Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella typhimurium

Abstract

Many bacteria secrete cellulose, which forms the structural basis for bacterial multicellular aggregates, termed biofilms. The cellulose synthase complex of Salmonella typhimurium consists of the catalytic subunits BcsA and BcsB and several auxiliary subunits that are encoded by two divergently transcribed operons, bcsRQABZC and bcsEFG. Expression of the bcsEFG operon is required for full-scale cellulose production, but the functions of its products are not fully understood. This work aimed to characterize the BcsG subunit of the cellulose synthase, which consists of an N-terminal transmembrane fragment and a C-terminal domain in the periplasm. Deletion of the bcsG gene substantially decreased the total amount of BcsA and cellulose production. BcsA levels were partially restored by the expression of the transmembrane segment, whereas restoration of cellulose production required the presence of the C-terminal periplasmic domain and its characteristic metal-binding residues. The high-resolution crystal structure of the periplasmic domain characterized BcsG as a member of the alkaline phosphatase/sulfatase superfamily of metalloenzymes, containing a conserved Zn-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alkaline phosphatase-like enzymes, which includes bacterial Zn-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and the Mn-dependent lipoteichoic acid synthase (phosphoglycerol transferase) LtaS. These enzymes use the phospholipids phosphatidylethanolamine and phosphatidylglycerol, respectively, as substrates. These data are consistent with the recently discovered phosphoethanolamine modification of cellulose by BcsG and show that its membrane-bound and periplasmic parts play distinct roles in the assembly of the functional cellulose synthase and cellulose production.

The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

“…For the first time, we show at the protein level that the C-terminal domain of BcsG is sufficient for pEtN transfer in vitro. Furthermore, Sun et al (27) demonstrated that the C-terminal domain of BcsG from Salmonella typhimurium is capable of cleaving pEtN-phospholipids, but evidence of transfer to a cellulose acceptor was not demonstrated. Our results further expand this data to include direct transfer of the pEtN group to cellooligosachharides by the BcsG C-terminal domain in the absence of Bcs proteins, which was only predicted from the model originally proposed (26).…”

“…The specific role of BcsG has been proposed as a pEtN transferase directly modifying the cellulose polymer, although its sufficiency to do so has not before been demonstrated. Sun et al have shown BcsG exhibits phospholipase activity, fulfilling at least part of its proposed role in pEtN cellulose biosynthesis (27). Furthermore, the structure of the C-terminal catalytic domain from Salmonella typhimurium was also reported by Sun et al (PDB id 5OLT) and site-directed mutagenesis studies of a limited set of residues reflected particular phenotypes pointing to the importance of bcsG to the pEtN cellulose biofilm.…”

“…We also demonstrate for the first time that the C-terminal domain of EcBcsG is sufficient for transfer of pEtN onto cellulose acceptors, which is in contrast to prior work that suggested other members of the cellulose synthetic complex were essential for activity (ie. BcsA, BcsB and/or BcsE) and that BcsG may be part of a pathway that modifies intermediates (eg., BcsA, other periplasmic proteins, peptidoglycan, osmoregulated periplasmic glucan) instead of cellulose directly (26,27).…”

“…Furthermore, the structure of the C-terminal catalytic domain from Salmonella typhimurium was also reported by Sun et al (PDB id 5OLT) and site-directed mutagenesis studies of a limited set of residues reflected particular phenotypes pointing to the importance of bcsG to the pEtN cellulose biofilm. In conjunction, the role of the BcsG N-terminal domain in assembly of the mature bacterial cellulose synthase was confirmed (27). However, the consequences of pEtNunmodified cellulose on the properties of the biofilm, and neither the complete function nor mechanism of BcsG as a cellulose pEtN transferase have yet been demonstrated.…”

Siccibacter turicensis from Kangaroo Scats: Possible Implication in Cellulose Digestion

“…The gene bcsC is involved in helping the biosynthetic release of cellulose by encoding a protein that forms the pore in the outer membrane [ 13 ]. Recently, the bcsG gene has been characterised as encoding a Zn 2+ -dependent phosphoethanolamine transferase, which has a crucial role in cellulose formation and maintenance of biofilm integrity [ 29 ]. However, the function of the gene bcsF is not understood yet.…”