Gutierrez J A, Crowley P J, Brown D P, Hillman J D, Youngman P, Bleiweis A S
Department of Oral Biology, University of Florida, Gainesville 32610, USA.
J Bacteriol. 1996 Jul;178(14):4166-75. doi: 10.1128/jb.178.14.4166-4175.1996.
New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.
构建了新载体，用于通过转座子Tn917高效诱变变形链球菌（pTV1-OK）中难以转化的菌株，并随后在大肠杆菌（pT21delta2TetM）中回收中断的基因。在本报告中，我们通过展示以下内容证明了Tn917对变形链球菌（JH1005）中难以转化的菌株进行诱变的实用性：（i）携带Tn9l7的广宿主范围质粒pWVO1的repA（Ts）衍生物pTV1-OK在允许温度（30℃）与非允许温度（45℃）下在JH1005中的条件复制；（ii）转座频率与枯草芽孢杆菌报道的频率相似（10^-5至10^-4），在温度转移至42至45℃后，90%至97%的红霉素抗性存活菌中质粒有效消除；（iii）通过Southern杂交分析确定Tn917插入的明显随机性，以及分离营养突变体、耐酸突变体和细菌素产生突变体的能力，频率范围为0.1%至0.7%。通过两种方法实现了转座子中断基因的回收：（i）在大肠杆菌中用回收载体pTV21delta2TetM进行标记拯救，pTV21delta2TetM是一种四环素抗性和氨苄青霉素敏感的Tn9l7-pBR322杂种；（ii）将Tn917突变体的基因组文库“鸟枪法”克隆到pUC19中。序列分析揭示了在与梭菌属fhs（66%同一性）、大肠杆菌dfp（43%同一性）、枯草芽孢杆菌ylxM-ffh（58%同一性）、icd（citC [69%同一性]）和argD（百分之61同一性）显示同源性的序列中的五个不同基因位点的插入。icd和argD中的插入导致营养需求；ylxM-ffh中的插入导致酸敏感性，而fhs和dfp中的插入导致酸敏感性和营养需求。本文描述了pTV1-OK的构建，并证明其可有效地用于将Tn917递送至变形链球菌中进行遗传分析，具有一定程度的随机性，并且染色体中的插入可以很容易地回收用于后续表征。这是链球菌属中成功进行Tn9l7诱变的首次发表报告。
