Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality.

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MPG2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting RG-II CMP-ß-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

Characterization of Ssc, an N-acetylgalactosamine-containing Staphylococcus aureus surface polysaccharide.

Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N-acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCE: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.

Permethylation as a strategy for high-molecular-weight polysaccharide structure analysis by nuclear magnetic resonance-Case study of Xylella fastidiosa extracellular polysaccharide.

Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Acetylation in Ionic Liquids Dramatically Increases Yield in the Glycosyl Composition and Linkage Analysis of Insoluble and Acidic Polysaccharides.

Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo ß-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal ß-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.

Structural characterization and biological activity of an α-glucan from the mollusk Marcia hiantina (Lamarck, 1818).

Marcia hiantina (Mollusca, Bivalvia) (Lamarck, 1818), is an edible clam mainly distributed along the tropical coastal regions. Recent researches have demonstrated that clams can possess compounds, including polysaccharides, with a wide range of biological actions including antioxidant, immunomodulatory and antitumor activities. Here an α-glucan was isolated from M. hiantina by hot water, purified by anion exchange chromatography, and its structure was characterized by a combination of multiple nuclear magnetic resonance (NMR) methods (1D 1H, 1H-1H COSY, 1H-1H TOCSY, 1H-1H NOESY, 1H-13C HSQC and 1H-13C HSQC-NOESY spectra), gas chromatography-mass spectrometry, and high performance size exclusion chromatography (HPSEC). The analysis from NMR, monosaccharide composition, methylation analyses and HPSEC combined with multi-angle light scattering (MALS) of M. hiantina-derived α-glycan confirmed a branched polysaccharide exclusively composed of glucose (Glc), mostly 4-linked in its backbone, branched occasionally at 6-positions, and having a molecular weight of ~ 570 kDa. The mollusk α-glucan was subjected to four cell-based assays: (i) viability of three cell lines (RAW264.7, HaCaT, and HT-29), (ii) activity on lipopolysaccharide (LPS)-induced prostaglandin production in RAW264.7 cells, (iii) inhibitory activities of in H2O2- and LPS-induced reactive oxygen species (ROS) production in HMC3 cells, and (iv) HaCaT cell proliferation. Results have indicated no cytotoxicity, potent inhibition of both H2O2- and LPS-induced ROS, and potent cell proliferative activity.

'Candidatus Liberibacter asiaticus'-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology.

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a ß-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

Evaluating the Utility of Permethylated Polysaccharide Solution NMR Data for Characterization of Insoluble Plant Cell Wall Polysaccharides.

Plant cell wall polysaccharide analysis encompasses the utilization of a variety of analytical tools, including gas and liquid chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. These methods provide complementary data, which enable confident structural proposals of the many complex polysaccharide structures that exist in the complex matrices of plant cell walls. However, cell walls contain fractions of varying solubilities, and a few techniques are available that can analyze all fractions simultaneously. We have discovered that permethylation affords the complete dissolution of both soluble and insoluble polysaccharide fractions of plant cell walls in organic solvents such as chloroform or acetonitrile, which can then be analyzed by a number of analytical techniques including MS and NMR. In this work, NMR structure analysis of 10 permethylated polysaccharide standards was undertaken to generate chemical shift data providing insights into spectral changes that result from permethylation of polysaccharide residues. This information is of especial relevance to the structure analysis of insoluble polysaccharide materials that otherwise are not easily investigated by solution-state NMR methodologies. The preassigned NMR chemical shift data is shown to be vital for NMR structure analysis of minor polysaccharide components of plant cell walls that are particularly difficult to assign by NMR correlation data alone. With the assigned chemical shift data, we analyzed the permethylated samples of destarched, alcohol-insoluble residues of switchgrass and poplar by two-dimensional NMR spectral profiling. Thus, we identified, in addition to the major polysaccharide components, two minor polysaccharides, namely, <5% 3-linked arabinoxylan (switchgrass) and <2% glucomannan (poplar). In particular, the position of the arabinose residue in the arabinoxylan of the switchgrass sample was confidently assigned based on chemical shift values, which are highly sensitive to local chemical environments. Furthermore, the high resolution afforded by the 1H NMR spectra of the permethylated switchgrass and poplar samples allowed facile relative quantitative analysis of their polysaccharide composition, utilizing only a few milligrams of the cell wall material. The concepts herein developed will thus facilitate NMR structure analysis of insoluble plant cell wall polysaccharides, more so of minor cell wall components that are especially challenging to analyze with current methods.

