Live cell imaging of <scp>SOS</scp> and prophage dynamics in isogenic bacterial populations

Helfrich, Stefan; Pfeifer, Eugen; Krämer, Christina; Sachs, Christian Carsten; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina; Frunzke, Julia

doi:10.1111/mmi.13147

Cited by 37 publications

(61 citation statements)

References 67 publications

(101 reference statements)

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“…To study the impact of cgpS expression on the activity of the CGP3 prophage, we overexpressed cgpS in a strain carrying a reporter construct (WT-P lys -eyfp ) indicative for the activation of CGP3 by the production of the yellow fluorescent protein eYFP under the control of a phage promoter (22). Upon induction with mitomycin C, the control strain carrying the empty plasmid displayed increased reporter activity.…”

Section: Resultsmentioning

confidence: 99%

“…For single-cell analysis an in-house developed microfluidic platform was used (22,35–37). Phase-contrast and fluorescence time-lapse imaging was performed at 6 min intervals.…”

Section: Methodsmentioning

confidence: 99%

“…The genome of this important industrial amino acid producer contains three cryptic prophages (20,21). Whereas CGP1 and CGP2 are highly degenerated, CGP3 comprises almost 6% of the entire genome (187 kb) and is inducible in an SOS-dependent manner (22,23). Even under non-inducing conditions, spontaneous prophage induction (SPI) was observed, preceded by a spontaneous activation of the SOS response in >60% of cases (20,22,23).…”

Section: Introductionmentioning

confidence: 99%

“…Whereas CGP1 and CGP2 are highly degenerated, CGP3 comprises almost 6% of the entire genome (187 kb) and is inducible in an SOS-dependent manner (22,23). Even under non-inducing conditions, spontaneous prophage induction (SPI) was observed, preceded by a spontaneous activation of the SOS response in >60% of cases (20,22,23). However, the precise regulatory control of CGP3 induction has not been studied thus far.…”

Section: Introductionmentioning

confidence: 99%

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Silencing of cryptic prophages inCorynebacterium glutamicum

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 99%

“…For single-cell analysis an in-house developed microfluidic platform was used (22,35–37). Phase-contrast and fluorescence time-lapse imaging was performed at 6 min intervals.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Silencing of cryptic prophages inCorynebacterium glutamicum

et al. 2016

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“…Analyses of each 100,000 cells of C. glutamicum WT and Δpup carrying pJC1-P ripA -crimson reporter constructs was performed as described previously (47).…”

Section: Methodsmentioning

confidence: 99%

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

Küberl

Polen

Bott

2016

Proc. Natl. Acad. Sci. U.S.A.

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The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.iron limitation | prokaryotic ubiquitin-like protein | ATPase ARC |

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Spatiotemporal microbial single‐cell analysis using a high‐throughput microfluidics cultivation platform

et al. 2015

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Cell-to-cell heterogeneity typically evolves due to a manifold of biological and environmental factors and special phenotypes are often relevant for the fate of the whole population but challenging to detect during conventional analysis. We demonstrate a microfluidic single-cell cultivation platform that incorporates several hundred growth chambers, in which isogenic bacteria microcolonies growing in cell monolayers are tracked by automated time-lapse microscopy with spatiotemporal resolution. The device was not explicitly developed for a specific organism, but has a very generic configuration suitable for various different microbial organisms. In the present study, we analyzed Corynebacterium glutamicum microcolonies, thereby generating complete lineage trees and detailed single-cell data on division behavior and morphology in order to demonstrate the platform's overall capabilities. Furthermore, the occurrence of spontaneously induced stress in individual C. glutamicum cells was investigated by analyzing strains with genetically encoded reporter systems and optically visualizing SOS response. The experiments revealed spontaneous SOS induction in the absence of any external trigger comparable to results obtained by flow cytometry (FC) analyzing cell samples from conventional shake flask cultivation. Our microfluidic setup delivers detailed single-cell data with spatial and temporal resolution; complementary information to conventional FC results. V C 2015 International Society for Advancement of Cytometry

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

Cited by 37 publications

References 67 publications

Silencing of cryptic prophages inCorynebacterium glutamicum

Silencing of cryptic prophages inCorynebacterium glutamicum

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

Spatiotemporal microbial single‐cell analysis using a high‐throughput microfluidics cultivation platform

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