The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

Structural diversity in salt excreting glands and salinity tolerance in Oryza coarctata, Sporobolus anglicus and Urochondra setulosa.

MAIN CONCLUSION: Unlike the bicellular glands characteristic of all known excreting grasses, unique single-celled salt glands were discovered in the only salt tolerant species of the genus Oryza, Oryza coarctata. Salt tolerance has evolved frequently in a large number of grass lineages with distinct difference in mechanisms. Mechanisms of salt tolerance were studied in three species of grasses characterized by salt excretion: C3 wild rice species Oryza coarctata, and C4 species Sporobolus anglicus and Urochondra setulosa. The leaf anatomy and ultrastructure of salt glands, pattern of salt excretion, gas exchange, accumulation of key photosynthetic enzymes, leaf water content and osmolality, and levels of some osmolytes, were compared when grown without salt, with 200 mM NaCl versus 200 mM KCl. Under salt treatments, there was little effect on the capacity for CO2 assimilation, while stomatal conductance decreased with a reduction in water loss by transpiration and an increase in water use efficiency. All three species accumulate compatible solutes but with drastic differences in osmolyte composition. Having high capacity for salt excretion, they have distinct structural differences in the salt excreting machinery. S. anglicus and U. setulosa have bicellular glands while O. coarctata has unique single-celled salt glands with a partitioning membrane system that are responsible for salt excretion rather than multiple hairs as previously suggested. The features of physiological responses and salt excretion indicate similar mechanisms are involved in providing tolerance and excretion of Na+ and K+.

Untargeted Metabolomic Investigation of Wheat Infected with Stinking Smut Tilletia caries.

Tilletia caries infection of wheat (Triticum aestivum) has become an increasing problem in organic wheat agriculture throughout the world. Little is known about how this pathogen alters host metabolism to ensure a successful infection. We investigated how T. caries allocates resources from wheat for its growth over the life cycle of the pathogen. An untargeted metabolomics approach that combined gas chromatography time-of-flight mass spectrometry and ultraperformance liquid chromatography tandem mass spectrometry platforms was used to determine which primary or specialized metabolite pathways are targeted and altered during T. caries infection. We found that T. caries does not dramatically alter the global metabolome of wheat but instead alters key metabolites for its own nutrient uptake and to antagonize host defenses by reducing wheat's sweet immunity response and other related pathways. Our results highlight metabolic characteristics needed for selecting wheat varieties that are resistant to T. caries infection for organic agriculture. In addition, several wheat metabolites were identified that could be used in developing a diagnostic tool for early detection of T. caries infection.

Biomolecular archaeology reveals ancient origins of indigenous tobacco smoking in North American Plateau.

Chemical analysis of residues contained in the matrix of stone smoking pipes reveal a substantial direct biomolecular record of ancient tobacco (Nicotiana) smoking practices in the North American interior northwest (Plateau), in an area where tobacco was often portrayed as a Euro-American-introduced postcontact trade commodity. Nicotine, a stimulant alkaloid and biomarker for tobacco, was identified via ultra-performance liquid chromatography-mass spectrometry in 8 of 12 analyzed pipes and pipe fragments from five sites in the Columbia River Basin, southeastern Washington State. The specimens date from 1200 cal BP to historic times, confirming the deep time continuity of intoxicant use and indigenous smoking practices in northwestern North America. The results indicate that hunting and gathering communities in the region, including ancestral Nez Perce peoples, established a tobacco smoking complex of wild (indigenous) tobacco well before the main domesticated tobacco (Nicotiana tabacum) was introduced by contact-era fur traders and settlers after the 1790s. This is the longest continuous biomolecular record of ancient tobacco smoking from a single region anywhere in the world-initially during an era of pithouse development, through the late precontact equestrian era, and into the historic period. This contradicts some ethnohistorical data indicating that kinnikinnick, or bearberry (Arctostaphylos uva-ursi) was the primary precontact smoke plant in the study area. Early use likely involved the management and cultivation of indigenous tobaccos (Nicotiana quadrivalvis or Nicotiana attenuata), species that are today exceedingly rare in the region and seem to have been abandoned as smoke plants after the entry of trade tobacco.

Extracellular ATP Shapes a Defense-Related Transcriptome Both Independently and along with Other Defense Signaling Pathways.

