Figure S1: Overexpression of Yqcg Leads to Cell Death and Chromosome Degradation

Supplemental Materials and Methods

DNA and RNA extraction

Cells (ME187) were grown in LB medium to an OD600 of ~0.6 and xylose was added to induce yqcG expression. Samples were collected before and after induction and were immediately frozen by liquid Nitrogen. DNA and RNA were extracted using the DNA purification kit Wizard Genomic (Promega) or FastRNA Pro Blue kit (MP Biomedicals), respectively. For RNA extraction cells were processed three times with the FastPrep-24 (MP Biomedicals) in setting 6.5 m/sec, for 45 sec each time, with 5 min of ice cooling between cycles. DNA and RNA concentrations were determined using NanoDrop 2000C (Thermo scientific), and sample integrity was evaluated by 1% agarose gel for DNA and capillary gel electrophoresis using the Agilent 2100 Bioanalyzer for RNA.

SDS-PAGE and Western blot analysis

Cells were grown in LB medium to an OD600 of ~0.6 and xylose was added to induce yqcG expression, andsamples were collected before and after induction. Samples were resuspended with lysis buffer [10mM TRIS pH 8, 10mM MgCl2, halt protease inhibitor cocktail x1 (Thermo scientific), 0.5 mg/ml lysozyme, 5µg/ml DNaseI], incubated at 37ºC for 10 minutes, and then incubated at 100ºC for 5 minutes with Laemmli sample buffer. Proteins were separated by SDS-PAGE 12.5% which was either stained with Instant Blue (Expendeon) or electroblotted onto a polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P; Millipore). For Immunoblot analysis of GFP and SigA proteins, membranes were blocked for 1 hour at room temperature (0.05% Tween-20, 5% skim milk in PBSx1 for GFP or TBSx1 for SigA). Blots werethen incubated for 1 hour at room temperature with polyclonal rabbit anti-GFP antibodies (1:20,000in 0.05% Tween-20, 5% skim milk in PBSx1) or overnight at 4°C with polyclonal rabbit anti-SigA antibodies (1:10,0000 in 0.05% Tween-20, 5% skim milk in TBSx1). Next, membranes were incubated for 1 hour at room temperature with peroxidase conjugated goat anti-rabbit secondary antibody (1:3,000 in 0.05% Tween-20, 5% skim milk in PBSx1 for GFP or 1:3,000 in TBSx1 for SigA). EZ-ECL kit (Biological Industries, Beit Haemek, Israel) was used for final detection.

Plasmid construction

pME38 which contains the 3’ region of manP fused to gfp, was constructed by amplifying the 3’ region of manP gene by PCR using the primers 275 and 276, which replaced the stop codon with XhoI site. The PCR-amplified DNA was digested with EcoRIand XhoI and was cloned into the EcoRI and XhoI sites of pKL168 (kan) (1),which contains the gfp coding sequence.

pME40 which contains the 3’ region of rplA fused to gfp, was constructed by amplifying the 3’ region of rplA gene by PCR using the primers 501 and 502, which replaced the stop codon with XhoI site. The PCR-amplified DNA was digested with EcoRIand XhoI and was cloned into the EcoRI and XhoI sites of pKL168 (kan) (1),which contains the gfp coding sequence.

References

1.Lemon KP, Grossman AD. 1998. Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science 282:1516-1519.