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Antibiotic use during pregnancy is associated with increased asthma risk in children. Since approximately 25% of women use antibiotics during pregnancy, it is important to identify the pathways involved in this phenomenon. We investigate how mother-to-offspring transfer of antibiotic-induced gut microbial dysbiosis influences immune system development along the gut-lung axis. Using a mouse model of maternal antibiotic exposure during pregnancy, we immunophenotyped offspring in early life and after asthma induction. In early life, prenatal-antibiotic exposed offspring exhibited gut microbial dysbiosis, intestinal inflammation (increased fecal lipocalin-2 and IgA), and dysregulated intestinal ILC3 subtypes. Intestinal barrier dysfunction in the offspring was indicated by a FITC-dextran intestinal permeability assay and circulating lipopolysaccharide. This was accompanied by increased T-helper (Th)17 cell percentages in the offspring's blood and lungs in both early life and after allergy induction. Lung tissue additionally showed increased percentages of RORγt T-regulatory (Treg) cells at both time points. Our investigation of the gut-lung axis identifies early-life gut dysbiosis, intestinal inflammation, and barrier dysfunction as a possible developmental programming event promoting increased expression of RORγt in blood and lung CD4 + T cells that may contribute to increased asthma risk., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Sponges are known for hosting diverse communities of microbial symbionts, but despite persistent interest in the sponge microbiome, most research has targeted marine sponges; freshwater sponges have been the focus of less than a dozen studies. Here, we used 16 S rRNA gene amplicon sequencing and shotgun metagenomics to characterize the microbiome of the freshwater sponge Ephydatia muelleri and identify potential indicators of sponge-microbe mutualism. Using samples collected from the Sooke, Nanaimo, and Cowichan Rivers on Vancouver Island, British Columbia, we show that the E. muelleri microbiome is distinct from the ambient water and adjacent biofilms and is dominated by Sediminibacterium, Comamonas, and unclassified Rhodospirillales. We also observed phylotype-level differences in sponge microbiome taxonomic composition among different rivers. These differences were not reflected in the ambient water, suggesting that other environmental or host-specific factors may drive the observed geographic variation. Shotgun metagenomes and metagenome-assembled genomes further revealed that freshwater sponge-associated bacteria share many genomic similarities with marine sponge microbiota, including an abundance of defense-related proteins (CRISPR, restriction-modification systems, and transposases) and genes for vitamin B12 production. Overall, our results provide foundational information on the composition and function of freshwater sponge-associated microbes, which represent an important yet underappreciated component of the global sponge microbiome., (© 2022. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before β-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase's specificity for 4-phospho-acetophenone ( k cat / K M = 34 ± 2 mM -1 s -1 ) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13 C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody "hog fuel" pile (53 and 62 °C zones). 13 C-Lignin degradation was monitored using IR-GCMS of 13 CO 2 , and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13 C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13 C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature., (© 2022. The Author(s).)

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Navas, Dexter, Liu, Levy-Booth, Cho, Jang, Mansfield, Renneckar, Mohn and Eltis.)

Bile salts are amphiphilic steroids with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile salt steroid skeleton resembles the well-studied pathway for other steroids, like testosterone, while specific differences occur during side chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ 1,4 - or Δ 4,6 -3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ 1,4 variant is well known from many model organisms, the Δ 4,6 variant involving a 7-hydroxysteroid dehydratase as a key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic, and functional analyses of the Δ 4,6 variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to that of the Δ 1,4 variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for the degradation of the C 5 carboxylic side chain encoding a CoA ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ 4,6 variant to be widespread among the Sphingomonadaceae , which was verified for three type strains which also have the predicted side chain degradation cluster. A second amidase in the side chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile salt hydrolases. This study identifies members of the Sphingomonadaceae that are remarkably well adapted to the utilization of bile salts via a partially distinct metabolic pathway. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

Background: Bacterial and fungal microbiotas are increasingly recognized as important in health and disease starting early in life. However, microbiota composition has not yet been investigated in most rural, low-resource settings, and in such settings, bacterial and fungal microbiotas have not been compared. Thus, we applied 16S and ITS2 amplicon sequencing, respectively, to investigate bacterial and fungal fecal microbiotas in rural Ghanaian children cross-sectionally from birth to 5 years of age. Corresponding maternal fecal and breast milk microbiotas were additionally investigated., Results: While bacterial communities differed systematically across the age spectrum in composition and diversity, the same was not observed for the fungal microbiota. We also identified a novel and dramatic change in the maternal postpartum microbiota. This change included much higher abundance of Escherichia coli and much lower abundance of Prevotella in the first vs. fourth week postpartum. While infants shared more bacterial taxa with their mother's stool and breast milk than with those of unrelated mothers, there were far fewer shared fungal taxa., Conclusion: Given the known ability of commensal fungi to influence host health, the distinct pattern of their acquisition likely has important health consequences. Similarly, the dynamics of mothers' bacterial microbiotas around the time of birth may have important consequences for their children's health. Both topics require further study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amenyogbe, Adu-Gyasi, Enuameh, Asante, Konadu, Kaali, Dosoo, Panigrahi, Kollmann, Mohn and Owusu-Agyei.)