Composition of the Holdfast Polysaccharide from Caulobacter crescentus.

Surface colonization is central to the lifestyles of many bacteria. Exploiting surface niches requires sophisticated systems for sensing and attaching to solid materials. Caulobacter crescentus synthesizes a polysaccharide-based adhesin known as the holdfast at one of its cell poles, which enables tight attachment to exogenous surfaces. The genes required for holdfast biosynthesis have been analyzed in detail, but difficulties in isolating analytical quantities of the adhesin have limited efforts to characterize its chemical structure. In this report, we describe a method to extract the holdfast from C. crescentus cultures and present a survey of its carbohydrate content. Glucose, 3-O-methylglucose, mannose, N-acetylglucosamine, and xylose were detected in our extracts. Our results provide evidence that the holdfast contains a 1,4-linked backbone of glucose, mannose, N-acetylglucosamine, and xylose that is decorated with branches at the C-6 positions of glucose and mannose. By defining the monosaccharide components in the polysaccharide, our work establishes a framework for characterizing enzymes in the holdfast pathway and provides a broader understanding of how polysaccharide adhesins are built.IMPORTANCE To colonize solid substrates, bacteria often deploy dedicated adhesins that facilitate attachment to surfaces. Caulobacter crescentus initiates surface colonization by secreting a carbohydrate-based adhesin called the holdfast. Because little is known about the chemical makeup of the holdfast, the pathway for its biosynthesis and the physical basis for its unique adhesive properties are poorly understood. This study outlines a method to extract the C. crescentus holdfast and describes the monosaccharide components contained within the adhesive matrix. The composition analysis adds to our understanding of the chemical basis for holdfast attachment and provides missing information needed to characterize enzymes in the biosynthetic pathway.

Comprehensive Monosaccharide Composition Analysis of Insoluble Polysaccharides by Permethylation To Produce Methyl Alditol Derivatives for Gas Chromatography/Mass Spectrometry.

Knowledge of the monosaccharide composition of plant and microbial cell wall polysaccharides is critical for the understanding of polysaccharide structure and function. Differences in the hydrolytic stability of the glycosidic bonds and in the susceptibility of monosaccharides to acid-catalyzed degradation cause inconsistency of signal response in the common glycosyl composition methods. In addition, many polysaccharides are insoluble, partially soluble, or form highly viscous gels in water, and this also hinders or even prevents detection by traditional methods. As a result, currently available methods for monosaccharide composition analysis lack accuracy and are limited to the soluble portions of biological samples or expose the polysaccharides to very harsh conditions, resulting in loss of less stable residues. Here we present a new approach to accomplish the monosaccharide composition analysis of polysaccharides, including those that are not or sparingly soluble, based on permethylation in DMSO as the initial derivatization step. Our key finding is that the permethylation solubilizes the polysaccharide before the hydrolysis step, so that differences in solubility are no longer a factor in the efficiency of the acid-catalyzed depolymerization. After the hydrolysis, the partially methylated monosaccharides are reduced to alditols and remethylated for GC/MS analysis. In addition to enabling composition analysis of insoluble polysaccharides, this method also has the advantages that it is comprehensive, allowing quantification of all types of sugars, including uronic acids, on the same column and gives consistent response factors for different monosaccharide classes.

Lipopolysaccharides Trigger Two Successive Bursts of Reactive Oxygen Species at Distinct Cellular Locations.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are an important microbe-associated molecular pattern (MAMP) that triggers immune responses in plants and animals. A previous genetic screen in Arabidopsis (Arabidopsis thaliana) identified LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE), a B-type lectin S-domain receptor kinase, as a sensor of LPS. However, the LPS-activated LORE signaling pathway and associated immune responses remain largely unknown. In this study, we found that LPS trigger biphasic production of reactive oxygen species (ROS) in Arabidopsis. The first transient ROS burst was similar to that induced by another MAMP, flagellin, whereas the second long-lasting burst was induced only by LPS. The LPS-triggered second ROS burst was found to be conserved in a variety of plant species. Microscopic observation of the generation of ROS revealed that the LPS-triggered second ROS burst was largely associated with chloroplasts, and functional chloroplasts were indispensable for this response. The lipid A moiety, the most conserved portion of LPS, appears to be responsible for the second ROS burst. Surprisingly, the LPS- and lipid A-triggered second ROS burst was only partially dependent on LORE. Together, our findings provide insight on the LPS-triggered ROS production and the associated signaling pathway.

