Abstract The ability of bacteria and fungi to degrade polymeric substrates is often assessed in liquid-based assays conducted by exposing polymers to planktonic cells, or cell-free supernatants or enzymes generated from them. These assessments miss the opportunity to specifically examine the role of biofilm formation and physiology in degradative processes. In a previous study, we examined the ability of cell-free supernatants from wildtype and hydrolase-deficient Pseudomonas protegens Pf-5 strains to degrade a commercial polyester polyurethane (PU). In this study, we developed the methodology to conduct spatial-temporal analysis of both biofilm colonization and degradation of polyester PU coatings using the same strains. Wild-type Pf-5 grew as confluent biofilms that produced tendrils containing viable cells; in contrast, Pf-5ΔpueAB, a mutant lacking PueA and PueB hydrolases, colonized the PU only by growing as a confluent biofilm but lacked tendril expansion. Degradation of PU by the wild-type hydrolases may alter the substrata, facilitating colonization by the biofilm. Chemical analysis by in situ Fourier transform infrared (FTIR) and Raman spectroscopies revealed the polyester block of the PU was preferentially degraded to varying degrees by the wild-type Pf-5 and mutant biofilms, and showed degradation due to PueB that was undetectable in liquid-based assays. Raman spectroscopy analysis of biofilms of Pf-5ΔpueAB grown on PU revealed a detectable, but the lowest, level of PU degradation relative to other strains. Degradation of PU by this mutant had previously been undetectable and this study revealed the existence of biofilm-associated hydrolytic mechanisms other than those driven by PueA and PueB. Taken together, our data suggest that both PueA and PueB hydrolases play a role in the colonization and degradation of a model polyester PU by P. protegens biofilms. This study demonstrates the importance of using biofilm-based assays to identify mechanisms of microbiologically-influenced degradation.

中文翻译：

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PueA 和 PueB 水解酶促进了 Pseudomonas protegens 生物膜对聚氨酯涂层的定植和降解

摘要 细菌和真菌降解聚合物底物的能力通常在通过将聚合物暴露于浮游细胞或无细胞上清液或由它们产生的酶进行的液基测定中进行评估。这些评估错过了专门检查生物膜形成和生理学在降解过程中的作用的机会。在之前的一项研究中，我们检查了来自野生型和水解酶缺陷型 Pseudomonas protegens Pf-5 菌株的无细胞上清液降解商业聚酯聚氨酯 (PU) 的能力。在这项研究中，我们开发了使用相同菌株对聚酯 PU 涂层的生物膜定植和降解进行时空分析的方法。野生型 Pf-5 生长为融合的生物膜，产生含有活细胞的卷须；相比之下，Pf-5ΔpueAB，一种缺乏 PueA 和 PueB 水解酶的突变体，仅通过生长为融合的生物膜而定植于 PU，但缺乏卷须扩张。野生型水解酶对 PU 的降解可能会改变基质，促进生物膜的定植。通过原位傅里叶变换红外 (FTIR) 和拉曼光谱进行的化学分析表明，PU 的聚酯嵌段优先被野生型 Pf-5 和突变生物膜不同程度地降解，并且显示出由于 PueB 导致的降解，在液体中无法检测到- 基于检测。对 PU 上生长的 Pf-5ΔpueAB 生物膜的拉曼光谱分析显示，相对于其他菌株，PU 降解水平可检测，但水平最低。这种突变体对 PU 的降解以前是无法检测到的，这项研究揭示了生物膜相关水解机制的存在，而不是由 PueA 和 PueB 驱动的水解机制。总之，我们的数据表明 PueA 和 PueB 水解酶在 P. protegens 生物膜对模型聚酯 PU 的定植和降解中发挥作用。这项研究证明了使用基于生物膜的测定来确定微生物影响降解机制的重要性。