Identification and characterization of novel factors needed for two aspects of Myxococcus xanthus physiology: social motility and osmoregulation
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Myxococcus xanthus is a non-pathogenic Gram-negative, delta proteobacterium. This bacterium has become an important model system for the study of complex bacterial phenomena such as cell-to-cell communication, motility, predation, developmental differentiation, and the production of pharmacologically interesting secondary metabolites. This thesis identifies and characterizes important structures that are involved in: I. motility and II. osmoregulation. I: Motility: M. xanthus cells use two different types of motility: Adventurous (A-) motility and social (S-) motility. A-motility is predominantly used on dry surfaces such as hard agar, while S-motility is the preferred mode of motility on moist surfaces like soft agar. So far the molecular motor of A-motility has not been identified with certainty, whereas the molecular motor for S-motility has been known for quite some time. For this motility, M. xanthus uses the power generated by the extension and retraction of type IV pili, proteinaceous filaments that propel the cells forward. Although several details of the molecular mechanisms of S-motility have been resolved, some outstanding questions still remain. First, it is proposed that type IV pili bind to the secreted carbohydrate-comprised extracellular matrix (ECM), which causes the retraction of the pilus, and thus, initiates the power stroke that propels the cell forward. However, the major pilin protein, PilA, does not have a predicted lectin domain and thus, it is difficult to imagine how exactly the pilus binds to the carbohydrate. Second, S-motility is a complex behavior, requiring both cell-to-cell contacts and cell-to-ECM contacts. How does PilA accomplish both of these tasks simultaneously since the two interactions certainly require very disparate interactions? Lastly, type IV pili from other bacterial species, such as Pseudomonas aeruginosa and Neisseria gonorrhoeae possess tip proteins, PilY1 and PilC1, respectively, which mediate binding to host cells or ECM components, as well as bacterial aggregation and piliation. No such tip protein has been identified for M. xanthus so far leaving the question unsolved whether a protein other than PilA mediates the interaction of the pilus with the environment. In my lab, a novel type IV pili isolation protocol for M. xanthus has been developed which allows purification of large quantities of pili. In these isolations, a minor, high molecular-weight (HMW) protein consistently co-purifies with the pili. Using mass spectrometry, this HMW protein was identified as MXAN_1365 (hereby referred to as PilY1), a homolog of the type IV pilus tip protein PilY1. Analysis of the M. xanthus genome shows that there exist two other paralogs, which I termed PilY2 and PilY3. Using gene deletions and motility assays, I show that M. xanthus uses not just one but two of these PilY proteins, PilY1 and PilY2, to achieve piliation, aggregation, and normal S-motility, while PilY2 is dominant for S-motility and aggregation. In contrast, the function of the third paralog, PilY3 in these processes is less clear and not thoroughly evident using conventional assays. Sequence analyses show that PilY1 has an N-terminal integrin-like domain, while PilY2 possesses an N-terminal lectin-like domain. Thus, I propose that the type IV pilus uses PilY1 to mediate cell-to-cell interactions while PilY2 is used for cell-to-ECM contacts. These important findings augment the field’s understanding of type IV pilus-mediated social motility in M. xanthus and highlight the diversity of molecular mechanisms used by these structures to facilitate interactions with a wide variety of surfaces. II: Osmoregulation: Osmoregulated periplasmic glucans (OPGs) are found in many Gram-negative bacteria. These glucans can exist as either linear oligomers of 6-8 glucose residues, as seen in E.coli, or as cyclic structures, as seen in Rhizobium. The main functional role described so far for periplasmic glucans is osmoprotection under low osmolarity of the external milieu. This protection is achieved through the attachment of various anionic groups to the oligomers that attract and bind water molecules keeping the periplasmic space and the cytoplasm hydrated. I describe here the discovery and characterization of a novel polysaccharide polymer in M. xanthus that is located in the periplasm and surprisingly appears to replace the oligomeric carbohydrates found in most bacteria. Chemical analyses show that the polysaccharide is composed of equal parts N-acetyl-glucosamine and glucose, as well as mannose and rhamnose, as opposed to OPGs, which are only made of glucose. Through linkage analysis and NMR, I have also determined the molecular structure of this polysaccharide. Electron microscopy of the isolated polymer reveals that it is composed of fibers forming a loose meshwork. Interestingly, this meshwork becomes highly hygroscopic when the cells are grown under high osmolarity conditions indicating that this polymer indeed acts as an OPG which under these conditions appears to change its composition to keep the periplasmic space and the cytoplasm well hydrated.