Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004.

The titer of the microbial fermentation products can be increased by enzyme engineering. l-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-l-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H2O2. This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.

Recent Advances in 2-Keto-l-gulonic Acid Production Using Mixed-Culture Fermentation and Future Prospects.

Vitamin C, also known as ascorbic acid, is an essential vitamin that cannot be synthesized by the human body and must be acquired through our diet. At present, the precursor of vitamin C, 2-keto-l-gulonic acid (2-KGA), is typically produced via a two-step fermentation process utilizing three bacterial strains. The second step of this traditional two-step fermentation method involves mixed-culture fermentation employing 2-KGA-producing bacteria (Ketogulonicigenium vulgare) along with associated bacteria. Because K. vulgare has defects in various metabolic pathways, associated bacteria are needed to provide key substances to promote K. vulgare growth and 2-KGA production. Unlike previous reviews where the main focus was the interaction between associated bacteria and K. vulgare, this Review presents the latest scientific research from the perspective of the metabolic pathways associated with 2-KGA production by K. vulgare and the mechanism underlying the interaction between K. vulgare and the associated bacteria. In addition, the dehydrogenases that are responsible for 2-KGA production, the 2-KGA synthesis pathway, strategies for simplifying 2-KGA production via a one-step fermentation route, and, finally, future prospects and research goals in vitamin C production are also presented.

Efficient production of 2-keto-L-gulonic acid from D-glucose in Gluconobacter oxydans ATCC9937 by mining key enzyme and transporter.

Direct production of 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) from D-glucose through 2,5-diketo-D-gluconic acid (2,5-DKG) is a promising alternative route. To explore the pathway of producing 2-KLG from D-glucose, Gluconobacter oxydans ATCC9937 was selected as a chassis strain. It was found that the chassis strain naturally has the ability to synthesize 2-KLG from D-glucose, and a new 2,5-DKG reductase (DKGR) was found on its genome. Several major issues limiting production were identified, including the insufficient catalytic capacity of DKGR, poor transmembrane movement of 2,5-DKG and imbalanced D-glucose consumption flux inside and outside of the host strain cells. By identifying novel DKGR and 2,5-DKG transporter, the whole 2-KLG biosynthesis pathway was systematically enhanced by balancing intracellular and extracellular D-glucose metabolic flux. The engineered strain produced 30.5 g/L 2-KLG with a conversion ratio of 39.0%. The results pave the way for a more economical large-scale fermentation process for vitamin C.

Allylpolyalkoxybenzene Inhibitors of Galactonolactone Oxidase from Trypanosoma cruzi.

Inhibition of biosynthetic pathways of compounds essential for Trypanosoma cruzi is considered as one of the possible action mechanisms of drugs against Chagas disease. Here, we investigated the inhibition of galactonolactone oxidase from T. cruzi (TcGAL), which catalyzes the final step in the synthesis of vitamin C, an antioxidant that T. cruzi is unable to assimilate from outside and must synthesize itself, and identified allylbenzenes from plant sources as a new class of TcGAL inhibitors. Natural APABs (apiol, dillapiol, etc.) inhibited TcGAL with IC50 = 20-130 µM. The non-competitive mechanism of TcGAL inhibition by apiol was established. Conjugation of APABs with triphenylphosphonium, which ensures selective delivery of biologically active substances to the mitochondria, increased the efficiency and/or the maximum percentage of TcGAL inhibition compared to nonmodified APABs.

Mechanism and linkage specificities of the dual retaining ß-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis.

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2→4) and ß-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L.

Identification and characterization of a deaminoneuraminic acid (Kdn)-specific aldolase from Sphingobacterium species.

Sialic acid (Sia) is a group of acidic sugars with a 9-carbon backbone, and classified into 3 species based on the substituent group at C5 position: N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). In Escherichia coli, the sialate aldolase or N-acetylneuraminate aldolase (NanA) is known to catabolize these Sia species into pyruvate and the corresponding 6-carbon mannose derivatives. However, in bacteria, very little is known about the catabolism of Kdn, compared with Neu5Ac. In this study, we found a novel Kdn-specific aldolase (Kdn-aldolase), which can exclusively degrade Kdn, but not Neu5Ac or Neu5Gc, from Sphingobacterium sp., which was previously isolated from a Kdn-assimilating bacterium. Kdn-aldolase had the optimal pH and temperature at 7.0-8.0 and 50 °C, respectively. It also had the synthetic activity of Kdn from pyruvate and mannose. Site-specific mutagenesis revealed that N50 residue was important for the Kdn-specific reaction. Existence of the Kdn-aldolase suggests that Kdn-specific metabolism may play a specialized role in some bacteria.

Structural analysis of the pseudaminic acid synthase PseI from Campylobacter jejuni.

