RUI: Understanding a Morphogenetic Biosurfactant in Streptomyces Coelicolor

Hofstra University, Hempstead NY

Investigators

Abstract

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.