ATP is not only an essential metabolite of cellular biochemistry but also acts as a signal in the extracellular milieu. In plants, extracellular ATP is monitored by the purinergic receptor P2K1. Recent studies have revealed that extracellular ATP acts as a damage-associated molecular pattern in plants, and its signaling through P2K1 is important for mounting an effective defense response against various pathogenic microorganisms. Biotrophic and necrotrophic pathogens attack plants using different strategies, to which plants respond accordingly with salicylate-based or jasmonate/ethylene-based defensive signaling, respectively. Interestingly, defense mediated by P2K1 is effective against pathogens of both lifestyles, raising the question of the level of interplay between extracellular ATP signaling and that of jasmonate, ethylene, and salicylate. To address this issue, we analyzed ATP-induced transcriptomes in wild-type Arabidopsis (Arabidopsis thaliana) seedlings and mutant seedlings defective in essential components in the signaling pathways of jasmonate, ethylene, and salicylate (classic defense hormones) as well as a mutant and an overexpression line of the P2K1 receptor. We found that P2K1 function is crucial for faithful ATP-induced transcriptional changes and that a subset of genes is more responsive in the P2K1 overexpression line. We also found that more than half of the ATP-responsive genes required signaling by one or more of the pathways for the classical defense hormones, with the jasmonate-based signaling being more critical than others. By contrast, the other ATP-responsive genes were unaffected by deficiencies in signaling for any of the classical defense hormones. These ATP-responsive genes were highly enriched for defense-related Gene Ontology terms. We further tested the ATP-induced genes in knockout mutants of transcription factors, demonstrating that MYCs acting downstream of the jasmonate receptor complex and calmodulin-binding transcription activators are nuclear transducers of P2K1-mediated extracellular ATP signaling.

Production of methoxylated flavonoids in yeast using ring A hydroxylases and flavonoid O-methyltransferases from sweet basil.

Numerous methoxylated flavonoids exhibit pronounced bioactivities. Their biotechnological production and diversification are therefore of interest to pharmaceutical and nutraceutical industries. We used a set of enzymes from sweet basil (Ocimum basilicum) to construct five strains of Saccharomyces cerevisiae producing 8- and/or 6-substituted, methoxylated flavones from their natural precursor apigenin. After identifying several growth parameters affecting the overall yields and flux, we applied optimized conditions and explored the ability of the generated strains to utilize alternative substrates. The yeast cells produced substantial amounts of 6-hydroxylated, methylated derivatives of naringenin and luteolin while the corresponding derivatives of flavonol kaempferol were only detected in trace amounts. Analysis of the intermediates and by-products of the different bioconversions suggested that the substrate specificity of both the hydroxylases and the flavonoid O-methyltransferases is imposing barriers on yields obtained with alternative substrates and highlighted steps that appear to represent bottlenecks en route to increasing the strains' efficiencies. Additionally, analysis of flavonoid localization during fermentation revealed unequal distribution with strong intracellular accumulation of a number of methylated flavonoids and extracellular enrichment of several pathway intermediates. This work establishes a platform for the production of complex methoxylated flavonoids and discusses strategies for its improvement.

Porcine Breast Extracellular Matrix Hydrogel for Spatial Tissue Culture.

Porcine mammary fatty tissues represent an abundant source of natural biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel and porous scaffolds from the extracted ECM proteins, the structural properties of the scaffolds (tissue matrix scaffold, TMS), and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.

Production of the antibiotic secondary metabolite solanapyrone A by the fungal plant pathogen Ascochyta rabiei during fruiting body formation in saprobic growth.

Fungi are noted producers of a diverse array of secondary metabolites, many of which are of pharmacological importance. However, the biological roles of the vast majority of these molecules during the fungal life cycle in nature remain elusive. Solanapyrones are polyketide-derived secondary metabolites produced by diverse fungal species including the plant pathogen Ascochyta rabiei. This molecule was originally thought to function as a phytotoxin facilitating pathogenesis of A. rabiei. Chemical profiling and gene expression studies showed that solanapyrone A was specifically produced during saprobic, but not parasitic growth of A. rabiei. Expression of the gene encoding the final enzymatic step in solanapyrone biosynthesis was specifically associated with development of the asexual fruiting bodies of the fungus on certain substrates. In confrontation assays with saprobic fungi that were commonly found in chickpea debris in fields, A. rabiei effectively suppressed the growth of all competing fungi, such as Alternaria, Epicoccum and Ulocladium species. Solanapyrone A was directly detected in the inhibitory zone using a MALDI-imaging mass spectrometry, and the purified compound showed significant antifungal activities against the potential saprobic competitors. These results suggest that solanapyrone A plays an important role for competition and presumably the survival of the fungus.