Campylobacter jejuni PseI is a pseudaminic acid synthase that condenses the 2,4-diacetamido-2,4,6-trideoxy-l-altrose sugar (6-deoxy AltdiNAc) and phosphoenolpyruvate to generate pseudaminic acid, a sialic acid-like 9-carbon backbone α-keto sugar. Pseudaminic acid is conjugated to cell surface proteins and lipids and plays a key role in the mobility and virulence of C. jejuni and other pathogenic bacteria. To provide insights into the catalytic mechanism of PseI, we performed a structural study on PseI. PseI forms a two-domain structure and assembles into a domain-swapped homodimer. The PseI dimer has two cavities, each of which accommodates a metal ion using conserved histidine residues. A comparative analysis of structures and sequences suggests that the cavity of PseI functions as an active site that binds the 6-deoxy AltdiNAc and phosphoenolpyruvate substrates and mediates their condensation. Furthermore, we propose the substrate binding-induced structural rearrangement of PseI and predict 6-deoxy AltdiNAc recognition residues that are specific to PseI.

Metabolic Incorporation of Azido-Sugars into LPS to Enable Live-Cell Fluorescence Imaging.

Metabolic labeling of lipopolysaccharides (LPS) with the exogenous azido analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) or Kdo-N3 allows for both live-cell and molecular analysis of the outer membrane composition and biosynthesis in different Gram-negative bacteria. Here, we describe Kdo-N3 incorporation into bacterial cells, followed by click labeling with a fluorescent dye. The fluorescently labeled LPS can be analyzed from lysed cells by SDS-PAGE and from intact cells by microscopy and flow cytometry. These methods have been applied to the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, which possess the sialic acid transporter NanT that is also capable of transporting exogenous Kdo and Kdo analogs into the cytoplasm for incorporation into nascent LPS.

Combined evolutionary and metabolic engineering improve 2-keto-L-gulonic acid production in Gluconobacter oxydans WSH-004.

The direct fermentation of the precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG), has been a long-pursued goal. Previously, a strain of Gluconobacter oxydans WSH-004 was isolated that produced 2.5 g/L 2-KLG, and through adaptive evolution engineering, the strain G. oxydans MMC3 could tolerate 300 g/L D-sorbitol. This study verified that the sndh-sdh gene cluster encoded two key dehydrogenases for the 2-KLG biosynthesis pathway in this strain. Then G. oxydans MMC3 further evolved through adaptive evolution to G. oxydans 2-KLG5, which can tolerate high concentrations of D-sorbitol and 2-KLG. Finally, by increasing the gene expression levels of the sndh-sdh and terminal oxidase cyoBACD in G. oxydans 2-KLG5, the 2-KLG accumulation in the 5-L fermenter increased to 45.14 g/L by batch fermentation. The results showed that combined evolutionary and metabolic engineering efficiently improved the direct production of 2-KLG from D-sorbitol in G. oxydans.

[Production of mucic acid from pectin-derived D-galacturonic acid: a review].

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.

Production of mucic acid from pectin-derived D-galacturonic acid: a review / 生物工程学报

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.

Evaluation of the Binding Mechanism of Human Defensin 5 in a Bacterial Membrane: A Simulation Study.

Human α-defensin 5 (HD5) is a host-defense peptide exhibiting broad-spectrum antimicrobial activity. The lipopolysaccharide (LPS) layer on the Gram-negative bacterial membrane acts as a barrier to HD5 insertion. Therefore, the pore formation and binding mechanism remain unclear. Here, the binding mechanisms at five positions along the bacterial membrane axis were investigated using Molecular Dynamics. (MD) simulations. We found that HD5 initially placed at positions 1 to 3 moved up to the surface, while HD5 positioned at 4 and 5 remained within the membrane interacting with the middle and inner leaflet of the membrane, respectively. The arginines were key components for tighter binding with 3-deoxy-d-manno-octulosonic acid (KDO), phosphates of the outer and inner leaflets. KDO appeared to retard the HD5 penetration.

The d-mannose/l-galactose pathway is the dominant ascorbate biosynthetic route in the moss Physcomitrium patens.

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d-mannose/l-galactose (d-Man/l-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d-galacturonate (d-GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d-Man/l-Gal pathway and an orthologous gene encoding aldonolactonase (ALase) - a key enzyme for the d-GalUA pathway - Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l-galactonic acid to the final precursor l-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l-Gal phosphorylase in the d-Man/l-Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d-Man/l-Gal pathway, not the d-GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.

Large-scale plasma-metabolome analysis identifies potential biomarkers of psoriasis and its clinical subtypes.

BACKGROUND: Psoriasis is an immune-mediated skin disease for which the crosstalk between genetic and environmental factors is responsible. To date, no definitive diagnostic criteria for psoriasis yet, and specific biomarkers are required. OBJECTIVE: We performed metabolome analysis to identify metabolite biomarkers of psoriasis and its subtypes such as psoriatic arthritis (PsA) and cutaneous psoriasis (PsC). METHODS: We constructed metabolomics profiling of 130 plasma samples (42 PsA patients, 50 PsC patients, and 38 healthy controls) using a nontargeted metabolomics approach. RESULTS: Psoriasis-control association tests showed that one metabolite (ethanolamine phosphate) was significantly increased in psoriasis samples than in the controls, whereas three metabolites decreased (false discovery rate [FDR] < 0.05; XA0019, nicotinic acid, and 20α-hydroxyprogesterone). In the association test between PsA and PsC, tyramine significantly increased in PsA than in PsC, whereas mucic acid decreased (FDR < 0.05). Molecular pathway analysis of the PsA-PsC association test identified enrichment of vitamin digestion and absorption pathway in PsC (P = 1.3 × 10-4). Correlation network analyses elucidated that a subnetwork centered on aspartate was constructed among the psoriasis-associated metabolites; meanwhile, the major subnetwork among metabolites with differences between PsA and PsC was primarily formed from saturated fatty acids. CONCLUSION: Our large-scale metabolome analysis highlights novel characteristics of plasma metabolites in psoriasis and the differences between PsA and PsC, which could be used as potential biomarkers of psoriasis and its clinical subtypes. These findings contribute to our understanding of psoriasis pathophysiology.

