Nicolette Goularte

All Publications

• Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-rich Structural Framework for Monarchs and Swallowtails. Journal of molecular biology Goularte, N. F., Kallem, T., Cegelski, L. 1800: 167456

  Abstract

  The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analysis of associated soluble proteins indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as key cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

  View details for DOI 10.1016/j.jmb.2022.167456

  View details for PubMedID 35045329

• Molecular organization of the E. coli cellulose synthase macrocomplex. Nature structural & molecular biology Acheson, J. F., Ho, R., Goularte, N. F., Cegelski, L., Zimmer, J. 2021; 28 (3): 310–18

  Abstract

  Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.

  View details for DOI 10.1038/s41594-021-00569-7

  View details for PubMedID 33712813

• Identification of a novel pyruvyltransferase using 13C solid-state NMR to analyze rhizobial exopolysaccharides. Journal of bacteriology Wells, D. H., Goularte, N. F., Barnett, M. J., Cegelski, L., Long, S. R. 2021: JB0040321

  Abstract

  The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPS), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts, and thus for formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation, and can facilitate invasion in the absence of succinoglycan on some host plants. Biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to: identify putative pyruvyltransferase and acetyltransferase genes; construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan; and analyze EPS from mutant bacterial strains. EPS samples were examined by 13C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enable detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains, with deletions in five candidate acyl/acetyltransferase genes (exoZ, exoH, SMb20810, SMb21188, SMa1016) and a putative pyruvyltransferase (wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through ectopic expression of plasmid-encoded wgaE. Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPS) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. Biosynthesis of galactoglucan is not as well characterized as succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into biosynthesis of an important S. meliloti EPS and expands knowledge of enzymes that modify polysaccharides.

  View details for DOI 10.1128/JB.00403-21

  View details for PubMedID 34606371

• Structural basis of the day-night transition in a bacterial circadian clock SCIENCE Tseng, R., Goularte, N. F., Chavan, A., Luu, J., Cohen, S. E., Chang, Y., Heisler, J., Li, S., Michael, A. K., Tripathi, S., Golden, S. S., LiWang, A., Partch, C. L. 2017; 355 (6330): 1174-1180

  Abstract

  Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post-adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

  View details for DOI 10.1126/science.aag2516

  View details for Web of Science ID 000396351200036

  View details for PubMedID 28302851

  View details for PubMedCentralID PMC5441561