Thermal swamps are unique ecosystems where geothermally warmed waters mix with decomposing woody biomass, hosting novel biogeochemical-cycling and lignin-degrading microbial consortia. Assembly of shotgun metagenome libraries resolved 351 distinct genomes from hot-spring (30-45 °C) and mesophilic (17 °C) sediments. Annotation of 39 refined draft genomes revealed metabolism consistent with oligotrophy, including pathways for degradation of aromatic compounds, such as syringate, vanillate, p-hydroxybenzoate, and phenol. Thermotolerant Burkholderiales, including Rubrivivax ssp., were implicated in diverse biogeochemical and aromatic transformations, highlighting their broad metabolic capacity. Lignin catabolism was further investigated using metatranscriptomics of sediment incubated with milled or Kraft lignin at 45 °C. Aromatic compounds were depleted from lignin-amended sediment over 148 h. The metatranscriptomic data revealed upregulation of des/lig genes predicted to specify the catabolism of syringate, vanillate, and phenolic oligomers in the sphingomonads Altererythrobacter ssp. and Novosphingobium ssp., as well as in the Burkholderiales genus, Rubrivivax. This study demonstrates how temperature structures biogeochemical cycling populations in a unique ecosystem, and combines community-level metagenomics with targeted metatranscriptomics to identify pathways with potential for bio-refinement of lignin-derived aromatic compounds. In addition, the diverse aromatic catabolic pathways of Altererythrobacter ssp. may serve as a source of thermotolerant enzymes for lignin valorization.

The microbiome plays a fundamental role in how the immune system develops and how inflammatory responses are shaped and regulated. The "gut-lung axis" is a relatively new term that highlights a crucial biological crosstalk between the intestinal microbiome and lung. A growing body of literature suggests that dysbiosis, perturbation of the gut microbiome, is a driving force behind the development, and severity of allergic asthma. Animal models have given researchers new insights into how gut microbe-derived components and metabolites, such as short-chain fatty acids (SCFAs), influence the development of asthma. While the full understanding of how SCFAs influence allergic airway disease remains obscure, a recurring theme of epigenetic regulation of gene expression in several immune cell compartments is emerging. This review will address our current understanding of how SCFAs, and specifically butyrate, orchestrates cell behavior, and epigenetic changes and will provide a detailed overview of the effects of these modifications on immune cells in the context of allergic airway disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yip, Hughes, Li, Cait, Hirst, Mohn and McNagny.)

The gut microbiome is a well-recognized modulator of host immunity, and its compositions differ between geographically separated human populations. Systemic innate immune responses to microbial derivatives also differ between geographically distinct human populations. However, the potential role of the microbiome in mediating geographically varied immune responses is unexplored. We here applied 16S amplicon sequencing to profile the stool microbiome and, in parallel, measured whole-blood innate immune cytokine responses to several pattern recognition receptor (PRR) agonists among 2-year-old children across biogeographically diverse settings. Microbiomes differed mainly between high- and low-resource environments and were not strongly associated with other demographic factors. We found strong correlations between responses to Toll-like receptor 2 (TLR2) and relative abundances of Bacteroides and Prevotella populations, shared among Canadian and Ecuadorean children. Additional correlations between responses to TLR2 and bacterial populations were specific to individual geographic cohorts. As a proof of concept, we gavaged germfree mice with human donor stools and found murine splenocyte responses to TLR stimulation were consistent with responses of the corresponding human donor populations. This study identified differences in immune responses correlating to gut microbiomes across biogeographically diverse settings and evaluated biological plausibility using a mouse model. This insight paves the way to guide optimization of population-specific interventions aimed to improve child health outcomes. IMPORTANCE Both the gut microbiome and innate immunity are known to differ across biogeographically diverse human populations. The gut microbiome has been shown to directly influence systemic immunity in animal models. With this, modulation of the gut microbiome represents an attractive avenue to improve child health outcomes associated with altered immunity using population-specific approaches. However, there are very scarce data available to determine which members of the gut microbiome are associated with specific immune responses and how these differ around the world, creating a substantial barrier to rationally designing such interventions. This study addressed this knowledge gap by identifying relationships between distinct bacterial taxa and cytokine responses to specific microbial agonists across highly diverse settings. Furthermore, we provide evidence that immunomodulatory effects of region-specific stool microbiomes can be partially recapitulated in germfree mice. This is an important contribution toward improving global child health by targeting the gut microbiome., (Copyright © 2021 Amenyogbe et al.)

