microfluidic-picoliter-bioreactor-for-microbial-single-cell-analysis

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

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Forschungszentrum Juelich GmbH

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition.

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression.

We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of haem homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study, ChrSA was shown to be crucial for haem resistance of C. glutamicum by activation of the putative haem-detoxifying ABC-transporter HrtBA in the presence of haem. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards haem. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA revealed that phosphorylated ChrA acts as an activator of hrtBA in the presence of haem. The haem oxygenase gene, hmuO, showed a decreased mRNA level in a chrSA deletion mutant but no significant binding of ChrA to the hmuO promoter was observed in vitro. In contrast, activation from P(hmuO) fused to eyfp was almost abolished in an hrrSA mutant, indicating that HrrSA is the dominant system for haem-dependent activation of hmuO in C. glutamicum. Remarkably, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralogous response regulator, whereas chrSA itself seemed to be repressed by HrrA. These data suggest a close interplay of HrrSA and ChrSA at the level of transcription and emphasize ChrSA as a second TCS involved in haem-dependent gene regulation in C. glutamicum, besides HrrSA.

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum.

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1-3). Recently, it was shown that the large lysogenic prophage CGP3 (∼187 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 have mainly focused 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 (Pint2, Plysin) fused to reporter genes encoding fluorescent proteins. Flow cytometric analysis revealed a spontaneous CGP3 activity in about 0.01-0.08% of the cells grown in standard minimal medium which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single cell level (ρ = 0.44-0.77). Furthermore, analysis of the ::PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 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.

Antonia Heyer

Institute of Bio- and Geosciences,
IBG-1: Biotechnology,
Institute of Bio- and Geosciences, IBG-1: Biotechnology

Forschungszentrum Juelich GmbH

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

Antonia Heyer

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Forschungszentrum Juelich GmbH

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

Institute of Bio- and Geosciences,
IBG-1: Biotechnology,
Institute of Bio- and Geosciences, IBG-1: Biotechnology