A (-)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum.

Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major bioactive neo-clerodane diterpenoid, remains virtually unknown. This investigation aimed to identify the enzyme that catalyzes the first reaction of salvinorin A biosynthesis, the formation of (-)-kolavenyl diphosphate [(-)-KPP], which is subsequently dephosphorylated to afford (-)-kolavenol. Peltate glandular trichomes were identified as the major and perhaps exclusive site of salvinorin accumulation in S. divinorum. The trichome-specific transcriptome was used to identify candidate diterpene synthases (diTPSs). In vitro and in planta characterization of a class II diTPS designated as SdKPS confirmed its activity as (-)-KPP synthase and its involvement in salvinorin A biosynthesis. Mutation of a phenylalanine into histidine in the active site of SdKPS completely converts the product from (-)-KPP into ent-copalyl diphosphate. Structural elements were identified that mediate the natural formation of the neo-clerodane backbone by this enzyme and suggest how SdKPS and other diTPSs may have evolved from ent-copalyl diphosphate synthase.

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of Scrophularia and Verbascum species used medicinally in North America.

INTRODUCTION: Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability (YLDs), such as arthritis and lower back pain. OBJECTIVES: We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America. METHODS: Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI. RESULTS: Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus. CONCLUSIONS: Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer.

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.

The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis.

Drastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon- and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway.

The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii.

The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and (13)C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700(+) reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic (13)CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates.

A Novel Type Pathway-Specific Regulator and Dynamic Genome Environments of a Solanapyrone Biosynthesis Gene Cluster in the Fungus Ascochyta rabiei.

Secondary metabolite genes are often clustered together and situated in particular genomic regions, like the subtelomere, that can facilitate niche adaptation in fungi. Solanapyrones are toxic secondary metabolites produced by fungi occupying different ecological niches. Full-genome sequencing of the ascomycete Ascochyta rabiei revealed a solanapyrone biosynthesis gene cluster embedded in an AT-rich region proximal to a telomere end and surrounded by Tc1/Mariner-type transposable elements. The highly AT-rich environment of the solanapyrone cluster is likely the product of repeat-induced point mutations. Several secondary metabolism-related genes were found in the flanking regions of the solanapyrone cluster. Although the solanapyrone cluster appears to be resistant to repeat-induced point mutations, a P450 monooxygenase gene adjacent to the cluster has been degraded by such mutations. Among the six solanapyrone cluster genes (sol1 to sol6), sol4 encodes a novel type of Zn(II)2Cys6 zinc cluster transcription factor. Deletion of sol4 resulted in the complete loss of solanapyrone production but did not compromise growth, sporulation, or virulence. Gene expression studies with the sol4 deletion and sol4-overexpressing mutants delimited the boundaries of the solanapyrone gene cluster and revealed that sol4 is likely a specific regulator of solanapyrone biosynthesis and appears to be necessary and sufficient for induction of the solanapyrone cluster genes. Despite the dynamic surrounding genomic regions, the solanapyrone gene cluster has maintained its integrity, suggesting important roles of solanapyrones in fungal biology.

Comparative proteomic analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale and different monocot species.