Background: The human skin microbiome is highly personalized, depending on, for example, body site, age, gender, and lifestyle factors. The temporal stability of an individual's skin microbiome-its resiliency and robustness over months and years-is also a personalized feature of the microbiome. The authors measured the temporal stability of the facial skin microbiome in a large cohort of subjects. In addition to measuring microbiome dynamics, they tracked facial skin condition using noninvasive, objective imaging and biophysical measures to identify significant facial features associated with temporal changes in microbiome diversity and composition., Methods: The authors used 16S ribosomal RNA amplicon sequencing to track cheek and forehead skin microbiome diversity and composition annually over a 2-year period (2017-2019) in 115 healthy adult men and women. Skin metadata included facial features, such as wrinkles, hyperpigmentation, porphyrins, and skin color tone, as well as biophysical parameters for stratum corneum barrier function, pH, hydration, and elasticity., Results: Across the subject population, the facial skin microbiome composition and diversity were relatively stable, showing minor variation over the 2-year period. However, for some subjects, composition, diversity, and relative abundance of specific organisms showed substantial changes from one year to the next, and these changes were associated with changes in stratum corneum barrier function and follicular porphyrins., Conclusions: For healthy people, facial skin microbiome diversity and composition are relatively stable from year to year. Tracking the temporal changes in the microbiome along with skin phenotypic changes allows for a deeper understanding of the skin microbiome's role in health and disease. These results should be helpful in the design of longer-term intervention trials with microbiome-based skin care treatments., (Copyright © 2020 by the American Society of Plastic Surgeons.)

Background: Vaccination remains one of the most effective means of reducing the burden of infectious diseases globally. Improving our understanding of the molecular basis for effective vaccine response is of paramount importance if we are to ensure the success of future vaccine development efforts., Methods: We applied cutting edge multi-omics approaches to extensively characterize temporal molecular responses following vaccination with hepatitis B virus (HBV) vaccine. Data were integrated across cellular, epigenomic, transcriptomic, proteomic, and fecal microbiome profiles, and correlated to final HBV antibody titres., Results: Using both an unsupervised molecular-interaction network integration method (NetworkAnalyst) and a data-driven integration approach (DIABLO), we uncovered baseline molecular patterns and pathways associated with more effective vaccine responses to HBV. Biological associations were unravelled, with signalling pathways such as JAK-STAT and interleukin signalling, Toll-like receptor cascades, interferon signalling, and Th17 cell differentiation emerging as important pre-vaccination modulators of response., Conclusion: This study provides further evidence that baseline cellular and molecular characteristics of an individual's immune system influence vaccine responses, and highlights the utility of integrating information across many parallel molecular datasets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Shannon, Blimkie, Ben-Othman, Gladish, Amenyogbe, Drissler, Edgar, Chan, Krajden, Foster, Kobor, Mohn, Brinkman, Le Cao, Scheuermann, Tebbutt, Hancock, Koff, Kollmann, Sadarangani and Lee.)

In both high- and low-income countries, HIV-negative children born to HIV-positive mothers (HIV exposed, uninfected [HEU]) are more susceptible to severe infection than HIV-unexposed, uninfected (HUU) children, with altered innate immunity hypothesized to be a cause. Both the gut microbiome and systemic innate immunity differ across biogeographically distinct settings, and the two are known to influence each other. And although the gut microbiome is influenced by HIV infection and may contribute to altered immunity, the biogeography of immune-microbiome correlations among HEU children have not been investigated. To address this, we compared the innate response and the stool microbiome of 2-y-old HEU and HUU children from Belgium, Canada, and South Africa to test the hypothesis that region-specific immune alterations directly correlate to differences in their stool microbiomes. We did not detect a universal immune or microbiome signature underlying differences between HEU versus HUU that was applicable to all children. But as hypothesized, population-specific differences in stool microbiomes were readily detected and included reduced abundances of short-chain fatty acid-producing bacteria in Canadian HEU children. Furthermore, we did not identify innate immune-microbiome associations that distinguished HEU from HUU children in any population. These findings suggest that maternal HIV infection is independently associated with differences in both innate immunity and the stool microbiome in a biogeographical population-specific way., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized, studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. Sampling included ~350 mL of blood, 12 microbiome samples, and lymph node fine needle aspirates obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, in vitro T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches-NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design., (Copyright © 2020 Ben-Othman, Cai, Liu, Varankovich, He, Blimkie, Lee, Gill, Novotny, Aevermann, Drissler, Shannon, McCann, Marty, Bjornson, Edgar, Lin, Gladish, Maclsaac, Amenyogbe, Chan, Llibre, Collin, Landais, Le, Reiss, Koff, Havenar-Daughton, Heran, Sangha, Walt, Krajden, Crotty, Sok, Briney, Burton, Duffy, Foster, Mohn, Kobor, Tebbutt, Brinkman, Scheuermann, Hancock, Kollmann and Sadarangani.)

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O -demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA , encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta -cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC , encoding the meta -cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL , encoding a CYP255A2 family P450 and an ortho -cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O -demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities ( k cat / K M ): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoA EP4 's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin., Competing Interests: The authors declare no competing interest.

