Focus on local food production and supply chains has heightened in recent years, as evidenced and amplified by the COVID-19 pandemic. This study aimed to assess the suitability of soft red winter (SRW) wheat breeding lines for local artisan bakers interested in locally sourced, strong gluten wheat for bread. Seventy-six genotyped SRW wheat breeding lines were milled into whole wheat flour and baked into small loaves. Bread aroma, flavor, and texture were evaluated by a sensory panel, and bread quality traits, including sedimentation volume, dough extensibility, and loaf volume, were measured to estimate heritability. SE-HPLC was performed on white flour, and breeding lines were characterized for different protein fraction ratios. Heritability of loaf volume was moderately high (h2 = 0.68), while heritability of sedimentation volume, a much easier trait to measure, was slightly lower (h2 = 0.55). Certain protein fraction ratios strongly related to loaf volume had high heritability (h2 = 0.7). Even though only a moderate heritability estimate of dough extensibility was found in our study, high positive correlations were found between this parameter and sedimentation volume (r = 0.6) and loaf volume (r = 0.53). This low-input and highly repeatable parameter could be useful to estimate dough functionality characteristics. Flavor and texture heritability estimates ranged from 0.16 to 0.37, and the heritability estimate of aroma was not significantly different from zero. However, the sensorial characteristics were significantly correlated with each other, suggesting that we might be able to select indirectly for aroma by selecting for flavor or texture characteristics. From a genome-wide association study (GWAS), we identified six SNPs (single nucleotide polymorphisms) associated with loaf volume that could be useful in breeding for this trait. Producing high-quality strong gluten flour in our high rainfall environment is a challenge, but it provides local growers and end users with a value-added opportunity.

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well‐defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans‐complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn‐seq (dTn‐seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans‐complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn‐seq to identify genes with secreted functions. We then applied dTn‐seq to identify an AcrAB efflux system as required for growth in metal‐limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition. While yersiniabactin is an essential virulence factor for several pathogens, the mechanism responsible for its secretion has eluded researchers. Droplet Tn‐Seq enabled the identification of an AcrAB efflux system necessary for yersiniabactin secretion. While yersiniabactin is an essential virulence factor for several pathogens, the mechanism responsible for its secretion has eluded researchers. Droplet TnSeq enabled the identification of an AcrAB efflux system necessary for yersiniabactin secretion.

Increasing interest in products made from rye grain underscores the need for developing improved rye (Secale cereale L.) cultivars. In the context of consumer appeal, there are specific questions about the sensory attributes of different rye cultivars. Unfortunately, there are very few studies that focus on the flavor and other sensory attributes of rye products. In order to estimate rye-derived aroma and flavor variability, we evaluated sensory attributes in rye flatbread of forty-seven rye entries, selected from commercial rye cultivars and international accessions that are adapted to our latitude. Additionally, we evaluated distillates from eleven rye cultivars and breeding lines. We found significant differences in flatbread aroma and flavor (P < 0.001) and significant overall distillate aroma differences (P < 0.05) among tested rye entries. In our flatbread screening, heritability of nutty (h2 = 0.37), salty (h2 = 0.57), grassy (h2 = 0.37), toasted (h2 = 0.55), and raw dough-like (h2 = 0.34) flavor notes, and sweet (h2 = 0.29), fermented (h2 = 0.34), musty (h2 = 0.33), grassy (h2 = 0.37) and toasted (h2 = 0.5) aroma notes were found significant; the number of distillates evaluated was too small to estimate this parameter. We developed rye distillate aroma and flavor prediction models based on sensory evaluation of easy and quick-to-prepare flatbread. This manuscript proposes the usage of flatbread to evaluate rye grain aroma and flavor for sensory research purposes and for flavor prediction of other rye products.

Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that YfeA is polyspecific and can alter its substrate specificity. In minimal-media experiments, YfeA crystals grown after iron supplementation showed a threefold increase in iron fluorescence emission over the iron fluorescence emission from YfeA crystals grown from nutrient-rich conditions, and YfeA crystals grown after manganese supplementation during overexpression showed a fivefold increase in manganese fluorescence emission over the manganese fluorescence emission from YfeA crystals grown from nutrient-rich conditions. In all experiments, the YfeA crystals produced the strongest fluorescence emission from zinc and could not be manipulated otherwise. Additionally, this report documents the discovery of a novel surface metal-binding site that prefers to chelate zinc but can also bind manganese. Flexibility across YfeA crystal forms in three loops and a helix near the buried metal-binding site suggest that a structural rearrangement is required for metal loading and unloading.

The FeoABC ferrous transporter is a wide-spread bacterial system. While the feoABC locus is regulated by a number of factors in the bacteria studied, we have previously found that regulation of feoABC in Yersinia pestis appears to be unique. None of the non-iron responsive transcriptional regulators that control expression of feoABC in other bacteria do so in Y. pestis. Another unique factor is the iron and Fur regulation of the Y. pestis feoABC locus occurs during microaerobic but not aerobic growth. Here we show that this unique iron-regulation is not due to a unique aspect of the Y. pestis Fur protein but to DNA sequences that regulate transcription. We have used truncations, alterations, and deletions of the feoA::lacZ reporter to assess the mechanism behind the failure of iron to repress transcription under aerobic conditions. These studies plus EMSAs and DNA sequence analysis have led to our proposal that the feoABC locus has two promoters: an upstream P1 promoter whose expression is relatively iron-independent but repressed under microaerobic conditions and the known downstream Fur-regulated P2 promoter. In addition, we have identified two regions that bind Y. pestis protein(s), although we have not identified these protein(s) or their function. Finally we used iron uptake assays to demonstrate that both FeoABC and YfeABCD transport ferrous iron in an energy-dependent manner and also use ferric iron as a substrate for uptake.

A number of bacterial pathogens require the ZnuABC Zinc (Zn2+) transporter and/or a second Zn2+ transport system to overcome Zn2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn2+in vitro under the conditions tested. However, we detect a significant increase in Zn2+-binding ability of filtered supernatants from a Ybt+ strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

First paragraphs:No matter the subject, Barry Estabrook is a writer whose experiences in the world of food and agriculture are wide and deep. Pig Tales: An Omnivore's Quest for Sustainable Meat is no excep­tion. The title gives his approach away: this is a collection of tales from his travels and interviews to understand pig nature and produc­tion. He con­tinually engages the reader by supply­ing deep back­stories for his most significant interviewees. It is obvious he connects personally with each one, is able to put them at ease and thus get at the truth of their situations.Pig Tales begins with a scene of courtroom drama in which Estabrook almost gets arrested for being himself attending a trial in in which neighbors maintain that the concentrated animal feeding operation (CAFO) built nearby emits such foul odors they cannot be outside on their own property. The judge in the case is leery of his presence because of his previous book, Tomatoland, and so has him removed—an action that propels Barry on his journey.

By reducing immune function, trace metal deficiencies may substantially contribute to the global burden of diarrhea, pneumonia, and malaria. Human activities may be contributing to trace metal deficiency in soils and plants by exacerbating the preponderance of cereals and cash crops that reduce food diversity and micronutrient intake. Adaptive strategies are needed to reverse these trends. Anthropogenic activities have led to increased toxic metal exposure, and effects on human hosts need clarification. Metal toxicities can also impair the immune system and hence increase the susceptibility to infectious pathogens. Climate change affects metal speciation and the build-up of trace elements in the human food chain, with as yet unknown outcomes on infectious disease. Food processing and the use of metallic nanomaterials can alter human exposure to metals in ways that can influence the host–pathogen competition for metals. The effects of metals on human health may also be mediated through modification of the epigenome, conferring drug resistance on pathogenic bacteria and enhancing/ reducing human tolerance to infectious parasites. The emerging metals cerium, gadolinium, lanthanum, and yttrium constitute another driver of change in metal exposure and may potentially modulate the immune system with unknown consequences for human health.

