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Bacillus anthracis lcp Genes Support Vegetative Growth, Envelope Assembly, and Spore Formation.

ABSTRACT: Bacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and B. anthracis S-layer-associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the secondary cell wall polysaccharide (SCWP). B. anthracis expresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1 to -4, lcpC, and lcpD), which when expressed in Staphylococcus aureus promote attachment of wall teichoic acid to peptidoglycan. Mutations in B. anthracis lcpB3 and lcpD cause aberrations in cell size and chain length that can be explained as discrete defects in SCWP assembly; however, the function of the other lcp genes is not known. By deleting combinations of lcp genes from the B. anthracis genome, we generated variants with single lcp genes. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid (lcpB2(++)), and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2(++), lcpC, or lcpD strains supported neither S-layer assembly nor spore formation. We propose a model whereby LCP enzymes fulfill partially overlapping functions in transferring SCWP molecules to discrete sites within the bacterial envelope.Products of genes essential for bacterial envelope assembly represent targets for antibiotic development. The LytR-CpsA-Psr (LCP) enzymes tether bactoprenol-linked intermediates of secondary cell wall polymers to the C6 hydroxyl of N-acetylmuramic acid in peptidoglycan; however, the role of LCPs as a target for antibiotic therapy is not defined. We show here that LCP enzymes are essential for the cell cycle, vegetative growth, and spore formation of Bacillus anthracis, the causative agent of anthrax disease. Furthermore, we assign functions for each of the six LCP enzymes, including cell size and shape, vegetative growth and sporulation, and S-layer and S-layer-associated protein assembly.

SUBMITTER: Liszewski Zilla M

PROVIDER: S-EPMC4626891 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

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Bacillus anthracis lcp Genes Support Vegetative Growth, Envelope Assembly, and Spore Formation.

Liszewski Zilla Megan M Lunderberg J Mark JM Schneewind Olaf O Missiakas Dominique D

Journal of bacteriology 20150921 23

<h4>Unlabelled</h4>Bacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and B. anthracis S-layer-associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the secondary cell wall polysaccharide (SCWP). B. anthracis expresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1 to -4, lcpC, and lcpD), which when expres ...[more]

PMID: 26391207

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Project description:In preliminary experiments, the absence of anthrose in our genetic Sterne knockout was found to decrease pagA expression in BHI broth containing glucose (Fig 5A and B). This contrasted with the large spike in pagA expression when the Sterne antC mutant is grown in protein rich HIB media which does not contain sugars. When exogenous anthrose was added during growth in BHI broth, expression from PpagA increased in both WT (purple line in Fig 5A) and antC Sterne (purple line in Fig 5B). Many bacteria respond to levels of metabolic intermediates and modulate gene expression accordingly. We hypothesized intracellular or extracellular anthrose levels could impact the vegetative cells through modification of gene expression during vegetative growth. Wildtype B. anthracis Sterne was grown in the presence and absence of exogenously added pure anthrose to avoid any possibility of confounding data in a deletion mutant. Bacteria were harvested at two timepoints post-addition of anthrose, 30 min and 2 h, and compared to diluent only treated cultures (Fig 5C). After 30 min treatment, there were 6 genes significantly upregulated more than two-fold while 17 genes were downregulated (Fig 5D). These genes were mainly involved in generating metabolic intermediates such as pyruvate and trehalose. The most upregulated gene was a putative membrane protein (log2FC=10.98) followed by a gene encoding a GerPF homologue at a log2FC of 10.98. Mutation of GerPF family proteins have been linked to a super-dormant spore phenotype[10]. The gene experiencing the highest downregulation was a putative lipoprotein (log2FC = -20.02). No genes located on pXO1 were detected at 30 min. A subset of differentially transcribed genes from the 30 min timepoint are listed in Fig 5F. After 2 h of anthrose treatment, 52 genes experienced significant up regulation, 18 of which were hypothetical proteins (Fig 5E). Forty-five genes experienced significant down regulation across replicates, 13 of which were hypothetical proteins.

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Bacillus anthracis lcp Genes Support Vegetative Growth, Envelope Assembly, and Spore Formation.

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Bacillus anthracis lcp Genes Support Vegetative Growth, Envelope Assembly, and Spore Formation.

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