engleski

Improvement of pBBRMCS plasmids, a very useful series of broad-host-range (bhr) cloning vectors

pBBR1MCS vector, a chloramphenicol (Cm) resistant bhr plasmid, contains 16 unique cloning sites within the lacZα gene and is stably retained in the absence of antibiotic selection. Four derivatives of the vector have been generated, with Cm resistance being replaced with four other resistance determinants. All of these vectors are small in size (<5.3 kb), contain unique cloning sites (MCS) within the lacZα gene, are mobilizable and compatible with various plasmid incompatibility groups. Plasmids were found to replicate in various hosts, such as Pseudomonas putida, Salmonella typhimurium and Vibrio cholerae, to name a few. In our work, we cloned the sgm, armA, rmtA, rtmB, rmtC and rmtD genes for Arm and npmA and kamB for Kam family of enzymes into Tc-resistant pBBR1MCS-3 plasmid. Arm and Kam methyltransferases are a group of enzymes found in either natural producers of aminoglycoside antibiotics or in clinical strains isolated from human and veterinary pathogens. They bind to bacterial ribosomes, thereby causing high-level resistance to aminoglycoside antibiotics. In the course of our experiments we have found that E. coli cells expressing ArmA and KamB methyltransferases from pBBR1MCS-3 plasmid have an unusually low minimal inhibitory concetration (MIC) of kanamycin, unlike the cells expressing all the other enzymes, with xpected high-level kanamycin MIC. In our cloning experiments, we cloned the genes in the KpnI (5’ end) and XhoI/XbaI (3’ end) restriction sites. KpnI restriction site is the first site available after the beginning of lacZα gene, and is 60 nucleotides away from the start codon. This results in the fusion of at least first 20 amino acids of the β-galactosidase α-peptide with any protein expressed from this vector. In order to overcome this problem, we introduced an NdeI restriction site than generates the START codon directly at the beginning of lacZα gene by site-directed mutagenesis in the pBBR1MCS-3 plasmid. We then cloned armA and kamB genes into improved plasmids and noticed that kanamycin MICs became very high, suggesting that the enzymes were now fully functional. We also introduced the NdeI restriction site in the same manner to plasmids pBBR1MCS-2, 4 and 5. In this work we present an enhanced pBBR1MCS vector series. We strongly believe that the intervention we made is of crucial importance for using these plasmids because of the unknown effect of at least 20 extra N-terminal amino acids fused to the protein of interest and derived from the original series of pBBRMCS plasmids.

pBBR1MCS vectors; lacZα fusion; mutagenesis; NdeI site

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