Metals play a central role in the outcome of host–pathogen interactions. Microbes must acquire metals for metabolic processes, with nearly a half of all enzymes requiring a metal cofactor for function, yet microbes can be poisoned by metals. The host innate immune defenses are thought to exploit these vulnerabilities to protect against invading pathogens, whereas microbes can respond by employing multiple strategies to maintain their metal homeostasis. An understanding of these microbial strategies combined with knowledge of the diverse metal challenges faced by different microbes in the various host niches could inform the development of much needed new approaches for combating infectious diseases. This chapter summarizes extensive discussions on the interplay of metal ions in host–microbe interactions, from the microbial perspective. The focus is on fi ve key areas, highlighted as requiring a greater understanding: (a) how we defi ne and determine metal availability, (b) the different levels and sources of metals available to microbes in different niches within the host, (c) the effect of the metal status of a pathogen, as derived from its prior environment, on its ability to establish an infection or the severity of disease, (d) the interplay between metals and the microbiota, and (e) how metal restriction and metal oversupply can kill or inhibit the growth of microbes. This chapter provides an overview of current understanding in these areas and raises a number of important open questions in need of future research. From “Trace Metals and Infectious Diseases,” Jerome O. Nriagu and Eric P. Skaar, eds. 2015. Strüngmann Forum Reports, vol. 16, series ed. J. Lupp. Cambridge, MA: MIT Press. ISBN 978-0-262-02919-3. 100 J. S. Cavet et al.

Experts explore the influence of trace metals on the pathogenesis of infectious diseases. Many parts of the world in which common infectious diseases are endemic also have the highest prevalence of trace metal deficiencies or rising rates of trace metal pollution. Infectious diseases can increase human susceptibility to adverse effects of metal exposure (at suboptimal or toxic levels), and metal excess or deficiency can increase the incidence or severity of infectious diseases. The co-clustering of major infectious diseases with trace metal deficiency or toxicity has created a complex web of interactions with serious but poorly understood health repercussions, yet has been largely overlooked in animal and human studies. This book focuses on the distribution, trafficking, fate, and effects of trace metals in biological systems. Its goal is to enhance our understanding of the relationships between homeostatic mechanisms of trace metals and the pathogenesis of infectious diseases. Drawing on expertise from a range of fields, the book offers a comprehensive review of current knowledge on vertebrate metal-withholding mechanisms and the strategies employed by different microbes to avoid starvation (or poisoning). Chapters summarize current, state-of-the-art techniques for investigating pathogen-metal interactions and highlight open question to guide future research. The book makes clear that improving knowledge in this area will be instrumental to the development of novel therapeutic measures against infectious diseases. Contributors M. Leigh Ackland, Vahid Fa Andisi, Angele L. Arrieta, Michael A. Bachman, J. Sabine Becker, Robert E. Black, Julia …