Background: The use of antibiotics during pregnancy is associated with increased allergic asthma risk in the offspring, and given that approximately 25% of pregnant women are prescribed antibiotics, it is important to understand the mechanisms contributing to this phenomenon. Currently, there are no studies that directly test this association experimentally. Our objective was to develop a mouse model in which antibiotic treatment during pregnancy results in increased offspring asthma susceptibility., Methods: Pregnant mice were treated daily from gestation day 8-17 with an oral solution of the antibiotic vancomycin, and three concentrations were tested. At weaning, offspring were subjected to an adjuvant-free experimental asthma protocol using ovalbumin as an allergen. The composition of the gut microbiome was determined in mothers and offspring with samples collected from five different time points; short-chain fatty acids were also analyzed in allergic offspring., Results: We found that maternal antibiotic treatment during pregnancy was associated with increased offspring asthma severity in a dose-dependent manner. Furthermore, maternal vancomycin treatment during pregnancy caused marked changes in the gut microbiome composition in both mothers and pups at several different time points. The increased asthma severity and intestinal microbiome changes in pups were also associated with significantly decreased cecal short-chain fatty acid concentrations., Conclusion: Consistent with the "Developmental Origins Hypothesis," our results confirm that exposure to antibiotics during pregnancy shapes the neonatal intestinal environment and increases offspring allergic lung inflammation., (© 2020 The Authors. Allergy published by John Wiley & Sons Ltd.)

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino - and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus , Mycobacterium , and Amycolatopsis , as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics. IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino - and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate., (Copyright © 2020 American Society for Microbiology.)

Background: Allergic disease is the most frequent chronic health issue in children and has been linked to early-life gut microbiome dysbiosis. Many lines of evidence suggest that microbially derived short-chain fatty acids, and particularly butyrate, can promote immune tolerance., Objective: We sought to determine whether bacterial butyrate production in the gut during early infancy is protective against the development of atopic disease in children., Methods: We used shotgun metagenomic analysis to determine whether dysbiosis in butyrate fermentation could be identified in human infants, before their developing allergic disease., Results: We found that the microbiome of infants who went on to develop allergic sensitization later in childhood lacked genes encoding key enzymes for carbohydrate breakdown and butyrate production., Conclusions: Our findings support the importance of microbial carbohydrate metabolism during early infancy in protecting against the development of allergies., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well-elucidated. We isolated Rhodococcus rhodochrous EP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 and Rhodococcus jostii RHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by the aphABCDEFGHIQRS genes that degrades 4-ethylphenol via the meta- cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by the aphAB genes, which were upregulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2,000 U/mg) but did not detectably transform phenol. Nevertheless, a Δ aphA mutant in RHA1 grew on 4-ethylphenol by compensatory upregulation of phenol hydroxylase genes ( pheA1-3 ). Deletion of aphC , encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption of pcaL in the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-alkylphenols are catabolized exclusively via meta- cleavage in rhodococci while phenol is subject to ortho- cleavage. A putative genomic island encoding aph genes was identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.

Despite recognition that biogeography and individuality shape the function and composition of the human skin microbiome, we know little about how extrinsic and intrinsic host factors influence its composition. To explore the contributions of these factors to skin microbiome variation, we profiled the bacterial microbiomes of 495 North American subjects (ages, 9 to 78 years) at four skin surfaces plus the oral epithelium using 16S rRNA gene amplicon sequencing. We collected subject metadata, including host physiological parameters, through standardized questionnaires and noninvasive biophysical methods. Using a combination of statistical modeling tools, we found that demographic, lifestyle, and physiological factors collectively explained 12 to 20% of the variability in microbiome composition. The influence of health factors was strongest on the oral microbiome. Associations between host factors and the skin microbiome were generally dominated by operational taxonomic units (OTUs) affiliated with the Clostridiales and Prevotella A subset of the correlations between microbial features and host attributes were site specific. To further explore the relationship between age and the skin microbiome of the forehead, we trained a Random Forest regression model to predict chronological age from microbial features. Age was associated mostly with two mutually coexcluding Corynebacterium OTUs. Furthermore, skin aging variables (wrinkles and hyperpigmented spots) were independently correlated to these taxa. IMPORTANCE Many studies have highlighted the importance of body site and individuality in shaping the composition of the human skin microbiome, but we still have a poor understanding of how extrinsic (e.g., lifestyle) and intrinsic (e.g., age) factors influence its composition. We characterized the bacterial microbiomes of North American volunteers at four skin sites and the mouth. We also collected extensive subject metadata and measured several host physiological parameters. Integration of host and microbial features showed that the skin microbiome was predominantly associated with demographic, lifestyle, and physiological factors. Furthermore, we uncovered reproducible associations between chronological age, skin aging, and members of the genus Corynebacterium Our work provides new understanding of the role of host selection and lifestyle in shaping skin microbiome composition. It also contributes to a more comprehensive appreciation of the factors that drive interindividual skin microbiome variation., (Copyright © 2019 Dimitriu et al.)

