Gutierrez Juan A, Crowley Paula J, Cvitkovitch Dennis G, Brady L Jeannine, Hamilton Ian R, Hillman Jeffrey D, Bleiweis Arnold S
Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA.
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Microbiology (Reading). 1999 Feb;145 ( Pt 2):357-366. doi: 10.1099/13500872-145-2-357.
The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.
变形链球菌是一种与龋齿相关的细菌病原体，其耐受菌斑pH值快速下降的能力（耐酸性）被认为是一种重要的毒力因子。为了研究这一特性，已分离并部分鉴定了变形链球菌JH1005株表现出酸敏感性的Tn917突变体。本文报道了其中一个突变体AS17的鉴定结果。初步序列分析表明，AS17中的转座子插入发生在一个双基因位点的基因间区域，该位点已被命名为sat，代表分泌和耐酸性。该位点与枯草芽孢杆菌的ylxM-ffh操纵子具有高度同源性。通过用变形链球菌基因组文库互补条件性大肠杆菌ffh突变体，克隆了sat+位点。互补克隆的测序鉴定出完整的ylxM和ffh基因以及一个与枯草芽孢杆菌proUlopuAC基因具有同源性的部分开放阅读框，该基因编码ProU/OpuA渗透调节甘氨酸甜菜碱转运系统的结合蛋白。RNA斑点杂交实验表明，突变体中ffh mRNA的稳态水平比亲本水平低约八倍。这表明sat-1::Tn917突变对ffh表达有部分极性效应。在酸休克（pH 5）时，发现野生型ffh mRNA水平与未应激（pH 7.5）水平相比增加了约四至八倍。在相同条件下，突变体水平保持不变。旨在研究突变体酸敏感性起源的实验表明，缺乏酸适应性/耐受性反应。用酸休克后的AS17纯化膜测量的质子泵出ATP酶（H+/ATP酶）比活性测定显示，与亲本菌株相比，水平低两倍。此外，发现AS17虽然能够在葡萄糖和多种其他糖底物中生长，但不能发酵山梨醇。这些发现表明，Ffh可能参与变形链球菌适应不断变化的环境条件过程中功能膜蛋白组成的维持。