This overview covers the role of the metal ions in infectious diseases, focusing on iron (Fe), copper (Cu), zinc (Zn), and, to a lesser extent, manganese (Mn) and the metalloid selenium (Se). In addition, recommended dietary allowances are addressed, as are metal-based drugs for the treatment of tropical diseases.The human organism binds essential metals such as iron, manganese, copper, and zinc to specific compounds (including proteins) in order to withhold these metals from invading pathogens ("nutritional immunity"); in this way, metal binding provides resistance to infection. Selenium status can also affect the host-pathogen interaction, but pathogens have mechanisms to counteract this protective potency. As alternative to a withdrawal of metals, microbes can be exposed to particularly high-and thus toxic-levels of metal ions. A secondary protective mechanism stems from the production (by host innate immune cells) of reactive oxygen and nitrogen species; this can also result in host tissue damage. In addition, the gasotransmitters nitric oxide (an oxidant) and carbon monoxide are indirectly involved in side effects (deprotection and protection, respectively, of bound heme) that result from the immune response.Host-mediated alteration of Fe homeostasis directly impacts on the proliferation of microbes. Depending on the type of pathogen, different regulatory mechanisms can be initiated. Limiting the availability of iron can be an efficient strategy to restrict extracellular bacteria, although such a strategy is detrimental for intracellular pathogens. Iron homeostasis is partly linked to Cu homeostasis. Copper deficiency predisposes mammals to infectious diseases, to some extent as a consequence of a lack of neutrophils induced by inadequate Cu availability or supply. Finally, there is a clear-cut correlation between bacterial infections and Zn removal from serum. More generally, Zn deficiency reduces immune defense against infections, chronic inflammatory disease, and reduced cellular activation, whereas high levels of zinc can hamper effective signal transduction.Due to the epidemic proportions of tropical diseases (e.g., leishmaniasis, Chagas disease, and malaria) and lack of effective treatment, drugs are being developed that are based on coordination compounds of metals, including copper, iron, ruthenium, and gold. These metals are coordinated to aromatic ligand systems that allow for a stabilization of the drug, during the drug's transport to its target, and eventually intercalation into DNA. For malaria, the increasing resistance of the malaria parasite against the classical drug chloroquine may be overcome by employing ferrocenyl derivatives of chloroquine.

Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent.

Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+-deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+-dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe3+ acquisition by Ybt, are not needed for Ybt-dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model.

Yersinia pestis, a gram-negative bacterium and member of the Enterobacteriaceae family, is the causative agent of bubonic, septicemic, and pneumonic plague. In general, increased levels of cyclic di-GMP (c-di-GMP) inside bacterial cells correlate with biofilm formation and expression of exopolysaccharide (EPS) and adherence factors, while decreased intracellular levels stimulate motility and a planktonic lifestyle. c-di-GMP homeostasis is carried out by GGDEF domain proteins, cyclic diguanylate cyclases (DGCs), which synthesize c-di-GMP, as well as EAL and HD-GYP domain proteins, c-di-GMP phosphodiesterases (PDEs), which degrade this nucleotide. Y. pestis was reported to express an Hms-dependent biofilm EPS, based on electron microscopy, reaction with antisera against poly-β-1,6-GlcNAc, and on the ability of cells to bind Congo red (CR), calcofluor white, and ruthenium red, each shown to stain various polysaccharides. A Y. pestis hmsP mutant overproduces biofilm but shows no increase in the levels of HmsHFRS proteins. All other potential DGCs and PDEs encoded by the Y. pestis KIM6+ genome do not compensate for hmsT or hmsP mutations, and the authors' analysis suggests that HmsT and Y3730 are the only two functional DGCs and that HmsP is the only functional PDE in Y. pestis. Mutations in speC (encoding ornithine decarboxylase), speA (encoding arginine decarboxylase), or both genes cause progressively lower intracellular levels of putrescine and a corresponding loss of crystal violet staining as a measure of adherence. Currently, only biofilm development is known to be controlled by c-di-GMP signaling in Y. pestis.

The second messenger molecule cyclic diguanylate is essential for Yersinia pestis biofilm formation that is important for blockage-dependent plague transmission from fleas to mammals. Two diguanylate cyclases (DGCs) HmsT and Y3730 (HmsD) are responsible for biofilm formation in vitro and biofilm-dependent blockage in the oriental rat flea Xenopsylla cheopis respectively. Here, we have identified a tripartite signalling system encoded by the y3729-y3731 operon that is responsible for regulation of biofilm formation in different environments. We present genetic evidence that a putative inner membrane-anchored protein with a large periplasmic domain Y3729 (HmsC) inhibits HmsD DGC activity in vitro while an outer membrane Pal-like putative lipoprotein Y3731 (HmsE) counteracts HmsC to activate HmsD in the gut of X. cheopis. We propose that HmsE is a critical element in the transduction of environmental signal(s) required for HmsD-dependent biofilm formation.