(A) Partial maps of the plasmids containing wecA. pAA11 and pAA12 contain the 3′ end of rho. E, EcoRI site. The putative hairpin structure involved in transcription termination of rho is indicated. pAA14, pAA15, and pAA16 do not include sequences upstream of the putative start codons of wecA. (B) Nucleotide sequence of the rho-wecA region of E. coli K-12 and deduced amino acid sequences (GenBank accession no. M76129) (22). Horizontal arrows indicate the putative transcriptional terminator of rho. Putative starting sites for translation of wecA (underlined) are GTG₃₁₂, GTG₄₁₁, and ATG₆₄₂. The 5′ ends of cloned wecA derivatives in pAA14, pAA15, and pAA16 are also indicated (vertical arrows). Amino acids involved in predicted transmembrane domains I, II, and III are indicated (shaded boxes).

Cell fractionation by sucrose density gradients of JM109DE3 cells containing pAA14 (A and C) and pAA16 (B and D), expressing WecA₃₆₇ and WecA₂₅₇, respectively. (A and B) Immunoblot analysis with MAb M2 of fractions collected from sucrose gradients. (C and D) Protein concentration (●) and NADH oxidase activity (□) profiles of the various fractions, as determined by measurement of A₃₄₀ (right vertical axis) and units of enzyme activity per milliliter (left vertical axis), respectively, are shown.

Construction of pAA8 (FLAG fusion cloning vector) and pAA11 (carrying the WecA-FLAG fusion). (A) DNA sequence of the oligonucleotide encoding the FLAG epitope tag. (B) DNA sequence of the multiple cloning site of pUC18 as described in the published sequence. The first 24 amino acids of the LacZ protein are indicated (shaded box). (C) Modifications of the pUC18 multiple cloning site giving rise to pAA8. A 41-bp fragment (underlined) encoding the FLAG epitope sequence (bold) followed by a stop codon was added into the HindIII site of pUC18. The FLAG sequence was shifted by 1 base (+1, base is italic) at the 5′ end. The reading frame of the lacZ gene was corrected by addition of 2 bases (+2, bases are italic) following the FLAG-encoding sequence. (D) Construction of WecA-FLAG fusion plasmid pAA11. The sequence of the C-terminal end of WecA (double underlining) and the last four amino acids of WecA (open box) are shown. Two base pairs (+2, italic) were added to correct the reading frame such that the FLAG epitope (bold) is fused in frame to the C terminus of WecA. The XbaI site in the multiple cloning site of pUC18 (B, asterisk) was changed to XhoI (asterisk), and this strategy also added 11 intervening amino acids between the end of WecA and the FLAG epitope tag.

(A) Detection of WecA derivatives tagged with the FLAG epitope by immunoblotting with MAb M2. JM109DE3 cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and processed as described in the text. Lanes: 1, pAA8; 2, pAA11; 3, pAA12; 4, pAA14; 5, pAA15; 6, pAA16. Arrows indicate polypeptides with higher molecular masses (barely visible in lanes 2 and 5) that may represent oligomers of WecA. The positions of the following molecular mass standards are shown: myosin (250 kDa), bovine serum albumin (98 kDa), glutamic acid dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa), carbonic anhydrase (36 kDa), and myoglobin (30 kDa). (B) Immunoblot of LPS samples prepared from MV501 (lanes 1 to 5 and 7) and VW187 (lane 6), which were reacted with anti-O7 antibodies. MV501 was transformed with the various wecA derivatives as follows: lane 1, pAA16; lane 2, pAA15; lane 3, pAA14; lane 4, pAA12; lane 5, pAA11.