Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum

Abstract

Proteins of the LCP (LytR, CpsA, Psr) family have been shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the Corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here, we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum, both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth-related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA gene resulted in severe growth defects and drastic morphological changes. Compared to the wild-type cell wall, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular, rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of a so-far-uncharacterized C-terminal domain (LytR_C domain) which is frequently found at the C terminus of the LCP domain in this prokaryotic phylum. IMPORTANCEAbout one-third of the world's population is infected with Mycobacterium tuberculosis, and multiple-antibiotic resistance provokes the demand for novel antibiotics. The special cell wall architecture of Corynebacteriales is critical for treatments because it is either a direct target or a barrier that the drug has to cross. Here, we present the analysis of LcpA and LcpB of the closely related Corynebacterium glutamicum, the first of which is an essential protein involved in cell wall biogenesis. Our work provides a comprehensive characterization of the impact of LCP proteins on cell wall biogenesis in this medically and biotechnologically important class of bacteria. Special focus is set on the two periplasmic LcpA domains and their contributions to physiological function.

Mycobacterium tuberculosis Rv2700 Contributes to Cell Envelope Integrity and Virulence

“…In mycobacteria, recent work indicates that the ligation of AG to PG is catalyzed by proteins in the LytR-Cps2A-Psr (LCP) family (3), which also ligate PG to wall teichoic acid and capsular polysaccharide in diverse Gram-positive bacteria (3)(4)(5)(6). Although wall teichoic acids, capsular polysaccharide, and AG are chemically diverse, they are attached to PG through a similar phosphodiester linkage, a function that is performed by LCP proteins.…”

“…Although wall teichoic acids, capsular polysaccharide, and AG are chemically diverse, they are attached to PG through a similar phosphodiester linkage, a function that is performed by LCP proteins. LCP proteins are widespread in firmicutes and actinobacteria (3). Most LCP proteins consist only of a predicted amino (N)-terminal transmembrane helix and the LCP domain itself, but in actinobacteria, roughly half of LCP proteins have an additional carboxyl (C)-terminal domain, termed a LytR_C domain, that is rarely found in firmicutes (3,7).…”

“…LCP proteins are widespread in firmicutes and actinobacteria (3). Most LCP proteins consist only of a predicted amino (N)-terminal transmembrane helix and the LCP domain itself, but in actinobacteria, roughly half of LCP proteins have an additional carboxyl (C)-terminal domain, termed a LytR_C domain, that is rarely found in firmicutes (3,7). Approximately 60% of all proteins with a LytR_C domain also include an LCP domain; the remainder have only the LytR_C domain and a predicted N-terminal helix, and we refer to these as LytR_C-only proteins.…”

“…Very few studies have addressed the function of LytR_C domains per se. One study has shown that a Corynebacterium glutamicum LCP-LytR_C protein requires its LytR_C domain (3), but its molecular function is unknown.…”

“…1B). CpsA1/Lcp1 and CpsA/CpsA2 are collectively essential in M. tuberculosis, and there is substantial evidence that these proteins and their orthologs in related species perform the essential function of AG-PG ligation (3)(4)(5)11). An earlier study (prior to the discovery of LCP proteins as AG-PG ligases) found that an M. tuberculosis virR mutant sheds cell envelope fragments, but that study focused on the immunological response to the virR mutant and did not further investigate the effects of VirR on cell envelope integrity (10).…”