Effects of Pseudoalteromonas marina flagellin on biofilm formationand settlement of Mytilus coruscus
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摘要: 大多数海洋无脊椎动物在发育过程中都经历浮游、底栖附着阶段，厚壳贻贝(Mytilus coruscus)作为海洋经济物种与大型污损生物，其附着机制受到广泛关注。为探究海洋细菌与厚壳贻贝附着的互作关系，选取了对厚壳贻贝稚贝附着具有较高诱导活性的海洋细菌—海假交替单胞菌(Pseudoalteromonas marina)，采用酸解超速离心法提取P. marina的鞭毛蛋白。将提取的鞭毛蛋白与琼脂糖溶液混合，形成凝胶直接刺激稚贝；再用提取的鞭毛蛋白处理P. marina 生物被膜进行稚贝附着实验。通过共聚焦激光扫描分析形成的生物被膜上生物量、细菌密度和胞外产物含量的变化。结果表明：P. marina 鞭毛蛋白与琼脂糖形成的混合凝胶可显著促进厚壳贻贝稚贝的附着；鞭毛蛋白处理的生物被膜对厚壳贻贝稚贝附着的诱导活性显著提高；生物被膜上的生物量、细菌密度、膜厚、胞外β-多糖、脂质和蛋白浓度都有所增加。研究表明，鞭毛蛋白可以直接调控厚壳贻贝稚贝的附着，也可通过改变P. marina 生物被膜的生物学特性，间接影响厚壳贻贝稚贝的附着，为探究细菌鞭毛蛋白与厚壳贻贝附着互作机制提供理论依据。Abstract: Most marine invertebrates undergo planktonic and benthic stages during growth. As a marine economic shellfish and macrofouling organism, Mytilus coruscus has attracted widespread attention. To explore the interaction between marine bacteria and the mussel settlement, Pseudoalteromonas marina, which has a high inducing activity for the plantigrade settlement of the M. coruscus was chosen and the flagellin was extracted by acid hydrolysis ultracentrifugation. The impact of extracted flagellin mixed with agarose solution to on plantigrade settlement was tested directly. The P. marina biofilms treated with extracted flagellin were used to investigate the inducing capacity on plantigrade settlement. Changes in biomass, bacterial density and extracellular products of the biofilms were analyzed by confocal laser scanning microscopy. The results showed that the mixed gel containing P. marina flagellin and agarose significantly promoted the settlement of plantigrades. In other treatment, the flagellin-treated biofilms also had a high inductivity of plantigrade settlement. Biomass, bacterial density, membrane thickness, extracellular β-polysaccharide, lipid and protein contents all increased. The study reveals that flagellin can directly regulate the settlement of plantigrades and control the inductivity by changing the biological characteristics of the P. marina biofilm indirectly. The study is valuable for clarifying the interaction between bacterial flagellin and plantigrade settlement of the M. coruscus.
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Key words:
- biofilm /
- flagellin /
- Mytilus coruscus /
- plantigrade /
- settlement
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图 1 鞭毛蛋白SDS-PAGE结果
M. Maker（购买自伯乐生物公司）； I. 目标鞭毛蛋白
Fig. 1 The SDS-PAGE of flagellin
M. Maker (purchased from Bole Biotech); I. target flagellin
图 2 鞭毛蛋白对稚贝附着的影响
Fig. 2 Effects of settlement of M. coruscus plantigrades on the flagellin
图 3 不同处理方式形成的生物被膜对稚贝附着的影响及细菌密度的分析
A. 鞭毛蛋白用不同方式处理P. marina细菌生物被膜后对厚壳贻贝稚贝的诱导作用; B. 鞭毛蛋白对P. marina 生物被膜上细菌密度的影响
Fig. 3 Influence of biofilms formed by different treatments on the settlement of plantigrades and the analysis of bacterial density
A. Percentages of settlement M. coruscus plantigrades on P. marina biofilm by flagellin is treated in different ways; B. effect of flagellin on bacterial density of P. marina biofilm
图 4 激光共聚焦扫描显微镜下鞭毛蛋白对P. marina 生物被膜上细菌分布与聚集状态的影响以及鞭毛蛋白对P. marina生物被膜膜厚膜厚的影响
A. 单一生物被膜上细菌密度；B. P. marina与鞭毛蛋白共同形成被膜上的细菌密度；C. P. marina生物被膜添加鞭毛蛋白后膜上细菌密度； D. 经鞭毛蛋白处理后的生物被膜膜厚分析
Fig. 4 Effect of flagellin on the distribution and aggregation of bacteria on P. marina biofilm by laser confocal scanning microscopy and the effect of flagellin on thickness of P. marina biofilm
A. Bacterial density on a single biofilm; B. the bacterial density on biofilm formed by P. marina and flagellin; C. the bacterial density on P. marina biofilm with flagellin; D. analysis of biofilm thickness after flagellin treatment
图 5 胞外产物含量
A. 鞭毛蛋白对P. marina 生物被膜上α-多糖、β-多糖、蛋白和脂类含量的影响；B. 鞭毛蛋白对P. marina 生物被膜上α-多糖、β-多糖、蛋白和脂类含量的统计及差异
Fig. 5 The extracellular polymeric substance
A. Effects of flagellin on the contents of α-polysaccharides, β-polysaccharides, proteins and lipids of P. marina biofilm; B. statistics and difference of α-polysaccharides, β-polysaccharides, proteins and lipids contents on P. marina biofilm by flagellin protein
表 1 P. marina细菌鞭毛蛋白质谱分析鉴定结果
Tab. 1 The mass spectrometry identification of flagellin extracted from P. marina
蛋白条带 蛋白名称 相似度/% 多肽/Da 独特性/% 分子量/Da A 4-fliC 61 30 14 33 469 2-fliC 56 27 15 33 464 3-fliC 55 21 12 33 442 B 2-fliC 42 15 7 33 464 4-fliC 42 14 4 33 469 3-fliC 39 13 8 33 442 
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