A new mini-transposon for in vivo protein epitope tagging: application to Burkholderia multivorans.
A short amino acid sequence coding for the mature Pseudomonas aeruginosa OprI lipoprotein was fused to a mini-Tn5 plasposon (mini-transposon with an origin of replication) with tetracycline resistance in order to generate in-frame fusion proteins after transposition. After conjugative transfer to Burkholderia multivorans, clones reacting with an anti-OprI mab were selected. In-frame OprI-tagged proteins were detected and identified for six clones. The six C-tagged proteins were detected by immunoblot. The different mutants had insertions into a histone H1-like coding gene, cspD, encoding a cold-shock protein, dsbC, encoding a putative outer membrane lipoprotein involved in thiol-disulfide exchange, paaE, a ferredoxin-NADPH reductase gene, a gene for the catabolism of propionate, and one encoding an unknown protein.
Kholti
Abdelaziz
A
Laboratory of Microbial Interactions, Vrije Universiteit Brussel, Department of Molecular and Cellular Interactions, Flanders Interuniversity Institute of Biotechnology, Pleinlaan 2, 1050 Brussels, Belgium.
Plesa
Maria
M
Cornelis
Pierre
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D019117:Burkholderia / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D004251:DNA Transposable Elements; D000939:Epitopes; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005819:Genetic Markers / Q000276:immunology; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D013753:Tetracycline Resistance
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D000939:Epitopes; D005819:Genetic Markers; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; C060507:outer membrane lipoprotein I, Bacteria