Operating Room Fires.

Operating room fires are rare but devastating events. Guidelines are available for the prevention and management of surgical fires; however, these recommendations are based on expert opinion and case series. The three components of an operating room fire are present in virtually all surgical procedures: an oxidizer (oxygen, nitrous oxide), an ignition source (i.e., laser, "Bovie"), and a fuel. This review analyzes each fire ingredient to determine the optimal clinical strategy to reduce the risk of fire. Surgical checklists, team training, and the specific management of an operating room fire are also reviewed.

Arabinoxyloglucan Oligosaccharides May Contribute to the Antiadhesive Properties of Porcine Urine after Cranberry Consumption.

Cranberry ( Vaccinium macrocarpon) juice is traditionally used for the prevention of urinary tract infections. Human urine produced after cranberry juice consumption can prevent Escherichia coli adhesion, but the antiadhesive urinary metabolites responsible have not been conclusively identified. Adult female sows were therefore fed spray-dried cranberry powder (5 g/kg/day), and urine was collected via catheter. Urine fractions were tested for antiadhesion activity using a human red blood cell (A+) anti-hemagglutination assay with uropathogenic P-fimbriated E. coli. Components were isolated from fractions of interest using Sephadex LH-20 gel filtration chromatography followed by HPLC on normal and reversed-phase sorbents with evaporative light scattering detection. Active urine fractions were found to contain a complex series of oligosaccharides but not proanthocyanidins, and a single representative arabinoxyloglucan octasaccharide was isolated in sufficient quantity and purity for full structural characterization by chemical derivatization and NMR spectroscopic methods. Analogous cranberry material contained a similar complex series of arabinoxyloglucan oligosaccharides that exhibited antiadhesion properties in preliminary testing. These results indicate that oligosaccharides structurally related to those found in cranberry may contribute to the antiadhesion properties of urine after cranberry consumption.

Salmonella enterica Serovar Typhi Lipopolysaccharide O-Antigen Modification Impact on Serum Resistance and Antibody Recognition.

Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase (gtr) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr-dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonellagtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines.

Thermoregulatory behavior and orientation preference in bearded dragons.

The regulation of body temperature is a critical function for animals. Although reliant on ambient temperature as a heat source, reptiles, and especially lizards, make use of multiple voluntary and involuntary behaviors to thermoregulate, including postural changes in body orientation, either toward or away from solar sources of heat. This thermal orientation may also result from a thermoregulatory drive to maintain precise control over cranial temperatures or a rostrally-driven sensory bias. The purpose of this work was to examine thermal orientation behavior in adult and neonatal bearded dragons (Pogona vitticeps), to ascertain its prevalence across different life stages within a laboratory situation and its interaction with behavioral thermoregulation. Both adult and neonatal bearded dragons were placed in a thermal gradient and allowed to voluntarily select temperatures for up to 8h to observe the presence and development of a thermoregulatory orientation preference. Both adult and neonatal dragons displayed a non-random orientation, preferring to face toward a heat source while achieving mean thermal preferences of ~ 33-34°C. Specifically, adult dragons were more likely to face a heat source when at cooler ambient temperatures and less likely at warmer temperatures, suggesting that orientation behavior counter-balances local selected temperatures but contributes to their thermoregulatory response. Neonates were also more likely to select cooler temperatures when facing a heat source, but required more experience before this orientation behavior emerged. Combined, these results demonstrate the importance of orientation to behavioral thermoregulation in multiple life stages of bearded dragons.

Operating Room Management at the Edge of Order and Chaos.

The operating room (OR) management literature tends to view management problems as having finite solutions and assumes that equilibrium exists in the intricate encounters that occur every day. In this article, we review complexity theory and assess its applicability to the strategic, tactical, and operational issues facing OR managers. By building on complexity theory and its assumptions, we also show that as complex systems, ORs resemble high-reliability organizations more than they resemble ultra-safe organizations. This distinction and the limitations of the current, linear modeling may have potential implications for the future of OR management research and practice. Opening the door to complexity, understanding the underpinnings of high-reliability organizations, and admitting that OR systems are complex adaptive systems, will lead to self-governing, transparent processes that envision the OR as a living, growing, sustainable human endeavor.