EF4 knockout E. coli cells exhibit lower levels of cellular biosynthesis under acidic stress

Running title: EF4 is essential for optimal growth under low pH stress

Proteins from E. coli WT (A) and ΔEF4 (KD) (B) cells were extracted and separated on a pH 3-10 IPG strip, followed by 12% SDS-PAGE and Coomassie Brilliant Blue R-250 staining. There were no detectable differences in spots between the gels.

MATERIALS AND METHODS
All chemicals used were purchased from Sigma-Aldrich unless otherwise indicated.

Bacterial strains and culture conditions
BW25113 (lacI q rrnB T14 △lacZ WJ16 hsdR514 △araBA-D AH33 △rhaBAD LD78 ) is a derivative of the F -, λ -, E. coli K-12 strain BD792 and was obtained from the Keio collection (National Institute of Genetics, Japan) (Baba et al., 2006). Plasmids pKD46 and pKD13 were kind gifts from Dr. Yong Tao of the Institute of Microbiology, CAS.
The ΔEF4 strain was generated as described previously with a few modifications (Baba et al., 2006). The gene was replaced by an in-frame kanamycin resistance gene using the following primer pair: 5'-GCCTGGTTAACCGGGCATTAAGGCACAATAATCATACTTATTCCGGGGA TCCGTCGACC-3' and 5'-TCAGGGCAAACATATTCGCCATGCCAACTCCTAAGGGTTTGTAGGCTGG AGCTGCTTCG-3' (underline sequence are homology arms). The kanamycin cassette was produced by PCR amplification from the plasmid pKD13, and the products were transformed into competent cell BW25113 containing pKD46. Recombinants were selected on LB (1% NaCl, 1% tryptone and 0.5% yeast extract) agar-kanamycin (50 µg/ml) plates and further screened by PCR. The kanamycin-resistance gene was Then Cells at pH 7 and pH 4 were further diluted ×10 with pH 7 medium once the 5 stationary phase was reached, and OD 580 was also detected at the indicated intervals.

Preparation and analysis of polysomes
A chloramphenicol-treated polysome fraction was prepared from E. coli WT and EF4 KO strains as well as rescued strains at pH 7 and pH 4 as described previously (Ishino et al., 2000) with some modifications. When the OD 580 reached 0.1, 0.4, 0.6, 0.8 and 1.0 respectively, the cells were treated with MgCl 2 (at a final concentration of 6 mM) and chloramphenicol (100 μg/ml) for 2 min, The cultures were immediately placed on ice before collection. Cells were spun down for 10 min at 4C and 1,600g. The pellet was resuspended in prechilled buffer (20 mM Hepes-KOH pH 7.6, 8.2 mM MgAc 2 , 80 mM NH 4 Ac, 4 mM -mercaptoethanol). Cells were lysed by freezing and thawing three times. Crude extracts were clarified by centrifugation for 1 h at 4C and 18,000g.
Typically 10 OD 260 U were loaded on linear 15-45% (w/v) sucrose gradients and spun using SW40 rotor (Beckman) for 3.5 h at 4°C and 160,000g (Karim, 2009). Gradients were manually collected by following the absorption trace at 260 nm, and the quantity of each gradient was calculated.

Assay for protein synthesis by measuring incorporation of 35 S-methionine
WT and KO cells at pH 4 and pH 7 were grown in LB medium at 37°C. When the OD 580 reached 0.6, 5 ml of the cultures were transferred to new 50 ml tubes, added with 4 μl EasyTag™ L-Methionine (0.5 mCi/ml) (PerkinElmer) and cultured at 37°C.
At the time points of 0, 3, 6, 10 min, 1 ml samples were removed and placed on ice containing 100 μl stop solution (100 mM Tris-HCl pH 7.5, 1mg/ml chloramphenicol, 10 mM methionine). All samples were then spun down and washed at least three 6 times with cold wash buffer (100 mM Tris-HCl pH 7.5, 0.1 mg/mL chloramphenicol, 1 mM methionine). The pellets were lysed in a boiling water bath for 20-30 min, and protein content was determined by BCA kit (Pierce). Then 1 μg protein was used for liquid scintillation counting.

Preparation of protein samples for 2-dimensional electrophoresis (2-DE)
E. coli cells were inoculated into LB medium and cultured overnight at 37°C.

SDS-PAGE and 2-DE
1.5 mg protein samples were separated by 12% SDS-PAGE, and then stained with Coomassie Brilliant Blue R-250. 0.7 mg protein samples were separated in the first dimension on a 24 cm, isoelectric focusing (IEF) tray with a pH 3-10 IPG gel strip (GE Healthcare) and by 12% SDS-PAGE in the second dimension. All samples were run at least twice to confirm reproducibility. Analytical gels were stained with Coomassie Brilliant Blue R-250 and then scanned with an UMAX Scanner