New shuttle vectors for Escherichia coli and Bacillus subtilis. III. Nucleotide sequence analysis of tetracycline resistance gene of pAM.ALPHA.1 and ori-177.

Abstract

A small plasmid pHY163PLK (2519 bp) was constructed from the shuttle vector pHY300PLK. pHY163PLK contains the replication origin of pACYC-177, the tetracycline resistance gene (tetal) of pAMal derived from Streptococcus faecalis and eight continuous unique cloning sites of a polylinker region (EcoRI, SmaI, BamHI, SalI, PstI, BglII, XbaI and HindIII), pHY163PLK, a copy-number mutant, has one base substitution in RNA I region compared with that of p15A which is the ancestor of ori-pHY163PLK.The tetal, which codes 458 amino acids (1374 bp), is highly homologous to the TcR genes of other Gram-positive bacteria but completely different from the TcR gene encoded by pBR322 in Gram-negative bacteria. The regulatory region of tetal (constitutive type) has an insertion of seven base pairs in the leader peptide region of a related inducible TcR

Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H+ antiporter of Staphylococcus aureus

“…The Tet(K) protein is closely related to the Tet(L) protein found among Gram-positive pathogens [6][7][8]. They confer resistance by the active efttux of tetracycline.…”

New shuttle vectors for Escherichia coli and Bacillus subtilis. IV The nucleotide sequence of pHY300PLK and some properties in relation to transformation.

“…The sequence from 3532 (HincII) to 4123 (BanI) presented in this figure has been determined by Sutcliffe (1978). About half of the present nucleotide sequence has been previously reported as pHY163PLK (Ishiwa and Shibahara 1985b). frequency of transformation in E. coli but shows only very low frequency transformation in B, subtilis. In contrast, the oligomeric form has a high transformation frequency in B, subtilis but only a low frequency in E, coli (Fig.…”

“…The nucleotide sequence was determined by the dideoxy chain termination method (Sanger 1981) as previously described (Ishiwa and Shibahara 1985b).…”

“…2) was constructed by combining the sequences of the three components; the nucleotide sequences of the replication function region derived from pAMal in pHY300PLK ( Fig. 1), pHY163PLK which is a derivative plasmid of pHY300PLK (Ishiwa and Shibahara 1985b), and the ApR gene from pBR322 (Sutcliffe 1978). The APR gene of pBR322 and pACYC177 originates from Tn3 (Chang and Cohen 1978;Sutcliffe 1978).…”

“…The NcoI2 site (2583 in Fig. 2) in the ORF al was interrupted by insertion of either one or two XhoI-linkers as described previously (Ishiwa and Shibahara 1985b), to produce pHY300.1PLK and pHY300.2PLK, respectively. The insertion of two XhoI-linkers in pHY300.2PLK created a new HaeIII site in pHY300.2PLK.…”