The Pacific coastal temperate rainforest (PCTR) is a global hot-spot for carbon cycling and export. Yet the influence of microorganisms on carbon cycling processes in PCTR soil is poorly characterized. We developed and tested a conceptual model of seasonal microbial carbon cycling in PCTR soil through integration of geochemistry, micro-meteorology, and eukaryotic and prokaryotic ribosomal amplicon (rRNA) sequencing from 216 soil DNA and RNA libraries. Soil moisture and pH increased during the wet season, with significant correlation to net CO 2 flux in peat bog and net CH 4 flux in bog forest soil. Fungal succession in these sites was characterized by the apparent turnover of Archaeorhizomycetes phylotypes accounting for 41% of ITS libraries. Anaerobic prokaryotes, including Syntrophobacteraceae and Methanomicrobia increased in rRNA libraries during the wet season. Putatively active populations of these phylotypes and their biogeochemical marker genes for sulfate and CH 4 cycling, respectively, were positively correlated following rRNA and metatranscriptomic network analysis. The latter phylotype was positively correlated to CH 4 fluxes (r = 0.46, p < 0.0001). Phylotype functional assignments were supported by metatranscriptomic analysis. We propose that active microbial populations respond primarily to changes in hydrology, pH, and nutrient availability. The increased microbial carbon export observed over winter may have ramifications for climate-soil feedbacks in the PCTR.

The intestinal microbiota plays an important role in development of the immune system and regulation of immune responses. This review summarizes the association between the intestinal microbiota and the development of allergic sensitization, eczema, and asthma in neonates and children. Overall, a greater relative abundance of Bacteroidaceae, Clostridiaceae, and Enterobacteriaceae and a lower relative abundance of Bifidobacteriaceae and Lactobacillaceae is associated with the development of allergic sensitization, eczema, or asthma. Reduced bacterial diversity can be associated with the development of allergic disease. The association between the composition of the intestinal microbiota and the development of allergic disease or asthma is less consistent in older children than in neonates, suggesting that early-life microbial exposure plays a more important role. Inconsistencies in the results reported from different studies might partly be explained by heterogeneity in design, study populations, diagnostic criteria, microbiota analysis methods, and reporting on different taxonomic levels. Larger studies that better account for antenatal and postnatal factors will further help determine specific microbial intestinal signatures associated with increased risk of allergy and asthma. This will enable the early identification of infants at high risk and facilitate novel strategies and interventions to prevent and treat these conditions, including modifying the intestinal microbiota early in life., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Delignification, or lignin-modification, facilitates the decomposition of lignocellulose in woody plant biomass. The extant diversity of lignin-degrading bacteria and fungi is underestimated by culture-dependent methods, limiting our understanding of the functional and ecological traits of decomposers populations. Here, we describe the use of stable isotope probing (SIP) coupled with amplicon and shotgun metagenomics to identify and characterize the functional attributes of lignin, cellulose and hemicellulose-degrading fungi and bacteria in coniferous forest soils from across North America. We tested the extent to which catabolic genes partitioned among different decomposer taxa; the relative roles of bacteria and fungi, and whether taxa or catabolic genes correlated with variation in lignocellulolytic activity, measured as the total assimilation of 13 C-label into DNA and phospholipid fatty acids. We found high overall bacterial degradation of our model lignin substrate, particularly by gram-negative bacteria (Comamonadaceae and Caulobacteraceae), while fungi were more prominent in cellulose-degradation. Very few taxa incorporated 13 C-label from more than one lignocellulosic polymer, suggesting specialization among decomposers. Collectively, members of Caulobacteraceae could degrade all three lignocellulosic polymers, providing new evidence for their importance in lignocellulose degradation. Variation in lignin-degrading activity was better explained by microbial community properties, such as catabolic gene content and community structure, than cellulose-degrading activity. SIP significantly improved shotgun metagenome assembly resulting in the recovery of several high-quality draft metagenome-assembled genomes and over 7500 contigs containing unique clusters of carbohydrate-active genes. These results improve understanding of which organisms, conditions and corresponding functional genes contribute to lignocellulose decomposition.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic Salmonella gastroenteritis model, Ab-mediated neutralization of IL-22 impaired intestinal epithelial barrier integrity and, consequently, exaggerated expression of proinflammatory cytokines. As disease normally resolved, neutralization of IL-22 caused luminal narrowing of the cecum-a feature reminiscent of fibrotic strictures seen in Crohn disease patients. Corresponding to the exaggerated immunopathology caused by IL-22 suppression, Salmonella burdens in the gut were reduced. This enhanced inflammation and pathogen clearance was associated with alterations in gut microbiome composition, including the overgrowth of Bacteroides acidifaciens Our findings thus indicate that IL-22 plays a protective role by limiting infection-induced gut immunopathology but can also lead to persistent pathogen colonization., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The mammalian gastrointestinal tract harbors a microbial community with metabolic activity critical for host health, including metabolites that can modulate effector functions of immune cells. Mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated T helper type 2 cell (Th2) responses, and are more susceptible to allergic lung inflammation. Here we show that dietary supplementation with short-chain fatty acids (SCFAs) ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells (DCs). Dysbiotic mice treated with SCFAs have fewer interleukin-4 (IL4)-producing CD4 + T cells and decreased levels of circulating immunoglobulin E (IgE). In addition, DCs exposed to SCFAs activate T cells less robustly, are less motile in response to CCL19 in vitro, and exhibit a dampened ability to transport inhaled allergens to lung draining nodes. Our data thus demonstrate that gut dysbiosis can exacerbate allergic lung inflammation through both T cell- and DC-dependent mechanisms that are inhibited by SCFAs.

