RUI: Understanding a Morphogenetic Biosurfactant in Streptomyces Coelicolor

RUI：了解天蓝色链霉菌中的形态发生生物表面活性剂

• 批准号: 0717852
• 负责人: Joanne Willey
• 金额: --
• 依托单位: Hofstra University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0717852&HistoricalAwards=false
• 关键词: RUI; Understanding; Morphogenetic; Biosurfactant; Streptomyces

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.

这个研究项目探索了一种不寻常和迷人的形态发生肽，称为SapB，在丝状土壤细菌天蓝色链霉菌的发育周期中所起的作用。这项研究还旨在让本科生分析这种多肽(小蛋白质)的结构和功能特征，以及这些微生物的生物学，从而向他们介绍假说驱动的科学。智力优势：天蓝色链霉菌的生活史以穿透土壤的底物菌丝(细丝)的形态分化为向上生长的气生菌丝为特征。SapB是为数不多的已知在这一过程中具有结构作用的分子之一。有人提出，SapB具有生物表面活性剂/洗涤剂的作用，可以降低菌落-空气界面的表面张力，从而促进新的气生菌丝向上出现。由于PI之前的NSF资助，确定SapB具有不寻常的l抗生素样结构，是发育调节的RAMS基因的高度修饰的产物。抗生素是一类核糖体合成的多肽抗生素，在成熟为功能性抗生素之前经过广泛和特异的修饰。有趣的是，虽然已知链霉菌可以制造许多抗生素，但SapB并没有明显的抗菌活性。然而，SapB可能除了具有生物表面活性外，还具有信号转导功能。这是因为观察到，当纯化的SapB被添加到天蓝色链霉菌Rams零突变体(气生菌丝形成延迟)中时，检测到伴随着完全分化恢复的基因表达的变化。目前尚不清楚SapB是否是信号本身，或者变化环境中的分子(或物理特征)是否触发了发育。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。假设SapB活动至少需要两个结构要素。这些包括肽N-末端的氨基酸，预测需要适当的修饰，以及抗生素特有的特征，包括某些丝氨酸和半胱氨酸残基。这些元件将通过定点突变而改变。突变的多肽将被提纯，它们的结构将通过质谱学验证，它们在天蓝色链球菌零突变体中表达时恢复正常发育的能力将被评估(Ii)以确定SapB是否只是一种表面活性物质，或者它是否也直接作为信号发挥作用。如果SapB同时具有这两种功能，就有可能构建保持一种活性而不保留另一种活性的突变多肽。因此，将测量在第(I)部分中构建的每个突变多肽的表面活性，并通过监测菌落形态、超微结构和微管蛋白样蛋白FtsZ的定位来评估每个多肽恢复表面活性缺乏突变体的形态分化的能力。(3)阐明SapB与细胞表面的相互作用。结合分析将使用放射性标记的SapB以及细胞壁和细胞膜的部分。如果SapB与质膜结合(暗示信号功能)，将进行实验以确定其结合伙伴；另一方面，如果如预测的那样与细胞壁结合，则将评估其结合脂质II的能力。更广泛的影响：该研究项目的设计是为了让本科生成为其成功不可或缺的一部分。已经开发了特定的调查部分，以使学生能够参与提出假设以及实验设计和执行。在她的职业生涯中，PI依靠了50多名本科生的才华，预计每年至少有三到四名自学本科生参与资助研究。此外，这项研究将支持博士后研究人员的活动，他将有机会在霍夫斯特拉接受教育科学家培训，霍夫斯特拉是一个主要的本科生机构。