Identification of a New Family of Enzymes with Potential \u3cem\u3eO\u3c/em\u3e-acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria

Background: The metabolism of the rigid bacterial cell wall heteropolymer peptidoglycan is a dynamic process requiring continuous biosynthesis and maintenance involving the coordination of both lytic and synthetic enzymes. The O-acetylation of peptidoglycan has been proposed to provide one level of control on these activities as this modification inhibits the action of the major endogenous lytic enzymes, the lytic transglycosylases. The O-acetylation of peptidoglycan also inhibits the activity of the lysozymes which serve as the first line of defense of host cells against the invasion of bacterial pathogens. Despite this central importance, there is a dearth of information regarding peptidoglycan O-acetylation and nothing has previously been reported on its de-acetylation. Results: Homology searches of the genome databases have permitted this first report on the identification of a potential family of O-Acetylpeptidoglycan esterases (Ape). These proteins encoded in the genomes of a variety of both Gram-negative and Gram-positive bacteria, including a number of important human pathogens such as species of Neisseria, Helicobacter, Campylobacter, and Bacillus anthracis, have been organized into three families based on amino acid sequence similarities with family 1 being further divided into three sub-families. The genes encoding these proteins are shown to be clustered with Peptidoglycan O-acetyltransferases (Pat) and in some cases, together with other genes involved in cell wall metabolism. Representative bacteria that encode the Ape proteins were experimentally shown to produce O-acetylated peptidoglycan. Conclusion: The hypothetical proteins encoded by the pat and ape genes have been organized into families based on sequence similarities. The Pat proteins have sequence similarity to Pseudomonas aeruginosa AlgI, an integral membrane protein known to participate in the O-acetylation of the exopolysaccaride, alginate. As none of the bacteria that harbor the pat genes produce alginate, we propose that the Pat proteins serve to O-acetylate peptidoglycan which is known to be a maturation event occurring in the periplasm. The Ape sequences have amino acid sequence similarity to the CAZy CE 3 carbohydrate esterases, a family previously known to be composed of only O-acetylxylan esterases. They are predicted to contain the α/β hydrolase fold associated with the GDSL and TesA hydrolases and they possess the signature motifs associated with the catalytic residues of the CE3 esterases. Specific signature sequence motifs were identified for the Ape proteins which led to their organization into distinct families. We propose that by expressing both Pat and Ape enzymes, bacteria would be able to obtain a high level of localized control over the degradation of peptidoglycan through the attachment and removal of O-linked acetate. This would facilitate the efficient insertion of pores and flagella, localize spore formation, and control the level of general peptidoglycan turnover

