Person: Leiman, Sara
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Leiman
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Sara
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Leiman, Sara
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Publication Reconstitution of Peptidoglycan Cross-Linking Leads to Improved Fluorescent Probes of Cell Wall Synthesis(American Chemical Society, 2014) Lebar, Matthew D.; May, Janine Margaret; Meeske, Alexander J.; Leiman, Sara; Lupoli, Tania J.; Tsukamoto, Hirokazu; Losick, Richard; Rudner, David; Walker, Suzanne; Kahne, DanielThe peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.Publication SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis(BioMed Central, 2014) Leiman, Sara; Arboleda, Laura C; Spina, Joseph S; McLoon, Anna LBackground: Bacteria often form multicellular, organized communities known as biofilms, which protect cells from a variety of environmental stresses. During biofilm formation, bacteria secrete a species-specific matrix; in Bacillus subtilis biofilms, the matrix consists of protein polymers and exopolysaccharide. Many domesticated strains of B. subtilis have a reduced ability to form biofilms, and we conducted a two-month evolution experiment to test whether laboratory culturing provides selective pressure against biofilm formation in B. subtilis. Results: Bacteria grown in two-month-long batch culture rapidly diversified their biofilm-forming characteristics, exhibiting highly diverse colony morphologies on LB plates in the initial ten days of culture. Generally, this diversity decreased over time; however, multiple types of colony morphology remained in our final two-month-old populations, both under shaking and static conditions. Notably, while our final populations featured cells that produce less biofilm matrix than did the ancestor, cells overproducing biofilm matrix were present as well. We took a candidate-gene approach to identify mutations in the strains that overproduced matrix and found point mutations in the biofilm-regulatory gene sinR. Introducing these mutations into the ancestral strain phenocopied or partially phenocopied the evolved biofilm phenotypes. Conclusions: Our data suggest that standard laboratory culturing conditions do not rapidly select against biofilm formation. Although biofilm matrix production is often reduced in domesticated bacterial strains, we found that matrix production may still have a fitness benefit in the laboratory. We suggest that adaptive specialization of biofilm-forming species can occur through mutations that modulate biofilm formation as in B. subtilis. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0301-8) contains supplementary material, which is available to authorized users.Publication Genetics and Regulation of Bacterial Biofilms(2015-03-24) Leiman, Sara; Kahne, Daniel; Garner, Ethan; Bernhardt, TomBacterial biofilm formation, the construction of dense, protective, multicellular communities, is a widely conserved behavior. In some bacteria, such as the Gram-positive model organism Bacillus subtilis, the genetics controlling biofilm formation are well understood. In other bacteria, however, including the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, the identities or roles of many biofilm genes remain unknown. Importantly, many proposed applications of biofilm research, particularly in the medical field, require knowledge not only of biofilm assembly but also of biofilm disassembly, the latter being a recent and underdeveloped area of study. It was previously reported that B. subtilis biofilms disassemble late in their life cycle due to the incorporation of four D-amino acids (D-leucine, D-methionine, D-tryptophan, and D-tyrosine, or D-LMWY) into peptidoglycan. It was further argued that D-LMWY specifically inhibits and disassembles the biofilms of diverse bacterial species, including B. subtilis and P. aeruginosa. Here I present a contrasting report. I describe how what had been perceived as D-LMWY-mediated biofilm inhibition is actually D-tyrosine-mediated toxicity. B. subtilis is sensitive to growth inhibition by D-tyrosine due to the absence of D-tyrosyl tRNATyr deacylase (Dtd), an enzyme that prevents the misincorporation of D-tyrosine and other D-amino acids into nascent proteins. By repairing the gene for Dtd, I was able to render B. subtilis resistant to both growth inhibition and biofilm inhibition by D-tyrosine and D-LMWY. In parallel, I recovered spontaneous mutants of B. subtilis that survive in the presence of D-LMWY. These isolates harbored mutations in pathways that regulate tRNATyr charging. Three of these mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNATyr synthetase (TyrRS), while a separate mutation improved the stereoselectivity of TyrRS. I concluded that these spontaneous D-LMWY resistance mutations were compensating for the absence of Dtd. In addition to my research on B. subtilis biofilm regulation, I demonstrated a new, non-destructive screening approach for identifying P. aeruginosa biofilm genes. Using this screen, I was able to recover a wide range of known biofilm genes as well as the new biofilm gene candidates ptsP, PA14_16550, and PA14_69700. These three genes are the focus of an ongoing study dedicated to characterizing P. aeruginosa biofilm formation, particularly as it relates to the secondary messenger cyclic di-GMP. In summary, this dissertation covers aspects of biofilm formation and dispersal in two bacterial species. My work offers mechanistic insight into D-amino acid resistance, resolves the relationship between D-amino acids and biofilms, and establishes a new tool for understanding the complexities of biofilm genetics and regulation.Publication D-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis(American Society for Microbiology, 2013) Leiman, Sara; May, J. M.; Lebar, M. D.; Kahne, Daniel; Kolter, R.; Losick, RichardThe soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.