Although previous studies, mostly based on microscopy analyses of a few groups of protists, have suggested that protists are abundant and diverse in litter and moss habitats, the overall diversity of moss and litter associated protists remains elusive. Here, high-throughput environmental sequencing was used to characterize the diversity and community structure of litter- and moss-associated protists along a gradient of soil drainage and forest primary productivity in a temperate rainforest in British Columbia. We identified 3262 distinct protist OTUs from 36 sites. Protists were strongly structured along the landscape gradient, with a significant increase in alpha diversity from the blanket bog ecosystem to the zonal forest ecosystem. Among all investigated environmental variables, calcium content was the most strongly associated with the community composition of protists, but substrate composition, plant cover and other edaphic factors were also significantly correlated with these communities. Furthermore, a detailed phylogenetic analysis of unicellular opisthokonts identified OTUs covering most lineages, including novel OTUs branching with Discicristoidea, the sister group of Fungi, and with Filasterea, one of the closest unicellular relatives to animals. Altogether, this study provides unprecedented insight into the community composition of moss- and litter-associated protists., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Forest ecosystems are critical to global biogeochemical cycles but under pressure from harvesting and climate change. We investigated the effects of organic matter (OM) removal during forest harvesting on the genetic potential of soil communities for biomass decomposition and nitrogen cycling in five ecozones across North America. We analyzed 107 samples, representing four treatments with varied levels of OM removal, at Long-Term Soil Productivity Study sites. Samples were collected more than ten years after harvesting and replanting and were analyzed via shotgun metagenomics. High-quality short reads totaling 1.2 Tbp were compared to the Carbohydrate Active Enzyme (CAZy) database and a custom database of nitrogen cycle genes. Gene profile variation was mostly explained by ecozone and soil layer. Eleven CAZy and nine nitrogen cycle gene families were associated with particular soil layers across all ecozones. Treatment effects on gene profiles were mainly due to harvesting, and only rarely to the extent of OM removal. Harvesting generally decreased the relative abundance of CAZy genes while increasing that of nitrogen cycle genes, although these effects varied among ecozones. Our results suggest that ecozone-specific nutrient availability modulates the sensitivity of the carbon and nitrogen cycles to harvesting with possible consequences for long-term forest sustainability.

Steroids are abundant growth substrates for bacteria in natural, engineered, and host-associated environments. This study analyzed the distribution of the aerobic 9,10-seco steroid degradation pathway in 346 publically available metagenomes from diverse environments. Our results show that steroid-degrading bacteria are globally distributed and prevalent in particular environments, such as wastewater treatment plants, soil, plant rhizospheres, and the marine environment, including marine sponges. Genomic signature-based sequence binning recovered 45 metagenome-assembled genomes containing a majority of 9,10-seco pathway genes. Only Actinobacteria and Proteobacteria were identified as steroid degraders, but we identified several alpha- and gammaproteobacterial lineages not previously known to degrade steroids. Actino- and proteobacterial steroid degraders coexisted in wastewater, while soil and rhizosphere samples contained mostly actinobacterial ones. Actinobacterial steroid degraders were found in deep ocean samples, while mostly alpha- and gammaproteobacterial ones were found in other marine samples, including sponges. Isolation of steroid-degrading bacteria from sponges confirmed their presence. Phylogenetic analysis of key steroid degradation proteins suggested their biochemical novelty in genomes from sponges and other environments. This study shows that the ecological significance as well as taxonomic and biochemical diversity of bacterial steroid degradation has so far been largely underestimated, especially in the marine environment. IMPORTANCE Microbial steroid degradation is a critical process for biomass decomposition in natural environments, for removal of important pollutants during wastewater treatment, and for pathogenesis of bacteria associated with tuberculosis and other bacteria. To date, microbial steroid degradation was mainly studied in a few model organisms, while the ecological significance of steroid degradation remained largely unexplored. This study provides the first analysis of aerobic steroid degradation in diverse natural, engineered, and host-associated environments via bioinformatic analysis of an extensive metagenome data set. We found that steroid-degrading bacteria are globally distributed and prevalent in wastewater treatment plants, soil, plant rhizospheres, and the marine environment, especially in marine sponges. We show that the ecological significance as well as the taxonomic and biochemical diversity of bacterial steroid degradation has been largely underestimated. This study greatly expands our ecological and evolutionary understanding of microbial steroid degradation., (Copyright © 2018 Holert et al.)

