The difficulty of cloning Streptococcus pneumoniae mal and ami loci in Escherichia coli: toxicity of malX and amiA gene products.
Stability problems are frequently encountered when cloning pneumococcal DNA in Escherichia coli multicopy plasmid vectors such as derivatives of ColE1. In this paper, we report our investigations of these problems using the pneumococcal mal and ami regions. We offer evidence that, in both cases, promoters are not the primary cause of cloning problems. Indeed, successful cloning of mal and ami promoters has been achieved with standard vectors (devoid of transcriptional terminators flanking the insertion site). Moreover, we show that the entire mal fragment can be introduced into an E. coli strain harboring a chromosomal mutation that reduces plasmid copy number. The cause of the cloning problems has been traced to the malX and amiA structural genes. Overexpression of these genes, which probably encode lipoproteins, could have deleterious effects on E. coli hosts, possibly as a result of impairing the protein export machinery.
Martin
B
B
Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, Université Paul Sabatier, Toulouse, France.
Alloing
G
G
Boucraut
C
C
Claverys
J P
JP
D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008074:Lipoproteins / Q000096:biosynthesis / Q000633:toxicity; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000633:toxicity*; D013296:Streptococcus pneumoniae / Q000235:genetics*
D004274:DNA, Recombinant; D008074:Lipoproteins; D011994:Recombinant Proteins