RUI: Understanding a Morphogenetic Biosurfactant in Streptomyces Coelicolor

这个研究项目探索了一种不寻常的和迷人的形态发生肽，称为SapB，在丝状土壤细菌链霉菌的发育周期中的作用。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，从而将他们引入假设驱动的科学。智力优势：S. coelicolor的生命周期以穿透土壤的基质菌丝（丝）向向上生长的气生菌丝的形态分化为特征。SapB是已知在这一过程中具有结构作用的少数分子之一。有人提出SapB作为一种生物表面活性剂/清洁剂，降低菌落-空气界面的表面张力，从而促进新的空气菌丝向上出现。由于PI先前的NSF资助，确定SapB具有不寻常的抗生素样结构，并且是发育调节的ramS基因的高度修饰产物。lantibiotic是一类核糖体合成的多肽抗生素，在成熟成为功能性抗生素之前需要经过广泛和特异性的修饰。有趣的是，尽管已知链菌可以制造许多抗生素，但SapB没有明显的抗菌活性。然而，SapB除了具有生物表面活性剂活性外，还可能具有信号功能。当纯化的SapB添加到S. coelicolor ramS零突变体（在空生菌丝形成中延迟）时，观察到基因表达的变化伴随着分化的完全恢复，这表明了这一点。目前尚不清楚SapB是信号本身，还是变化环境中的分子（或物理特征）触发了发育。因此，拟议研究的目标包括：(i)确定使SapB发挥作用的具体结构特征。据推测，SapB活性至少需要两个结构要素。这些包括肽的n端氨基酸，这些氨基酸被预测为正确修饰所必需，以及包括某些丝氨酸和半胱氨酸残基的抗生素特异性特征。这些元素将通过定点诱变而改变。突变肽将被纯化，它们的结构将被质谱验证，当在S. coelicolor ramS零突变体中表达时，它们恢复正常发育的能力将被评估。（ii）确定SapB是否仅仅是一种表面活性剂，或者它是否也直接作为信号起作用。如果SapB在两种能力中都起作用，那么可能构建保留一种活性而不保留另一种活性的突变肽。因此，将测量第(1)部分构建的每个突变肽的表面活性，并通过监测菌落形态、超微结构和微管蛋白样蛋白FtsZ的定位来评估每个肽在缺乏表面活性剂的零突变体中恢复形态分化的能力。（iii）阐明SapB与细胞表面的相互作用。结合试验将使用放射性标记的SapB和细胞壁和膜组分进行。如果SapB与质膜结合（提示信号功能），将进行实验以确定其结合伙伴；另一方面，如果SapB如预测的那样与细胞壁结合，将评估其结合脂质II的能力。更广泛的影响：研究计划的设计使本科生将成为其成功的组成部分。具体的研究组成部分已经开发，使学生参与制定假设和实验设计和执行。在她的职业生涯中，PI依靠了50多名本科生的才能，并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，这项研究将支持博士后研究人员的活动，他们将有机会在霍夫斯特拉大学（主要是本科生机构）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.