藻际环境铁载体细菌的筛选及群体感应信号对其合成的调节作用

Isolation of siderophore-producing bacteria from the phycosphere and quorum sensing role in siderphore biosynthesis

铁载体是海洋微生物获取铁元素的重要代谢因子,现有的一些研究推测海洋微生物铁代谢功能的发挥依赖群体效应,但海洋微生物群体行为的介导方式及其对铁载体功能的调节还未见揭示.为深入了解微生物的铁代谢特征,以藻际微生物为例,从中分离筛选产铁载体的细菌,并探讨群体感应信号(QS)对其合成的调控作用.利用稀释涂布法从甲藻赤潮样品中分离得到5株产铁载体的细菌,16Sr RNA基因序列鉴定为新鞘氨醇杆菌(Novosphingobium sp.)、鞘氨醇单胞菌(Sphingomonas sp.)、寡养单胞菌(Stenotrophomonas sp.)、嗜水气单胞菌(Aeromonas hydr ophi la)和根瘤菌(R hizobiumar enae).铬天青S(CAS)检测法结合化学显色反应分析显示,细菌分泌的铁载体主要包括羧酸盐、异羟肟酸、儿茶酚和肠杆菌素.薄层色谱法(TLC)检测到4株细菌具有明显产群体感应信号的能力,信号分子主要为高丝氨酸内酯(AHLs)AHL-C8、AHL-C8-O以及AHL-10.为验证群体感应信号对铁载体的调节能力,以新鞘氨醇杆菌为代表构建AHL突变株,通过突变株与野生株的比较试验,发现AHL的缺失明显减少了铁载体的表达量,证实铁载体的分泌受到AHL信号的正向调节.考虑铁元素是藻际环境重要的微量元素,本研究结果提示微生物利用群体感应信号介导铁载体的表达,可能在藻菌关系中扮演着互利共生的作用.

Siderophores are important metabolic factors for iron chelation in marine microorganisms.Some studies have speculated that the iron metabolic function of marine microorganisms depends on population effects.However,the factors that mediate microbial group behavior and the regulation of siderophore function have not been uncovered.To comprehensively understand iron metabolic characteristics in marine environments,we isolated siderophore-producing bacteria from the phycosphere niche.Next,we explored the regulation of siderophore synthesis by quorum sensing(QS)signals.The results show that five siderophore-producing bacteria were isolated from the dinoflagellate algal bloom samples.After 16S rRNA sequencing,the isolates were identified as Novosphingobium sp.,Sphingomonas sp.,Stenotrophomonas sp.,Aeromonas hydrophila,and Rhizobium arenae.Chrome azurol plate culture experiments and chemical color reaction analysis showed that the main siderophores secreted by the bacteria included carboxylates,hydroxamates,catechols,enterobactin,and enterobactin-like substances.Among the five bacteria,four had the ability to produce QS signals(i.e.,acyl-homoserine lactone,AHL)as revealed by thin-layer chromatography.The AHL signaling molecules produced were AHL-C8,AHL-C8-O,and AHL-C10.To verify the ability of AHL to regulate siderophores,representative AHL-defective mutants were constructed from Novosphingobium sp.Comparisons between the defective mutant and wild-type strains revealed that the AHL-defective mutant strain significantly reduced siderophore production,confirming that siderophore secretion is positively regulated by the AHL signal.Considering iron as an important trace element in the phycosphere environment,our results suggest that microorganisms use quorum-sensing signals to mediate the expression of siderophores,which may be an important factor in the mutually beneficial relationships between algae and bacteria.