RUI: Understanding a Morphogenetic Biosurfactant in Streptomyces Coelicolor

该研究项目探索了一种不寻常且令人着迷的形态发生肽（称为 SapB）在丝状土壤细菌天蓝色链霉菌发育周期中的作用。该研究还旨在让本科生参与分析这种肽（小蛋白质）的结构和功能特征以及这些微生物的生物学，从而向他们介绍假设驱动的科学。智力价值：天蓝色链球菌的生命周期以基底菌丝（丝）的形态分化为特征，基底菌丝穿透土壤，形成向上生长的气生菌丝。 SapB 是已知在此过程中具有结构作用的少数分子之一。有人提出，SapB 起到生物表面活性剂/去污剂的作用，降低菌落-空气界面的表面张力，从而促进新气生菌丝的向上出现。由于 PI 之前获得 NSF 资助，确定 SapB 具有不寻常的羊毛硫抗生素样结构，并且是发育调控 ramS 基因的高度修饰产物。羊毛硫抗生素是一类核糖体合成的肽抗生素，在成熟为功能性抗生素之前经过广泛和特定的修饰。有趣的是，尽管已知链霉菌可以产生多种抗生素，但 SapB 没有明显的抗菌活性。然而，SapB 除了其生物表面活性剂活性外，还可能具有信号传导功能。观察结果表明，当将纯化的 SapB 添加到 S. coelicolor ramS 无效突变体（其气生菌丝的形成被延迟）时，检测到基因表达的变化，伴随着分化的完全恢复。目前尚不清楚 SapB 本身是否是信号，或者变化的环境中的分子（或物理特征）是否触发发育。因此，拟议研究的目标包括 (i) 确定使 SapB 发挥功能的特定结构特征。据推测，SapB 活性至少需要两个结构元件。这些包括预计正确修饰所需的肽 N 末端的氨基酸，以及包括某些丝氨酸和半胱氨酸残基的羊毛硫抗生素特异性特征。这些元素将通过定点诱变而改变。将纯化突变肽，通过质谱验证其结构，并评估其在 S. coelicolor ramS 无效突变体中表达时恢复正常发育的能力 (ii) 以确定 SapB 是否仅仅是表面活性剂或是否也直接充当信号。如果 SapB 具有两种功能，那么就有可能构建保留一种活性但不保留另一种活性的突变肽。因此，将测量部分(i)中构建的每个突变肽的表面活性，并且将通过监测集落形态、超微结构和微管蛋白样蛋白FtsZ的定位来评估每个肽在表面活性剂缺陷的无效突变体中恢复形态分化的能力。 (iii) 阐明 SapB 与细胞表面之间的相互作用。将使用放射性标记的 SapB 以及细胞壁和膜组分进行结合测定。如果 SapB 与质膜结合（表明具有信号传导功能），将进行实验来鉴定其结合伙伴；另一方面，如果如预测的那样 SapB 与细胞壁结合，则将评估其结合脂质 II 的能力。更广泛的影响：该研究计划的设计使本科生成为其成功不可或缺的一部分。已经开发了具体的研究部分，以使学生能够参与假设的制定以及实验设计和执行。在她的职业生涯中，PI 依靠了 50 多名本科生的才能，预计每年至少有三到四名独立学习的本科生参与资助的研究。此外，这项研究还将支持博士后研究员的活动，该研究员将有机会在霍夫斯特拉（主要是本科院校）接受教育科学家培训。

This research program explores the role of an unusual and fascinating morphogenetic peptide, called SapB, in the developmental cycle of the filamentous soil bacterium Streptomyces coelicolor. The research is also designed to engage undergraduate students in the analyses of structural and functional features of this peptide (small proteins) as well as the biology of these microbes, thereby introducing them to hypothesis-driven science. Intellectual merit: The life cycle of S. coelicolor features the morphological differentiation of substrate hyphae (filaments), which penetrate the soil, into upwardly growing aerial hyphae. SapB is one of the few molecules known to have a structural role in this process. It has been proposed that SapB functions as a biosurfactant/detergent, reducing the surface tension at the colony-air interface thereby facilitating the upward emergence of new aerial hyphae. As a result of the PI's previous NSF funding, it was determined that SapB has an unusual lantibiotic-like structure and is the highly modified product of the developmentally regulated ramS gene. Lantibiotics are a class of ribosomally synthesized peptide antibiotics that undergo extensive and specific modification prior to maturation into a functional antibiotic. Interestingly, although the streptomyctes are known to make many antibiotics, SapB has no demonstrable antimicrobial activity. However, SapB may have a signaling function in addition to its biosurfactant activity. This is suggested by the observation that when purified SapB is added to S. coelicolor ramS null mutants (which are delayed in the formation of aerial hyphae) a change in gene expression is detected that accompanies complete restoration of differentiation. It is unclear if SapB is the signal per se, or if a molecule (or physical feature) in the changed environment triggers development. The objectives of the proposed research thus include (i) To identify specific structural features that render SapB functional. It is hypothesized that there are at least two structural elements needed for SapB activity. These include amino acids in the N-terminus of the peptide that are predicted to be required for proper modification, and lantibiotic-specific features that include certain serine and cysteine residues. These elements will be changed by site directed mutagenesis. Mutant peptides will be purified, their structure verified by mass spectrometry, and their capacity to restore normal development when expressed in a S. coelicolor ramS null mutant will be evaluated (ii) To determine if SapB is solely a surfactant or if it also functions directly as a signal. If SapB functions in both capacities, it may be possible to construct mutant peptides that retain one activity but not the other. Thus the surface activity of each mutant peptide constructed in part (i) will be measured, and the capacity of each peptide to restore morphological differentiation in a surfactant-deficient null mutant will be evaluated by monitoring colony morphology, ultrastructure, and the localization of the tubulin-like protein FtsZ. (iii) To elucidate the interaction between SapB and the cell surface. Binding assays will be performed using radiolabeled SapB and cell wall and membrane fractions. If SapB associates with the plasma membrane (suggesting a signaling function), experiments will be performed to identify its binding partner; on the other hand if, as predicted SapB associates with the cell wall, its capacity to bind lipid II will be assessed. Broader impact: The research program has been designed so that undergraduate students will be an integral part to its success. Specific investigational components have been developed to enable the participation of students in the formulation of hypotheses and experimental design and execution. During the course of her career, the PI has relied on the talents of over 50 undergraduate students and anticipates the involvement of at least three or four independent study undergraduate students each year of funded research. Also, this research will support the activities of a postdoctoral researcher who will have the opportunity to train as an educator-scientist while at Hofstra, a principally undergraduate institution.