Characterizing the cellulose-modifying enzyme BcsG from Escherichia coli

Microbial biofilms are communities of microorganisms that exhibit co-operative behaviour, producing a matrix of exopolysaccharide that enmeshes the community. The well-studied human pathogens Escherichia coli and Salmonella entericaproduce a biofilm matrix comprised chiefly of the biopolymer cellulose, along with amyloid protein fibers termed curli. This biofilm matrix confers surface adherence and acts as a protective barrier to disinfectants, antimicrobials, environmental stressors, and host immune responses. Pertaining to this research, the bcsEFG operon, conserved in the Enterobacteriaceae, encodes an inner membrane-spanning complex responsible for the addition of a phosphoethanolamine (pEtN) modification to microbial cellulose, essential for extracellular matrix assembly and biofilm architecture. Furthermore, E. coli deficient in bcsGproduce a biofilm matrix lacking structural integrity and the self-assembling architecture observed in wild-type E. coli. The presence of a pEtN-substituted cellulose matrix was shown to be important in bladder epithelial colonization by uropathogenic E. coli, further suggesting its role as a virulence factor in etiological agents of urinary tract infections observed in the clinic. The purpose of this research was to characterize theEcBcsG enzyme, a putative pEtN transferase, by resolving its structure, understanding its role in pEtN substitution of the cellulose matrix in E. coli, and by elucidating its catalytic mechanism to enable future efforts in drug discovery. All these research objectives were achieved, shedding light on the biochemical basis of pEtN cellulose in E. coli. The de novo X-ray crystal structure of the C-terminal catalytic domain of EcBcsG was solved using the single-wavelength anomalous diffraction (SAD) technique phased on L-selenomethionine substituted EcBcsG crystals and revealed EcBcsG folds as a zinc-dependent phosphotransferase belonging to the pEtN transferase family. TheEcBcsG active site was mapped using functional complementation, revealing EcBcsG shares a partially conserved active site with other known pEtN transferase family members, including enzymes responsible for resistance to cationic antimicrobial peptides (CAMPs). Using mock cosubstrates para-nitrophenyl phosphoethanolamine (p-NPPE) and cellooligosaccharides, the specific activity of EcBcsG was measured to be 11.45 +/- 0.51 nmol min-1mg-1in the presence of the cellulo-pentasaccharide. The kinetic parameters were measured as Km= 1.673 ±0.382 mM, kcat= 6.876 x 10-3± 4.14 x 10-4s-1, and kcat/Km= 4.110±0.970 M s-1. The enzymatic product of this reaction was identified and confirmed in vitro. Finally, the covalent phospho-enzyme intermediate was isolated, providing evidence for an EcBcsG catalytic mechanism. The structural and functional model of the cellulose modifying complex, resulting from this work, provides new insight into the biochemical basis for the biofilm matrix of E. coli and other Enterobacteriaceae.The research presented here offers opportunities in structure-based drug discovery and other efforts in inhibiting microbial biofilms and including the possibility of limiting the resilience of biofilm-forming pathogens. Additionally, further understanding of bcsEFG-directed phosphoethanolamine cellulose production may enable biosynthetic engineering of new cellulosic materials or confer the advantages of phosphoethanolamine cellulose in new organisms

Structural Investigation of BcsC: Insight into Periplasmic Transport During Cellulose Export

A biofilm can be defined by a community of microbes coexisting within a self-produced protective polymeric matrix. Exopolysaccharide (EPS) is a key component in biofilms and a contributor to their virulence and pathogenicity. The cellulose bacterial synthesis complex is one such EPS system that is found in many Enterobacteriaceae,including Escherichia coli and Salmonella spp., and is responsible for the production and secretion of the EPS cellulose. BcsC is the periplasmic protein responsible for the export of the exopolysaccharide cellulose and was the focus of this research. Sequence homology comparisons and structural predictions between BcsC, and the previously characterized alginate export proteins AlgK and AlgE indicate similar roles in facilitating the translocation of EPS across the bacterial cell wall. However, there are discrepancies between the systems, such as the purpose of several additional tetratricopeptide regions (TPRs) contained within BcsC compared to AlgK. To better understand the role that BcsC plays in cellulose export structural characterization of this protein was pursued. Six protein constructs that together cover overlapping portions of BcsCs TPR region were successfully expressed and purified, four of which were further analyzed with SAXS and screened for crystal formation. SAXS data was merged with a pre-existing protein data bank file of BcsCTPR 1-6 to identify similar regions and provided conceptual renderings as to the orientation and size of the protein. Promising crystal hits from BcsCTPR 12-21and BcsCTPR 1-15 were obtained, optimized and sent for X-ray diffraction, with resolution results between 12 and 2.8 Å. A complete dataset for BcsCTPR 1-15 has since been collected and structure solution is ongoing through a combination of molecular replacement and selenomethionine (SeMet) labelling techniques. Preliminary SeMet crystals are promising, but currently appear thinner than native crystals and additional optimization may be required before suitable X-ray diffraction data can be obtained

Identification of a new family of enzymes with potential O-acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria

