Identification of a Temperature-Sensitive Mutation in the ribE Gene of an Escherichia coli Keio Collection Strain

Experimental Section

Media, Chemicals, and Other Reagents. Cells were grown in LB containing 1% Bacto Tryptone (LPS solution, Daejeon, Korea), 0.5% yeast extract (BD, Pont de Claix, France), and 1% NaCl (Junsei, Tokyo, Japan) with or without antibiotics at 50 μg/mL kanamycin (EMD Chemicals, San Diego, USA), 100 μg/mL ampicillin (Duchefa biochemie, Haarlem, Netherlands), and 34 μg/mL chloramphenicol (Sigma, St. Louis, USA). Cells were also grown in M9 medium (42.2 mM Na2HPO4, 22 mM KH2PO4, 18.7 mM NH4Cl, and 8.6 mM NaCl) containing 2 mM MgSO4, 0.1 mM CaCl2, and 0.4% glucose. When necessary, the media were supplemented with riboflavin (Sigma, St. Louis, USA). Oligonucleotides were obtained from Genotech (Daejeon, Korea). Enzymes were acquired from Enzynomics (Daejeon, Korea), unless indicated otherwise. Taq polymerase (Solgent, Daejeon, Korea) was used for PCR. A DNA-spin plasmid (Intron, Hiden, Germany) kit was used to isolate plasmid DNAs. DNA ladder markers were from Enzynomics.

Bacterial Strains and Plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. The Keio dbpA deletion strain was from the Keio collection constructed from E. coli K-12 strain BW25113 (lacZq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBA-DAH33 ΔrhaBADCD78).2 A dbpA::kan cassette was transferred from the Keio dbpA deletion strain to BW25113 to generate the new deletion strain via P1 transduction.2,21 The dbpA deletion was confirmed via polymerase chain reaction (PCR) using primers k1 (5′-CAGTCATAGCCGAATAGCCT-3′) and d1 (5′- CAGCGTCTACCCTTTAAGAG-3′). Corresponding plasmids from the ASKA library were used for ectopic expression of DbpA or RibE.22 pCA24N was used as a control plasmid in experiments employing ASKA plasmids.

Identification of Genes Complementing Growth Defects of the Keio dbpA Deletion Strain. We constructed a pBR322- based genomic library with chromosomal DNA from the E. coli strain, MG1655. Partial Sau3AI-digested DNA fragments of 1.5-5 kb were inserted into the BamHI site of pBR322 to generate the genomic library. The Keio dbpA deletion strain was transformed with the E. coli genomic library. In total 23,000 transformants were screened for cell growth at 43 °C. Plasmid DNAs were purified from the positive clones and subjected to sequence analysis using two pBR322-sequencing primers (5′-CTTGGAGCCACTATCGAC-3′) and reverse primer (5′-GGTGATGTCGGCGATATAGG-3′) to obtain sequence information from both ends of DNA inserted into each plasmid.

Complementation with Ectopic Expression of DbpA and RibE. Cells of the Keio dbpA deletion strain were transformed with a DbpA or RibE expression plasmid from the ASKA library and subjected to complementation assays. Complementation tests were performed by examining the growth of cells containing the DbpA or RibE expression plasmid on LB plates supplemented with 34 μg/mL chloramphenicol in the absence or presence of IPTG at 43 °C.

Growth Analysis on Riboflavin-Complemented Media. The plate spotting assay was performed to verify the growth of the Keio dpbA deletion strain. Tenfold serial dilutions of overnight cultures were prepared and each 5 μL of the dilutions was plated onto agar plates containing a series of concentrations of riboflavin ranging from 50 to 800 mg/L. Cells were grown for 16 h and 30 h at 43 °C on LB rich and M9 minimal media, respectively.