The rhizome is responsible for the invasiveness and competitiveness of many plants with great economic and agricultural impact worldwide. Besides its value as an invasive organ, the rhizome plays a role in the establishment and massive growth of forage, providing biomass for biofuel production. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development, and function in plants. In this work, we characterized the proteome of rhizome apical tips and elongation zones from different species using a GeLC-MS/MS (one-dimensional electrophoresis in combination with liquid chromatography coupled online with tandem mass spectrometry) spectral-counting proteomics strategy. Five rhizomatous grasses and an ancient species were compared to study the protein regulation in rhizomes. An average of 2200 rhizome proteins per species were confidently identified and quantified. Rhizome-characteristic proteins showed similar functional distributions across all species analyzed. The over-representation of proteins associated with central roles in cellular, metabolic, and developmental processes indicated accelerated metabolism in growing rhizomes. Moreover, 61 rhizome-characteristic proteins appeared to be regulated similarly among analyzed plants. In addition, 36 showed conserved regulation between rhizome apical tips and elongation zones across species. These proteins were preferentially expressed in rhizome tissues regardless of the species analyzed, making them interesting candidates for more detailed investigative studies about their roles in rhizome development.

HMGA1 drives metabolic reprogramming of intestinal epithelium during hyperproliferation, polyposis, and colorectal carcinogenesis.

Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.

Unexpected roles for ancient proteins: flavone 8-hydroxylase in sweet basil trichomes is a Rieske-type, PAO-family oxygenase.

Most elucidated hydroxylations in plant secondary metabolism are catalyzed by oxoglutarate- or cytochrome P450-dependent oxygenases. Numerous hydroxylations still evade clarification, suggesting that they might be performed by alternative enzyme types. Here, we report the identification of the flavone 8-hydroxylase (F8H) in sweet basil (Ocimum basilicum L.) trichomes as a Rieske-type oxygenase. Several features of the F8H activity in trichome protein extracts helped to differentiate it from a cytochrome P450-catalyzed reaction and identify candidate genes in the basil trichome EST database. The encoded ObF8H proteins share approximately 50% identity with Rieske-type protochlorophyllide a oxygenases (PTC52) from higher plants. Homology cloning and DNA blotting revealed the presence of several PTC52-like genes in the basil genome. The transcripts of the candidate gene designated ObF8H-1 are strongly enriched in trichomes compared to whole young leaves, indicating trichome-specific expression. The full-length ObF8H-1 protein possesses a predicted N-terminal transit peptide, which directs green fluorescent protein at least in part to chloroplasts. The F8H activity in crude trichome protein extracts correlates well with the abundance of ObF8H peptides. The purified recombinant ObF8H-1 displays high affinity for salvigenin and is inactive with other tested flavones except cirsimaritin, which is 8-hydroxylated with less than 0.2% relative activity. The efficiency of in vivo 8-hydroxylation by engineered yeast was improved by manipulation of protein subcellular targeting. blast searches showed that occurrence of several PTC52-like genes is rather common in sequenced plant genomes. The discovery of ObF8H suggests that Rieske-type oxygenases may represent overlooked candidate catalysts for oxygenations in specialized plant metabolism.

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants.

We developed a porcine dermal explant model to determine the extent to which Staphylococcus aureus biofilm communities deplete oxygen, change pH, and produce damage in underlying tissue. Microelectrode measurements demonstrated that dissolved oxygen (DO) in biofilm-free dermal tissue was 4.45 ± 1.17 mg/liter, while DO levels for biofilm-infected tissue declined sharply from the surface, with no measurable oxygen detectable in the underlying dermal tissue. Magnetic resonance imaging demonstrated that biofilm-free dermal tissue had a significantly lower relative effective diffusion coefficient (0.26 ± 0.09 to 0.30 ± 0.12) than biofilm-infected dermal tissue (0.40 ± 0.12 to 0.48 ± 0.12; P < 0.0001). Thus, the difference in DO level was attributable to biofilm-induced oxygen demand rather than changes in oxygen diffusivity. Microelectrode measures showed that pH within biofilm-infected explants was more alkaline than in biofilm-free explants (8.0 ± 0.17 versus 7.5 ± 0.15, respectively; P < 0.002). Cellular and nuclear details were lost in the infected explants, consistent with cell death. Quantitative label-free shotgun proteomics demonstrated that both proapoptotic programmed cell death protein 5 and antiapoptotic macrophage migration inhibitory factor accumulated in the infected-explant spent medium, compared with uninfected-explant spent media (1,351-fold and 58-fold, respectively), consistent with the cooccurrence of apoptosis and necrosis in the explants. Biofilm-origin proteins reflected an extracellular matrix-adapted lifestyle of S. aureus. S. aureus biofilms deplete oxygen, increase pH, and induce cell death, all factors that contribute to impede wound healing.