Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12

Abstract

DNA from the marine bacterium Alteromonas haloplanktis 214 was partially digested with Sau 3A and inserted into the Bam HI site of the cloning vector pBR322. The ligation mixture was used to transform Escherichia coli HB101. The gene bank plasmid preparation obtained was used to transform Escherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to contain A. haloplanktis DNA and to be capable of complementing the appropriate mutation in E. coli EO2717. Restriction maps showed that each of the plasmids was different.

Cloning in Escherichia coli K-12 of a Na+-dependent transport system from a marine bacterium

“…One of the mutants negative for D-alanine transport isolated, E. coli RM1, was transformed with the A. haloplanktis gene bank hybrid plasmid preparation used previously (13) by the transformation procedure of Maniatis et al (14). To screen for transport-positive transformants, the cells in the transformation mixture were washed with buffered salts solution and spread at a concentration of 105 cells per plate on the surface of M9-D-alanine agar medium containing ampicillin.…”

“…DNA manipulations. Plasmid and chromosomal DNA were isolated as described previously (13). DNA-DNA hybridizations were carried out by the method of Southern (22) by standard procedures (14).…”

“…DNA-DNA hybridizations were carried out by the method of Southern (22) by standard procedures (14). Conditions used for restriction enzyme digestions, ligations, and transformations of bacterial cells bave been described previously (13). Electroelution of restriction fragments from agarose gels was carried out by the procedure of Dretzen et al (6).…”

“…A transport system serving for the entry of D-alanine, D-serine, and glycine is also found in Escherichia coli (3,20,26), but in this organism the accumulation of these amino acids is energized by an electrochemical potential of protons (1,15). In a previous report, MacLeod et al (13) described the preparation of a gene bank of A. haloplanktis chromosomal DNA and showed that various biosynthetic genes in the gene bank could be expressed in E. coli. In the present study, we isolated mutants of E. coli defective for D-alanine transport and used the gene bank to obtain transformants of one of these which transport D-alanine by a Na+-dependent mechanism.…”

Overexpression of the Alanine Carrier Protein Gene from Thermophilic Bacterium PS3 in Escherichia coli

“…The plasmid encoding the MBP-ACP fusion protein was designated as pAC6268. E. coli strain RM1 (kindly donated by Prof. MacLeod, R.A., McGill Univ., Canada), which lacks active alanine and glycine transport activity (11), was used as the host strain and transformed with various plasmids for the transport assay in this study. For overexpression of MBP-ACP, the E. coli TB1 strain (New England Biolabs), harboring expression plasmid pAC6268, was used.…”