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

Highlights

• BcsG subunit of cellulose synthase is required for full-scale cellulose production.

• BcsG affects cellulose production via at least two distinct molecular mechanisms.

• Transmembrane part of BcsG is required for proper production of the BcsA subunit.

• The periplasmic domain of BcsG has the alkaline phosphatase superfamily structure.

• Crystal structure of the BcsG periplasmic domain shows a single active-site Zn ion.

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 Zn²⁺-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alkaline phosphatase-like enzymes, which includes bacterial Zn²⁺-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and the Mn²⁺-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.

Introduction

Bacterial cellulose, an exopolysaccharide with versatile biological roles, is produced by a variety of phylogenetically diverse bacteria [1]. In many of them, cellulose is required for biofilm formation that mediates environmental persistence, stress protection, and an anti-virulence phenotype [2], [3], [4], [5]. Cellulose production is also important for microbial cell–cell interactions including bacterial–fungal interactions, adherence to surfaces, slowing-down of cell motility, interaction with amyloid fibers and protection against disinfectants [6], [7], [8], [9], [10]. Cellulose is a seemingly simple biopolymer that consists of glucose monomers bound into linear β-(1–4)-glucan chains and is resistant against hydrolysis by alkali and most strong acids. Despite this simple structure, biosynthesis of cellulose in different bacteria is carried out by at least three distinct operon classes, which are characterized by different auxiliary and accessory genes [1], [11], [12], [13], [14] to produce macromolecules of amazingly different properties.

The core genes of all characterized cellulose biosynthesis operons code for the cellulose synthase catalytic subunit BcsA, an inner-membrane protein with a cytosolic domain containing the active site, which together with BcsB, a periplasmic protein with a single BcsA-interacting C-terminal transmembrane (TM) domain, forms the enzymatically active cellulose synthase (Fig. S1; [15], [16], [17]). The active site of BcsA is blocked by a gating loop and requires second messenger cyclic diguanosine monophosphate (c-di-GMP) binding to the C-terminal PilZ domain to allow substrate access. In addition, bcsZ gene, encoding a periplasmic endoglucanase, is typically located either within the cellulose biosynthesis operon or in close vicinity [5]. Further on, bcsC, a gene predicted to encode an outer membrane pore, is part of class I and II bcs operons [1]. The function of various accessory genes, often specific to certain cellulose biosynthesis operons, is starting to become unraveled. For example, in class II operons that are found in many beta- and gamma-proteobacteria, the bcsEFG operon is adjacent to the bcsABZC operon. BcsE was recently shown to be a novel c-di-GMP receptor required for optimal cellulose biosynthesis in Salmonella enterica serovar Typhimurium (hereafter S. typhimurium) and Escherichia coli [18]. Bacterial two-hybrid assays have shown a strong interaction of the E. coli BcsG with the cellulose synthase subunit BcsA and the BcsF protein [13], [19]. Mutating the bcsG gene in E. coli and Salmonella. resulted in severely disturbed cellulose synthesis, indicating a role of this protein in maintaining wild-type levels of cellulose [13], [18], [20]. More recently, BcsG was shown to participate in a chemical modification of the growing glucan chain in E. coli and S. typhimurium that results in production of cellulose with a phosphoethanolamine group added to every other glucosyl residue [19].

In this work, we further investigate the role(s) of the BcsG protein in cellulose biosynthesis and report the high-resolution crystal structure of its periplasmic domain. The crystal structure confirms that this domain is a member of the alkaline phosphatase/sulfatase (AlkP) enzyme superfamily, related to the membrane-anchored phosphoethanolamine and phosphoglycerol transferases. Mutational analyses demonstrated that the Ser278 residue, which is conserved in the BcsG family, is required for the catalytic activity of BcsG in vitro and optimal cellulose biosynthesis in vivo. However, the protein scaffold is required for production of wild type levels of the cellulose synthase subunit BcsA. Thus, this work shows that BcsG is a multifunctional protein with at least two functions involved not only in transfer of a phosphoethanolamine headgroup from phospholipids, but also in maintenance of inner-membrane protein production.