Department of Experimental and Clinical Biomedical Sciences, University of Florence, Italy.
Department of Agriculture, Food, Environment and Forestry, University of Florence, Italy.
Int J Biol Macromol. 2021 May 15;179:279-291. doi: 10.1016/j.ijbiomac.2021.02.189. Epub 2021 Mar 3.
Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) biovar 3 involved all global interest since 2008. We have found that in Psa3 genome, similarly to other P. syringae, there are three putative genes, lscα, lscβ and lscγ, coding for levansucrases. These enzymes, breaking the sucrose moiety and releasing glucose can synthetize the fructose polymer levan, a hexopolysaccharide that is well known to be part of the survival strategies of many different bacteria. Considering lscα non-coding because of a premature stop codon, in the present work we cloned and expressed the two putatively functional levansucrases of Psa3, lscβ and lscγ, in E. coli and characterized their biochemical properties such as optimum of pH, temperature and ionic strength. Interestingly, we found completely different behaviour for both sucrose splitting activity and levan synthesis between the two proteins; lscγ polymerizes levan quickly at pH 5.0 while lscβ has great sucrose hydrolysis activity at pH 7.0. Moreover, we demonstrated that at least in vitro conditions, they are differentially expressed suggesting two distinct roles in the physiology of the bacterium.
细菌性溃疡病是由丁香假单胞菌 pv.actinidiae(Psa)生物变种 3 引起的,自 2008 年以来引起了全球关注。我们发现,在 Psa3 基因组中,与其他丁香假单胞菌类似,有三个假定的基因 lscα、lscβ 和 lscγ,编码蔗糖转化酶。这些酶可以打破蔗糖部分并释放葡萄糖,合成果糖聚合物蔗聚糖,这是一种众所周知的许多不同细菌生存策略的一部分。由于提前出现终止密码子,我们认为 lscα 是非编码的,在本研究中,我们在大肠杆菌中克隆和表达了 Psa3 的两个假定功能的蔗糖转化酶 lscβ 和 lscγ,并对其生化特性进行了研究,如最适 pH 值、温度和离子强度。有趣的是,我们发现这两种蛋白质在蔗糖分解活性和蔗聚糖合成方面表现出完全不同的行为;lscγ 在 pH 5.0 时快速聚合蔗聚糖,而 lscβ 在 pH 7.0 时具有很强的蔗糖水解活性。此外,我们证明至少在体外条件下,它们的表达不同,这表明它们在细菌生理学中具有两种不同的作用。