Bacterial Metabolism During Biofilm Growth Investigated by 13C Tracing
Bacterial Metabolism During Biofilm Growth Investigated by 13C Tracing
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DOI：
10.3389/fmicb.2018.02657
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发表时间：
2018-11
影响因子：
5.2
通讯作者：
Ni Wan;Hao Wang;C. K. Ng;Manisha Mukherjee;D. Ren;B. Cao;Yinjie J. Tang
中科院分区：
文献类型：
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作者：
Ni Wan;Hao Wang;C. K. Ng;Manisha Mukherjee;D. Ren;B. Cao;Yinjie J. Tang
本研究通过显微成像、基因表达分析和13 C标记研究了铜绿假单胞菌PAO 1在生物膜形成过程中的代谢。首先，采用动态标记来研究新鲜生物膜（厚度40 - 60微米）中的葡萄糖利用率。葡萄糖-6-P的标记周转时间表明生物膜的代谢明显慢于细胞。其次，在连续管式生物膜反应器或摇瓶中培养PAO 1。然后根据蛋白质氨基酸的同位素异构体模式对PAO 1进行13 C-代谢通量分析。结果表明，PAO 1生物膜细胞在生长过程中保持了其渗透模式的通量特征。(1)葡萄糖可通过促进NAD（P）H供应的两种循环途径（TCA循环和Entner-Doudoroff-Embden-Meyerhof-Parnas循环）降解。(2)回补途径（包括丙酮酸分流）增加通量可塑性。(3)生物膜生长表型不需要显著的细胞内通量重新布线（生物膜和非稳态通量网络之间的变化，通过葡萄糖摄取率归一化为100%，小于20%）。(4)转录分析表明，在新鲜的生物膜细胞的关键分解代谢基因的表达水平相当的反硝化细胞。最后，PAO 1，希瓦氏菌oneidensis（作为对照组），和他们的c-di-GMP transconjugates（具有不同的生物膜形成能力）在生物膜反应器或反渗透条件下进行13 C-标记。氨基酸标记差异分析表明，希瓦氏菌通量网络在生物膜形成过程中的变化比PAO 1更灵活。
This study investigated the metabolism of Pseudomonas aeruginosa PAO1 during its biofilm development via microscopy imaging, gene expression analysis, and 13C-labeling. First, dynamic labeling was employed to investigate glucose utilization rate in fresh biofilms (thickness 40∼60 micrometer). The labeling turnover time of glucose-6-P indicated biofilm metabolism was substantially slower than planktonic cells. Second, PAO1 was cultured in continuous tubular biofilm reactors or shake flasks. Then 13C-metabolic flux analysis of PAO1 was performed based on the isotopomer patterns of proteinogenic amino acids. The results showed that PAO1 biofilm cells during growth conserved the flux features as their planktonic mode. (1) Glucose could be degraded by two cyclic routes (the TCA cycle and the Entner-Doudoroff-Embden-Meyerhof-Parnas loop) that facilitated NAD(P)H supplies. (2) Anaplerotic pathways (including pyruvate shunt) increased flux plasticity. (3) Biofilm growth phenotype did not require significant intracellular flux rewiring (variations between biofilm and planktonic flux network, normalized by glucose uptake rate as 100%, were less than 20%). (4) Transcription analysis indicated that key catabolic genes in fresh biofilm cells had expression levels comparable to planktonic cells. Finally, PAO1, Shewanella oneidensis (as the comparing group), and their c-di-GMP transconjugants (with different biofilm formation capabilities) were 13C-labeled under biofilm reactors or planktonic conditions. Analysis of amino acid labeling variances from different cultures indicated Shewanella flux network was more flexibly changed than PAO1 during its biofilm formation.