Bombyx mori can synthesize ascorbic acid through the l-gulose pathway to varying degrees depending on developmental stage.

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Crystal structure of the sugar acid-binding protein CxaP from a TRAP transporter in Advenella mimigardefordensis strain DPN7T.

Recently, CxaP, a sugar acid substrate binding protein (SBP) from Advenella mimigardefordensis strain DPN7T , was identified as part of a novel sugar uptake strategy. In the present study, the protein was successfully crystallized. Although several SBP structures of tripartite ATP-independent periplasmic transporters have already been solved, this is the first structure of an SBP accepting multiple sugar acid ligands. Protein crystals were obtained with bound d-xylonic acid, d-fuconic acid d-galactonic and d-gluconic acid, respectively. The protein shows the typical structure of an SBP of a tripartite ATP-independent periplasmic transporter consisting of two domains linked by a hinge and spanned by a long α-helix. By analysis of the structure, the substrate binding site of the protein was identified. The carboxylic group of the sugar acids interacts with Arg175, whereas the coordination of the hydroxylic groups at positions C2 and C3 is most probably realized by Arg154 and Asn151. Furthermore, it was observed that 2-keto-3-deoxy-d-gluconic acid is bound in protein crystals that were crystallized without the addition of any ligand, indicating that this molecule is prebound to the protein and is displaced by the other ligands if they are available. DATABASE: Structural data of CxaP complexes are available in the worldwide Protein Data Bank (https://www.rcsb.org) under the accession codes 7BBR (2-keto-3-deoxy-d-gluconic acid), 7BCR (d-galactonic acid), 7BCN (d-xylonic acid), 7BCO (d-fuconic acid) and 7BCP (d-gluconic acid).

Molecular insights into effector binding by DgoR, a GntR/FadR family transcriptional repressor of D-galactonate metabolism in Escherichia coli.

Several GntR/FadR transcriptional regulators govern sugar acid metabolism in bacteria. Although effectors have been identified for a few sugar acid regulators, the mode of effector binding is unknown. Even in the overall FadR subfamily, there are limited details on effector-regulator interactions. Here, we identified the effector-binding cavity in Escherichia coli DgoR, a FadR subfamily transcriptional repressor of D-galactonate metabolism that employs D-galactonate as its effector. Using a genetic screen, we isolated several dgoR superrepressor alleles. Blind docking suggested eight amino acids corresponding to these alleles to form a part of the effector-binding cavity. In vivo and in vitro assays showed that these mutations compromise the inducibility of DgoR without affecting its oligomeric status or affinity for target DNA. Taking Bacillus subtilis GntR as a representative, we demonstrated that the effector-binding cavity is similar among FadR subfamily sugar acid regulators. Finally, a comparison of sugar acid regulators with other FadR members suggested conserved features of effector-regulator recognition within the FadR subfamily. Sugar acid metabolism is widely implicated in bacterial colonization and virulence. The present study sets the basis to investigate the influence of natural genetic variations in FadR subfamily regulators on their sensitivity to sugar acids and ultimately on host-bacterial interactions.

Structural and Biosynthetic Diversity of Nonulosonic Acids (NulOs) That Decorate Surface Structures in Bacteria.

Nonulosonic acids (NulOs) are a diverse family of 9-carbon α-keto acid sugars that are involved in a wide range of functions across all branches of life. The family of NulOs includes the sialic acids as well as the prokaryote-specific NulOs. Select bacteria biosynthesize the sialic acid N-acetylneuraminic acid (Neu5Ac), and the ability to produce this sugar and its subsequent incorporation into cell-surface structures is implicated in a variety of bacteria-host interactions. Furthermore, scavenging of sialic acid from the environment for energy has been characterized across a diverse group of bacteria, mainly human commensals and pathogens. In addition to sialic acid, bacteria have the ability to biosynthesize prokaryote-specific NulOs, of which there are several known isomers characterized. These prokaryotic NulOs are similar in structure to Neu5Ac but little is known regarding their role in bacterial physiology. Here, we discuss the diversity in structure, the biosynthesis pathways, and the functions of bacteria-specific NulOs. These carbohydrates are phylogenetically widespread among bacteria, with numerous structurally unique modifications recognized. Despite the diversity in structure, the NulOs are involved in similar functions such as motility, biofilm formation, host colonization, and immune evasion.