Bacillus anthracis lcpGenes Support Vegetative Growth, Envelope Assembly, and Spore Formation
ABSTRACTBacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins andB. anthracisS-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. anthracisexpresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1to -4,lcpC, andlcpD), which when expressed inStaphylococcus aureuspromote attachment of wall teichoic acid to peptidoglycan. Mutations inB. anthracislcpB3andlcpDcause aberrations in cell size and chain length that can be explained as discrete defects in SCWP assembly; however, the function of the otherlcpgenes is not known. By deleting combinations oflcpgenes from theB. anthracisgenome, we generated variants with singlelcpgenes.B. anthracisexpressinglcpB3alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly. Strains expressinglcpB1orlcpB4displayed defects in cell size and shape, S-layer assembly, and spore formation yet sustained vegetative growth. In contrast, thelcpB2strain was unable to grow unless the gene was expressed from a multicopy plasmid (lcpB2++), and variants expressinglcpCorlcpDdisplayed severe defects in growth and cell shape. ThelcpB2++,lcpC, orlcpDstrains 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.IMPORTANCEProducts 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 ofN-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 ofBacillus 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.