Abstract
Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of Bacillus anthracis and Paenibacillus alvei. The P. alvei SCWP, consisting of pyruvylated disaccharide repeats [→4)-β-D-GlcNAc-(1→3)-4,6-Pyr-β-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in vitro in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of β-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the P. alvei TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of P. alvei was included in this in vitro reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.
Highlights
The cell surface influences the physicochemical properties of a bacterium, its physiology, life-style, and fitness in a competitive habitat
The functional coupling of S-layer homology (SLH) domain containing proteins (SLH proteins) and secondary cell wall polymer (SCWP) pyruvylation is substantiated by the finding that several bacteria – including, e.g., Bacillus anthracis (Forsberg et al, 2012), Bacillus cereus strains (Forsberg et al, 2011), Lysinibacillus sphaericus (Ilk et al, 1999), Thermoanaerobacterium thermosulfurigenes (May et al, 2006), and Paenibacillus alvei (Schäffer et al, 2000), all synthesize a suite of SLH proteins, contain pyruvate in their cell wall and have an ortholog of the CsaB enzyme predicted to catalyze the transfer of pyruvate ketal to β-D-ManNAc (Mesnage et al, 2000)
The current study focuses on the predicted UDP-ManNAc transferase TagA and the predicted pyruvyltransferase CsaB from the P. alvei SCWP biosynthesis gene locus
Summary
The cell surface influences the physicochemical properties of a bacterium, its physiology, life-style, and fitness in a competitive habitat. Several Gram-positive bacteria employ SLH domain-mediated sorting of cell surface proteins and concomitantly engage a pyruvylated SCWP ( known as cell wall glycopolymer) as a cell wall ligand. Pyruvate ketal linked to β-DManNAc is regarded an indispensable and ancestral epitope in this cell surface display mechanism (Mesnage et al, 2000; Cava et al, 2004; Kern et al, 2010). Experimental data on CsaB is available for B. anthracis (Mesnage et al, 2000; Kern et al, 2010) and Thermus thermophilus (Cava et al, 2004), where csaB deficient mutants revealed a drastically reduced pyruvic acid content (by ∼98%) in comparison to the parent strain supporting pyruvyl transfer activity of CsaB
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