Eugen Pfeifer

Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations

Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram‐positive Corynebacterium glutamicum ATCC 13032. Time‐lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA‐independent SPI. Whereas approx. 20% of SOS‐induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time‐resolved, quantitative description of SPI as general phenomenon of bacterial populations.

A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

Adaptive laboratory evolution of Corynebacterium glutamicum towards higher growth rates on glucose minimal medium

In this work, we performed a comparative adaptive laboratory evolution experiment of the important biotechnological platform strain Corynebacterium glutamicum ATCC 13032 and its prophage-free variant MB001 towards improved growth rates on glucose minimal medium. Both strains displayed a comparable adaptation behavior and no significant differences in genomic rearrangements and mutation frequencies. Remarkably, a significant fitness leap by about 20% was observed for both strains already after 100 generations. Isolated top clones (UBw and UBm) showed an about 26% increased growth rate on glucose minimal medium. Genome sequencing of evolved clones and populations resulted in the identification of key mutations in pyk (pyruvate kinase), fruK (1-phosphofructokinase) and corA encoding a Mg2+ importer. The reintegration of selected pyk and fruK mutations resulted in an increased glucose consumption rate and ptsG expression causative for the accelerated growth on glucose minimal medium, whereas corA mutations improved growth under Mg2+ limiting conditions. Overall, this study resulted in the identification of causative key mutations improving the growth of C. glutamicum on glucose. These identified mutational hot spots as well as the two evolved top strains, UBw and UBm, represent promising targets for future metabolic engineering approaches.

Silencing of cryptic prophages in Corynebacterium glutamicum

DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum. CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.

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

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 106 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 (PdivS) 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.