Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.

中文翻译：

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铜绿假单胞菌生物膜的碳饥饿选择分散不敏感突变体

生物膜会随着特定的环境因素而分散，例如氧气浓度降低、营养浓度变化和一氧化氮暴露。有趣的是，生物膜在这些条件下并没有完全分散，这通常归因于生物膜的生理异质性。然而，我们的研究结果表明，遗传异质性在营养缺乏后生物膜中铜绿假单胞菌的非分散种群中也起着重要作用。在这项研究中，12.2% 的生物膜在连续饥饿诱导的分散 4 天后未能分散。从分散相以及剩余的生物膜中回收细胞。对于 96 小时饥饿的生物膜，发现生物膜中存在皱纹小菌落变体 (RSCV)，但在分散流出物中未观察到。相比之下，野生型和小菌落变体（SCV）在扩散阶段大量被发现。这些变体的基因组测序表明，大多数基因在与生物膜形成相关的基因中具有单核苷酸突变，例如在 wspF、pilT、fha1 和 aguR 中。这些突变的互补恢复了饥饿诱导的生物膜扩散。因为 c-di-GMP 与生物膜的形成和扩散有关，我们将 c-di-GMP 报告基因引入野生型铜绿假单胞菌中，并在饥饿诱导的扩散前后监测绿色荧光蛋白 (GFP) 的表达。分散后，微集落更小，GFP 强度显着更亮，表明微集落内每个细胞的 c-di-GMP 相对浓度也增加了。此外，只有 RSCV 显示出增加的 c-di-GMP，而野生型和SCV与亲本株没有区别。这表明，虽然饥饿可以诱导生物膜的扩散，但它也导致对过度产生 c-di-GMP 并且无法响应扩散线索饥饿而分散的突变体的强烈选择。