Department of Biological Sciences, Binghamton University, Binghamton, New York, USA.
J Bacteriol. 2013 Aug;195(16):3531-42. doi: 10.1128/JB.01156-12. Epub 2013 May 31.
Dispersion is a process used by bacteria to successfully transit from a biofilm to a planktonic growth state and to spawn novel communities in new locales. Alterations in bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) levels have been shown to be associated with biofilm dispersal in a number of different bacteria. The signaling molecule nitric oxide (NO) is known to induce biofilm dispersion through stimulation of c-di-GMP-degrading phosphodiesterase (PDE) activity. However, no c-di-GMP modulating enzyme directly involved in NO-induced dispersion has yet been described in the opportunistic pathogen Pseudomonas aeruginosa. Here, we characterized MucR (PA1727) and NbdA (PA3311, NO-induced biofilm dispersion locus A), two membrane-bound proteins with identical domain organization consisting of MHYT-GGDEF-EAL, with respect to their role in NO-induced dispersion. Inactivation of mucR impaired biofilm dispersion in response to NO and glutamate, whereas inactivation of nbdA only impaired biofilm dispersion upon exposure to NO. A specific role of NbdA in NO-induced dispersion was supported by increased PDE activity, resulting in decreased c-di-GMP levels in biofilms expressing nbdA upon exposure to NO, a response that was absent in the ΔnbdA strain. Moreover, increased PDE activity was mainly due to a transcriptional activation of nbdA upon addition of NO. Biochemical analyses of recombinant protein variants lacking the membrane-anchored MHYT domain support NbdA being an active PDE. In contrast, MucR displayed both diguanylate cyclase and PDE activity in vitro, which seemed regulated in a growth-dependent manner in vivo. This is the first description of a PDE specifically involved in NO-induced biofilm dispersion in P. aeruginosa.
分散是细菌成功从生物膜过渡到浮游生长状态并在新环境中产生新群落的过程。在许多不同的细菌中，双（3′-5′）-环二鸟苷酸（c-di-GMP）水平的改变与生物膜分散有关。已知信号分子一氧化氮（NO）通过刺激 c-di-GMP 降解磷酸二酯酶（PDE）活性来诱导生物膜分散。然而，在机会性病原体铜绿假单胞菌中，尚未描述任何直接参与 NO 诱导分散的 c-di-GMP 调节酶。在这里，我们对 MucR（PA1727）和 NbdA（PA3311，NO 诱导的生物膜分散位点 A）进行了表征，这两种膜结合蛋白具有相同的结构域组织，由 MHYT-GGDEF-EAL 组成，与它们在 NO 诱导的分散中的作用有关。mucR 的失活会损害生物膜对 NO 和谷氨酸的分散，而 nbdA 的失活仅在暴露于 NO 时才会损害生物膜的分散。nbdA 在 NO 诱导的分散中的特定作用得到了支持，因为在表达 nbdA 的生物膜中，PDE 活性增加，导致 c-di-GMP 水平降低，而在ΔnbdA 菌株中没有这种反应。此外，NO 的添加主要导致 nbdA 的转录激活，从而增加 PDE 活性。缺乏膜锚定 MHYT 结构域的重组蛋白变体的生化分析支持 NbdA 是一种活性 PDE。相比之下，MucR 在体外显示出双鸟苷酸环化酶和 PDE 活性，这在体内似乎以生长依赖性方式受到调节。这是首次描述铜绿假单胞菌中特定参与 NO 诱导生物膜分散的 PDE。
