LeBlanc D J, Lee L N, Inamine J M
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Antimicrob Agents Chemother. 1991 Sep;35(9):1804-10. doi: 10.1128/AAC.35.9.1804.
Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with the following resistance phenotypes: Tcr Emr Spr, Tcr Emr, Tcr Spr, and Tcr only but never Emr or Spr only. The genetic determinant encoding resistance to spectinomycin was cloned in Streptococcus sanguis Challis from pDL55, a 26-kb plasmid harbored by a Tcr Spr transconjugant. Subcloning experiments yielded a 1.1-kb ClaI-NdeI fragment that encoded very high-level Spr in S. sanguis (10 mg/ml) and Escherichia coli (50 mg/ml). Cell extracts of cultures obtained from Spr strains expressed adenylating activity for spectinomycin but not for streptomycin, indicating that Spr was due to an AAD(9) activity. The nucleotide base sequence of the 1.1-kb ClaI-NdeI fragment contained a single 750-base open reading frame. The protein predicted from the open reading frame consisted of 250 amino acids and had a calculated size of approximately 28,000 daltons, similar to the size estimated from maxicell analysis (29,000 daltons). The deduced amino acid sequence of the streptococcal AAD(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3")(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S. sanguis or E. coli are presented.
粪肠球菌LDR55是一株从人类临床分离得到的菌株，对四环素（Tcr）、红霉素（Emr）以及高水平（大于2000微克/毫升）的壮观霉素（Spr）具有抗性，但对链霉素不具有抗性。菌株LDR55与粪肠球菌OG1-RF之间的滤膜杂交产生了具有以下抗性表型的接合子：Tcr Emr Spr、Tcr Emr、Tcr Spr和仅Tcr，但从未出现仅Emr或仅Spr的情况。编码对壮观霉素抗性的遗传决定因子从pDL55克隆到血链球菌Challis中，pDL55是一个由Tcr Spr接合子携带的26kb质粒。亚克隆实验产生了一个1.1kb的ClaI-NdeI片段，该片段在血链球菌（10毫克/毫升）和大肠杆菌（50毫克/毫升）中编码非常高水平的Spr。从Spr菌株获得的培养物的细胞提取物对壮观霉素表现出腺苷酸化活性，但对链霉素没有，这表明Spr抗性是由于AAD(9)活性。1.1kb的ClaI-NdeI片段的核苷酸碱基序列包含一个单一的750碱基开放阅读框。从开放阅读框预测的蛋白质由250个氨基酸组成，计算大小约为28000道尔顿，与从大细胞分析估计的大小（29000道尔顿）相似。将链球菌AAD(9)的推导氨基酸序列与葡萄球菌转座子Tn554编码的AAD(9)的氨基酸序列进行了比较。这两种蛋白质共享约39%的氨基酸同一性，当包括保守氨基酸变化时，同一性扩展到53%。当将链球菌蛋白与大肠杆菌的AAD(3")(9)蛋白进行比较时，基于实际氨基酸和保守替换，同一性程度分别为27%和47%。本文介绍了从粪肠球菌中克隆壮观霉素AAD(9)决定因子及其核苷酸碱基序列分析，该决定因子转移到血链球菌或大肠杆菌中时会导致高水平的Spr抗性。
