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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

The exo-β- N -acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

Endo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the β-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-β-N-acetylmuramidases, which catalyze an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

One fold, many functions—M23 family of peptidoglycan hydrolases

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Razew, Alicja↗
DOE OSTI2021

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A

Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (V H 3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific V H 3-IgM via a mechanism requiring CD4 + T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun’s lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Finally, sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural basis of peptidoglycan endopeptidase regulation

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past

Abstract Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The NtrYX Two-Component System Regulates the Bacterial Cell Envelope

ABSTRACT Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides , loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales

ABSTRACT The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria . Using Rhodobacter sphaeroides , we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides ’ divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆ RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria . Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C . difficile , MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn 2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn 2+ , which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn 2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Molecular Dynamics Simulation of Atomic Interactions in the Vancomycin Binding Site

Vancomycin is a glycopeptide antibiotic produced by Amycolaptopsis orientalis used to treat serious infections by Grampositive pathogens including methicillin-resistant Staphylococcus aureus. Vancomycin inhibits cell wall biosynthesis by targeting lipid II, which is the membrane-bound peptidoglycan precursor. The heptapeptide aglycon structure of vancomycin binds to the D-Ala-D-Ala of the pentapeptide stem structure in lipid II. The third residue of vancomycin aglycon is asparagine, which is not directly involved in the dipeptide binding. Nonetheless, asparagine plays a crucial role in substrate recognition, as the vancomycin analogue with asparagine substituted by aspartic acid (V D ) shows a reduction in antibacterial activities. To characterize the function of asparagine, binding of vancomycin and its aspartic-acid-substituted analogue V D to L-Lys-D-Ala-D-Ala and L-Lys-D-Ala-D-Lac was investigated using molecular dynamic simulations. Binding interactions were analyzed using root-mean-square deviation (RMSD), two-dimensional (2D) contour plots, hydrogen bond analysis, and free energy calculations of the complexes. The analysis shows that the aspartate substitution introduced a negative charge to the binding cleft of V D , which altered the aglycon conformation that minimized the repulsive lone pair interaction in the binding of a depsipeptide. Our findings provide new insight for the development of novel glycopeptide antibiotics against the emerging vancomycin-resistant pathogens by chemical modification at the third residue in vancomycin to improve its binding affinity to the D-Ala-D-Lac-terminated peptidoglycan in lipid II found in vancomycin-resistant enterococci and vancomycinresistant S. aureus.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB

In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1998

Phylogenetic mapping of bacterial morphology

The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

NASA Discipline Exobiology↗