BACKGROUND: The metabolism of the rigid bacterial cell wall heteropolymer peptidoglycan is a dynamic process requiring continuous biosynthesis and maintenance involving the coordination of both lytic and synthetic enzymes. The O-acetylation of peptidoglycan has been proposed to provide one level of control on these activities as this modification inhibits the action of the major endogenous lytic enzymes, the lytic transglycosylases. The O-acetylation of peptidoglycan also inhibits the activity of the lysozymes which serve as the first line of defense of host cells against the invasion of bacterial pathogens. Despite this central importance, there is a dearth of information regarding peptidoglycan O-acetylation and nothing has previously been reported on its de-acetylation. RESULTS: Homology searches of the genome databases have permitted this first report on the identification of a potential family of O-Acetylpeptidoglycan esterases (Ape). These proteins encoded in the genomes of a variety of both Gram-negative and Gram-positive bacteria, including a number of important human pathogens such as species of Neisseria, Helicobacter, Campylobacter, and Bacillus anthracis, have been organized into three families based on amino acid sequence similarities with family 1 being further divided into three sub-families. The genes encoding these proteins are shown to be clustered with Peptidoglycan O-acetyltransferases (Pat) and in some cases, together with other genes involved in cell wall metabolism. Representative bacteria that encode the Ape proteins were experimentally shown to produce O-acetylated peptidoglycan. CONCLUSION: The hypothetical proteins encoded by the pat and ape genes have been organized into families based on sequence similarities. The Pat proteins have sequence similarity to Pseudomonas aeruginosa AlgI, an integral membrane protein known to participate in the O-acetylation of the exopolysaccaride, alginate. As none of the bacteria that harbor the pat genes produce alginate, we propose that the Pat proteins serve to O-acetylate peptidoglycan which is known to be a maturation event occurring in the periplasm. The Ape sequences have amino acid sequence similarity to the CAZy CE 3 carbohydrate esterases, a family previously known to be composed of only O-acetylxylan esterases. They are predicted to contain the α/β hydrolase fold associated with the GDSL and TesA hydrolases and they possess the signature motifs associated with the catalytic residues of the CE3 esterases. Specific signature sequence motifs were identified for the Ape proteins which led to their organization into distinct families. We propose that by expressing both Pat and Ape enzymes, bacteria would be able to obtain a high level of localized control over the degradation of peptidoglycan through the attachment and removal of O-linked acetate. This would facilitate the efficient insertion of pores and flagella, localize spore formation, and control the level of general peptidoglycan turnover

Expression, Purification, Crystallization and Preliminary X-Ray Analysis of \u3cem\u3ePseudomonas aeuginosa\u3c/em\u3e AlgX

AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a=46.4, b=120.6, c=86.9Å, β=95.7°. The cyrstals exhibited the symmetry of space group P21 and diffracted to a minimimum d-spacing of 2.1Å. On the basis of the Matthews coefficient (VM=2.25Å3 Da-1), two molecules were estimated to be present in the asymmetric unit

Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation

P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation

<i>Pseudomonas aeruginosa</i> AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well-known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro, and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.</p

Structural Investigation of BcsC: Insight into Periplasmic Transport During Cellulose Export

A biofilm can be defined by a community of microbes coexisting within a self-produced protective polymeric matrix. Exopolysaccharide (EPS) is a key component in biofilms and a contributor to their virulence and pathogenicity. The cellulose bacterial synthesis complex is one such EPS system that is found in many Enterobacteriaceae,including Escherichia coli and Salmonella spp., and is responsible for the production and secretion of the EPS cellulose. BcsC is the periplasmic protein responsible for the export of the exopolysaccharide cellulose and was the focus of this research. Sequence homology comparisons and structural predictions between BcsC, and the previously characterized alginate export proteins AlgK and AlgE indicate similar roles in facilitating the translocation of EPS across the bacterial cell wall. However, there are discrepancies between the systems, such as the purpose of several additional tetratricopeptide regions (TPRs) contained within BcsC compared to AlgK. To better understand the role that BcsC plays in cellulose export structural characterization of this protein was pursued. Six protein constructs that together cover overlapping portions of BcsCs TPR region were successfully expressed and purified, four of which were further analyzed with SAXS and screened for crystal formation. SAXS data was merged with a pre-existing protein data bank file of BcsCTPR 1-6 to identify similar regions and provided conceptual renderings as to the orientation and size of the protein. Promising crystal hits from BcsCTPR 12-21and BcsCTPR 1-15 were obtained, optimized and sent for X-ray diffraction, with resolution results between 12 and 2.8 Å. A complete dataset for BcsCTPR 1-15 has since been collected and structure solution is ongoing through a combination of molecular replacement and selenomethionine (SeMet) labelling techniques. Preliminary SeMet crystals are promising, but currently appear thinner than native crystals and additional optimization may be required before suitable X-ray diffraction data can be obtained

