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
The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (ϳ187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (P int2 and P lysin ) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A P recA -eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (ϳ0.2%) exhibited a spontaneous induction of the SOS response. Correlation of P recA to the activity of downstream SOS genes (P divS and P recN ) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of P recA and CGP3 promoter fusions displayed a positive correlation at the single-cell level ( ؍ 0.44 to 0.77). Furthermore, analysis of the P recA -eyfp/P int2 -e2-crimson strain during growth revealed the highest percentage of spontaneous P recA and P int2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages. G enome sequencing projects have revealed a large amount of prophage DNA in bacterial genomes. Although not all prophage DNA accounts for functional prophages, because it includes degenerated phage remnants, this DNA can have a marked impact on bacterial physiology (1). The biotechnological platform organism Corynebacterium glutamicum is a Gram-positive, biotin-auxotroph soil bacterium that is used for the industrial production of more than four million tons of L-glutamate and L-lysine per year (2, 3). As revealed by whole-genome sequencing, C. glutamicum ATCC 13032 possesses three prophages that are integrated into its genome (CGP1 to -3), of which CGP1 and CGP2 are probably degenerated phage remnants (4-6). Previous studies showed that the large prophage CGP3 (187 kb) retains the ability to be excised from the genome and exist as a circular DNA molecule. Interestingly, a small number of wild-type cells showed a much higher copy number of circular phage DNA per cell than the average of the population (5).Recent studies in Shewanella oneidensis (7) and Streptococcus pneumoniae (8) have provided evidence that sacrificing a small number of cells by spontaneous prophage-induced lysis is beneficial to the remainder of the population. For these species, genomic DNA released into the extracellular space following cell lysis s...
A series of reticulated Arabidopsis thaliana mutants were previously described. All mutants show a reticulate leaf pattern, namely green veins on a pale leaf lamina. They have an aberrant mesophyll structure but an intact layer of bundle sheath cells around the veins. Here, we unravel the function of the previously described reticulated EMS-mutant dov1 (differential development of vascular associated cells 1). By positional cloning, we identified the mutated gene, which encodes glutamine phosphoribosyl pyrophosphate aminotransferase 2 (ATase2), an enzyme catalyzing the first step of purine nucleotide biosynthesis. dov1 is allelic to the previously characterized cia1-2 mutant that was isolated in a screen for mutants with impaired chloroplast protein import. We show that purine-derived total cytokinins are lowered in dov1 and crosses with phytohormone reporter lines revealed differential reporter activity patterns in dov1. Metabolite profiling unraveled that amino acids that are involved in purine biosynthesis are increased in dov1. This study identified the molecular basis of an established mutant line, which has the potential for further investigation of the interaction between metabolism and leaf development.
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