication at the CGP3 locus in  C. glutamicum . Finally, the workflow was applied to unravel iron-triggered prophage induction in early exponential cultures. Here, an up-shift in iron levels resulted in a heterogeneous response of an SOS (P  divS  ) reporter. RNA-sequencing of the induced subpopulation confirmed induction of the SOS response triggering also activation of the CGP3 prophage. The fraction of CGP3-induced cells was enhanced in a mutant lacking the iron regulator DtxR suffering from enhanced iron uptake. Altogether, these findings demonstrate the potential of the established workflow to gain insights into the phenotypic dynamics of bacterial populations.

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Cytometry meets next-generation sequencing – RNA-Seq of sorted subpopulations reveals regional replication and iron-triggered prophage induction in Corynebacterium glutamicum