The growing demand for renewable, carbon-neutral materials and energy is leading to intensified forest land-use. The long-term ecological challenges associated with maintaining soil fertility in managed forests are not yet known, in part due to the complexity of soil microbial communities and the heterogeneity of forest soils. This study determined the long-term effects of timber harvesting, accompanied by varied organic matter (OM) removal, on bacterial and fungal soil populations in 11- to 17-year-old reforested coniferous plantations at 18 sites across North America. Analysis of highly replicated 16 S rRNA gene and ITS region pyrotag libraries and shotgun metagenomes demonstrated consistent changes in microbial communities in harvested plots that included the expansion of desiccation- and heat-tolerant organisms and decline in diversity of ectomycorrhizal fungi. However, the majority of taxa, including the most abundant and cosmopolitan groups, were unaffected by harvesting. Shifts in microbial populations that corresponded to increased temperature and soil dryness were moderated by OM retention, which also selected for sub-populations of fungal decomposers. Biogeographical differences in the distribution of taxa as well as local edaphic and environmental conditions produced substantial variation in the effects of harvesting. This extensive molecular-based investigation of forest soil advances our understanding of forest disturbance and lays the foundation for monitoring long-term impacts of timber harvesting.

The scarcity of long-term data on soil microbial communities in the decades following timber harvesting limits current understanding of the ecological problems associated with maintaining the productivity of managed forests. The high complexity of soil communities and the heterogeneity of forest and soil necessitates a comprehensive approach to understand the role of microbial processes in managed forest ecosystems. Here, we describe a curated collection of well replicated, multi-faceted data from eighteen reforested sites in six different North American ecozones within the Long-term Soil Productivity (LTSP) Study, without detailed analysis of results or discussion. The experiments were designed to contrast microbial community composition and function among forest soils from harvested treatment plots with varying intensities of organic matter removal. The collection includes 724 bacterial (16S) and 658 fungal (ITS2) amplicon libraries, 133 shotgun metagenomic libraries as well as stable isotope probing amplicon libraries capturing the effects of harvesting on hemicellulolytic and cellulolytic populations. This collection serves as a foundation for the LTSP Study and other studies of the ecology of forest soil and forest disturbance., Competing Interests: The authors declare no competing financial interests.

Soil management is vital for maintaining the productivity of commercial forests, yet the long-term impact of timber harvesting on soil microbial communities remains largely a matter of conjecture. Decomposition of plant biomass, comprised mainly of lignocellulose, has a broad impact on nutrient cycling, microbial activity and physicochemical characteristics of soil. At "Long-term Soil Productivity Study" sites in California dominated by Ponderosa pine, we tested whether clear-cut timber harvesting, accompanied by varying degrees of organic matter (OM) removal, affected the activity and structure of the cellulose-degrading microbial populations 16 years after harvesting. Using a variety of experimental approaches, including stable isotope probing with 13 C-labeled cellulose in soil microcosms, we demonstrated that harvesting led to a decrease in net respiration and cellulolytic activity. The decrease in cellulolytic activity was associated with an increased relative abundance of thermophilic, cellulolytic fungi (Chaetomiaceae), coupled with a decreased relative abundance of cellulolytic bacteria, particularly members of Opitutaceae, Caulobacter , and Streptomycetaceae. In general, harvesting led to an increase in stress-tolerant taxa (i.e., also non-cellulolytic taxa), though our results indicated that OM retention mitigated population shifts via buffering against abiotic changes. Stable-isotope probing improved shotgun metagenome assembly by 20-fold and enabled the recovery of 10 metagenome-assembled genomes of cellulolytic bacteria and fungi. Our study demonstrates the putative cellulolytic activity of a number of uncultured taxa and highlights the mineral soil layer as a reservoir of uncharacterized diversity of cellulose-degraders. It also and contributes to a growing body of research showing persistent changes in microbial community structure in the decades following forest harvesting.

