Population Heterogeneity inCorynebacterium glutamicumATCC 13032 Caused by Prophage CGP3

Abstract

The genome of Corynebacterium glutamicum type strain ATCC 13032 (accession number BX927147) contains three prophages, CGP1, CGP2, and CGP3. We recently observed that many genes within the CGP3 prophage region have increased mRNA levels in a dtxR deletion mutant that lacks the master regulator of iron homeostasis (J. Wennerhold and M. Bott, J. Bacteriol. 188:2907-2918, 2006). Here, we provide evidence that this effect is due to the increased induction of the prophage CGP3 in the dtxR mutant, possibly triggered by DNA damage caused by elevated intracellular iron concentrations. Upon induction, the CGP3 prophage region is excised from the genome and forms a circular double-stranded DNA molecule. Using quantitative real-time PCR, an average copy number of about 0.1 per chromosome was determined for circular CGP3 DNA in wild-type C. glutamicum. This copy number increased about 15-fold in the dtxR mutant. In order to visualize the CGP3 DNA within single cells, a derivative of the wild type was constructed that contained an array of tet operators integrated within the CGP3 region and a plasmid-encoded YFP-TetR fusion protein. As expected, one to two fluorescent foci that represented the chromosomally integrated CGP3 prophage were detected in the majority of cells. However, in a small fraction (2 to 4%) of the population, 4 to 10 CGP3 DNA molecules could be observed in a single cell. Interestingly, the presence of many CGP3 copies in a cell often was accompanied by an efflux of chromosomal DNA, indicating the lysis of the corresponding cell. However, evidence for the formation of functional infective CGP3 phage particles could not be obtained.

Analysis of SOS-Induced Spontaneous Prophage Induction in Corynebacterium glutamicum at the Single-Cell Level

“…In recent studies we observed a spontaneous excision of the prophage CGP3 in a small number of C. glutamicum cells cultivated in shake flasks with CGXII minimal medium (5). Transcriptome analysis revealed an upregulation of CGP3 genes (cg1890 to cg2071) upon induction of the SOS response by addition of the DNA-cross-linking antibiotic mitomycin C (A. Heyer and J. Frunzke, personal communication).…”

“…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).…”

“…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)(5)(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.…”

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

“…To demonstrate the applicability of the PBLR for fluorescence based time-lapse studies, we investigated the fluorescence emission of a C. glutamicum strain producing a plasmid-encoded YFP-TetR fusion protein under control of the Ptac promoter (pEKEx2-yfp-tetR) 18,22 . In the presence of low inducer (IPTG) concentrations, expression from Ptac is known to lead to significant cell-to-cell variation in isogenic bacterial populations.…”

Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

“…In ATCC 13032, CGP1 and CGP2 are rather small (13.5 kbp and 3.9 kbp, respectively) and appear to be highly degenerated. CGP3 (187.3 kbp) is one of the largest known prophages, constituting almost 6% of the entire C. glutamicum genome, and previous studies revealed spontaneous induction of CGP3 in a small fraction of wild-type cells even under standard cultivation conditions (23). A mutant lacking the master regulator of iron homeostasis (C. glutamicum ⌬dtxR) also exhibited increased CGP3 gene expression (24).…”