Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes.
To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used lambda-Red recombination to precisely and efficiently position PCR-generated DNA targeting cassettes containing a fluorescent protein gene and an antibiotic resistance marker, at the C-termini of the CDSs of interest, creating in-frame fusions under the control of their native promoters. We incorporated cre/loxP and flpe/frt technology to enable multiple rounds of chromosomal tagging events to be performed sequentially with minimal disruption to the target locus, thus allowing sets of proteins to be co-localized within the cell. The visualization of labeled proteins in live E. coli cells using fluorescence microscopy revealed a striking variety of distributions including: membrane and nucleoid association, polar foci and diffuse cytoplasmic localization. Fifty of the fifty-two independent targeting experiments performed were successful, and 21 of the 23 selected CDSs could be fluorescently visualized. Our results show that E. coli has an organized and dynamic proteome, and demonstrate that this approach is applicable for tagging and (co-) localizing CDSs on a genome-wide scale.
Watt
Rory M
RM
Open Laboratory of Chemical Biology, The Institute of Molecular Technology for Drug Discovery and Synthesis, Department of Chemistry, The University of Hong Kong, Hong Kong SAR, China.
Wang
Jing
J
Leong
Meikid
M
Kung
Hsiang-fu
HF
Cheah
Kathryn S E
KS
Liu
Depei
D
Danchin
Antoine
A
Huang
Jian-Dong
JD
D002876:Chromosomes, Bacterial / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis; D016153:Genes, Suppressor; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D008716:Methionine Adenosyltransferase / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D011995:Recombination, Genetic; D014166:Transferases / Q000032:analysis / Q000235:genetics
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; C078379:pyrH protein, E coli; D014166:Transferases; C519385:MetK protein, E coli; D008716:Methionine Adenosyltransferase