Characterizing the cellulose-modifying enzyme BcsG from Escherichia coli

Microbial biofilms are communities of microorganisms that exhibit co-operative behaviour, producing a matrix of exopolysaccharide that enmeshes the community. The well-studied human pathogens Escherichia coli and Salmonella entericaproduce a biofilm matrix comprised chiefly of the biopolymer cellulose, along with amyloid protein fibers termed curli. This biofilm matrix confers surface adherence and acts as a protective barrier to disinfectants, antimicrobials, environmental stressors, and host immune responses. Pertaining to this research, the bcsEFG operon, conserved in the Enterobacteriaceae, encodes an inner membrane-spanning complex responsible for the addition of a phosphoethanolamine (pEtN) modification to microbial cellulose, essential for extracellular matrix assembly and biofilm architecture. Furthermore, E. coli deficient in bcsGproduce a biofilm matrix lacking structural integrity and the self-assembling architecture observed in wild-type E. coli. The presence of a pEtN-substituted cellulose matrix was shown to be important in bladder epithelial colonization by uropathogenic E. coli, further suggesting its role as a virulence factor in etiological agents of urinary tract infections observed in the clinic. The purpose of this research was to characterize theEcBcsG enzyme, a putative pEtN transferase, by resolving its structure, understanding its role in pEtN substitution of the cellulose matrix in E. coli, and by elucidating its catalytic mechanism to enable future efforts in drug discovery. All these research objectives were achieved, shedding light on the biochemical basis of pEtN cellulose in E. coli. The de novo X-ray crystal structure of the C-terminal catalytic domain of EcBcsG was solved using the single-wavelength anomalous diffraction (SAD) technique phased on L-selenomethionine substituted EcBcsG crystals and revealed EcBcsG folds as a zinc-dependent phosphotransferase belonging to the pEtN transferase family. TheEcBcsG active site was mapped using functional complementation, revealing EcBcsG shares a partially conserved active site with other known pEtN transferase family members, including enzymes responsible for resistance to cationic antimicrobial peptides (CAMPs). Using mock cosubstrates para-nitrophenyl phosphoethanolamine (p-NPPE) and cellooligosaccharides, the specific activity of EcBcsG was measured to be 11.45 +/- 0.51 nmol min-1mg-1in the presence of the cellulo-pentasaccharide. The kinetic parameters were measured as Km= 1.673 ±0.382 mM, kcat= 6.876 x 10-3± 4.14 x 10-4s-1, and kcat/Km= 4.110±0.970 M s-1. The enzymatic product of this reaction was identified and confirmed in vitro. Finally, the covalent phospho-enzyme intermediate was isolated, providing evidence for an EcBcsG catalytic mechanism. The structural and functional model of the cellulose modifying complex, resulting from this work, provides new insight into the biochemical basis for the biofilm matrix of E. coli and other Enterobacteriaceae.The research presented here offers opportunities in structure-based drug discovery and other efforts in inhibiting microbial biofilms and including the possibility of limiting the resilience of biofilm-forming pathogens. Additionally, further understanding of bcsEFG-directed phosphoethanolamine cellulose production may enable biosynthetic engineering of new cellulosic materials or confer the advantages of phosphoethanolamine cellulose in new organisms