The role of LytR-CpsA-Psr enzymes in the lifecycle of Bacillus anthracis /

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Bibliographic Details
Author / Creator:Zilla, Megan Liszewski, author.
Ann Arbor : ProQuest Dissertations & Theses, 2015
Description:1 electronic resource (123 pages)
Format: E-Resource Dissertations
Local Note:School code: 0330
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Other authors / contributors:University of Chicago. degree granting institution.
Notes:Advisors: Dominique Missiakas Committee members: Juliane Bubeck Wardenburg; Balaji Manicassamy; Olaf Schneewind; Anne I. Sperling.
Dissertation Abstracts International, Volume: 77-02(E), Section: B.
Summary:The biogenesis of bacterial wall polymers often occurs in stages whereby diffusible and soluble substrates are first synthesized in the cytosol but polymerized at the cis side of the plasma membrane. Next, membrane-bound "insoluble" intermediates are flipped across the lipid bilayer for subsequent assembly and display on the surface of Gram-positive bacteria. For example, diffusible UDP-linked sugars such as UDP-GluNac and UDP-ManAc are synthesized in the cytosol but formation of the GluNac-ManAc disaccharide that primes the synthesis of wall teichoic acid (WTA) is catalyzed on the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan. Substrates of LCP enzymes include the WTA of Bacillus subtilis and Staphylococcus aureus as well as capsular polysaccharides (CPS) of S. aureus and several streptococcal species.
Bacillus anthracis replicates as chains of vegetative cells via controlled separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the pyruvylated and acetylated secondary cell wall polysaccharide (SCWP). SCWP is assembled on undecaprenol-phosphate and polymerized at the membrane. B. anthracis does not synthesize WTA and capsular polysaccharides, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We made mutations in each of the B. anthracis lcp genes and found that an lcpD mutant displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. We also generated strain variants that express only one lcp gene. Expression of lcpB2 alone was not sufficient to support growth unless the gene was provided on a multicopy plasmid ( lcpB2++). Variants expressing lcpB4, lcpC or lcpD alone displayed severe defects in growth and cell shape. Variants expressing lcpB2++, lcpC or lcpD alone were unable to support S-layer assembly. The deposition of S-layer proteins was drastically altered in all strains expressing a single lcp gene with one exception, lcpB3. lcpB3 was the sole lcp gene supporting spore formation, whereas strains expressing lcpB2++, lcpC or lcpD alone failed to generate spores. We propose a model whereby LcpB3 represents the housekeeping LCP, anchoring SCWP along the cell envelope of vegetative bacilli. LcpB2, LcpC and LcpD attach SCWP at the poles or septa, enabling BSL murein hydrolase separation of daughter cells. LcpB4 appears to anchor specialized SCWP along the cylindrical axis to guide asymmetric cell division during sporulation. LcpB1 is presumed to anchor SCWP polymer with discrete structure to support sub-cellular compartmentalization in B. anthracis..
We also examine the biogenesis of the unusual and rare capsular hyaluronic acid (HA) polymer encoded by bacteria of the B. cereus lato group. We use chemical extraction to decipher whether HA polymers are tethered to the cell wall and compare HA encapsulation of wild type bacteria and isogenic lcp variants. The data supports a model whereby the HA polymer is tethered to peptidoglycan and points to several LCP enzymes as catalysts responsible for this covalent association.