Processes governing the fixation, partitioning, and mineralization of carbon in soils are under increasing scrutiny as we develop a more comprehensive understanding of global carbon cycling. Here we examined fixation by Douglas-fir seedlings and transfer to associated ectomycorrhizal fungi, soil microbes, and full-sibling or nonsibling neighbouring seedlings. Stable isotope probing with 99% 13 C-CO 2 was applied to trace 13 C-labelled photosynthate throughout plants, fungi, and soil microbes in an experiment designed to assess the effect of relatedness on 13 C transfer between plant pairs. The fixation and transfer of the 13 C label to plant, fungal, and soil microbial tissue was examined in biomass and phospholipid fatty acids. After a 6 d chase period, c. 26.8% of the 13 C remaining in the system was translocated below ground. Enrichment was proportionally greatest in ectomycorrhizal biomass. The presence of mesh barriers (0.5 or 35 μm) between seedlings did not restrict 13 C transfer. Fungi were the primary recipients of 13 C-labelled photosynthate throughout the system, representing 60-70% of total 13 C-enriched phospholipids. Full-sibling pairs exhibited significantly greater 13 C transfer to recipient roots in two of four Douglas-fir families, representing three- and fourfold increases (+ c. 4 μg excess 13 C) compared with nonsibling pairs. The existence of a root/mycorrhizal exudation-hyphal uptake pathway was supported., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Asthma is a chronic disease of the airways affecting one in ten children in Westernized countries. Recently, our group showed that specific bacterial genera in early life are associated with atopy and wheezing in 1-year-old children. However, little is known about the link between the early life gut microbiome and the diagnosis of asthma in preschool age children. To determine the role of the gut microbiota in preschool age asthma, children up to 4 years of age enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) study were classified as asthmatic (n=39) or matched healthy controls (n=37). 16S rRNA sequencing and quantitative PCR (qPCR) were used to analyse the composition of the 3-month and 1-year gut microbiome of these children. At 3 months the abundance of the genus, Lachnospira (L), was decreased (P=0.008), whereas the abundance of the species, Clostridium neonatale (C), was increased (P=0.07) in asthmatics. Quartile analysis of stool composition at 3-months revealed a negative association between the ratio of these two bacteria (L/C) and asthma risk by 4 years of age [quartile 1: odds ratio (OR)=15, P=0.02, CI (confidence interval)= 1.8-124.7; quartile 2: OR=1.0, ns; quartile 3: OR=0.37, ns]. We conclude that opposing shifts in the relative abundances of Lachnospira and C. neonatale in the first 3 months of life are associated with preschool age asthma, and that the L/C ratio may serve as a potential early life biomarker to predict asthma development., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Unlabelled: Steroids are ubiquitous in natural environments and are a significant growth substrate for microorganisms. Microbial steroid metabolism is also important for some pathogens and for biotechnical applications. This study delineated the distribution of aerobic steroid catabolism pathways among over 8,000 microorganisms whose genomes are available in the NCBI RefSeq database. Combined analysis of bacterial, archaeal, and fungal genomes with both hidden Markov models and reciprocal BLAST identified 265 putative steroid degraders within only Actinobacteria and Proteobacteria, which mainly originated from soil, eukaryotic host, and aquatic environments. These bacteria include members of 17 genera not previously known to contain steroid degraders. A pathway for cholesterol degradation was conserved in many actinobacterial genera, particularly in members of the Corynebacterineae, and a pathway for cholate degradation was conserved in members of the genus Rhodococcus. A pathway for testosterone and, sometimes, cholate degradation had a patchy distribution among Proteobacteria. The steroid degradation genes tended to occur within large gene clusters. Growth experiments confirmed bioinformatic predictions of steroid metabolism capacity in nine bacterial strains. The results indicate there was a single ancestral 9,10-seco-steroid degradation pathway. Gene duplication, likely in a progenitor of Rhodococcus, later gave rise to a cholate degradation pathway. Proteobacteria and additional Actinobacteria subsequently obtained a cholate degradation pathway via horizontal gene transfer, in some cases facilitated by plasmids. Catabolism of steroids appears to be an important component of the ecological niches of broad groups of Actinobacteria and individual species of Proteobacteria., Importance: Steroids are ubiquitous growth substrates for environmental and pathogenic bacteria, and bacterial steroid metabolism has important pharmaceutical and health applications. To date, the genetics and biochemistry of microbial steroid degradation have mainly been studied in a few model bacteria, and the diversity of this metabolism remains largely unexplored. Here, we provide a bioinformatically derived perspective of the taxonomic distribution of aerobic microbial steroid catabolism pathways. We identified several novel steroid-degrading bacterial groups, including ones from marine environments. In several cases, we confirmed bioinformatic predictions of metabolism in cultures. We found that cholesterol and cholate catabolism pathways are highly conserved among certain actinobacterial taxa. We found evidence for horizontal transfer of a pathway to several proteobacterial genera, conferring testosterone and, sometimes, cholate catabolism. The results of this study greatly expand our ecological and evolutionary understanding of microbial steroid metabolism and provide a basis for better exploiting this metabolism for biotechnology., (Copyright © 2016 Bergstrand et al.)

Forest ecosystems need to be sustainably managed, as they are major reservoirs of biodiversity, provide important economic resources and modulate global climate. We have a poor knowledge of populations responsible for key biomass degradation processes in forest soils and the effects of forest harvesting on these populations. Here, we investigated the effects of three timber-harvesting methods, varying in the degree of organic matter removal, on putatively hemicellulolytic bacterial and fungal populations 10 or more years after harvesting and replanting. We used stable-isotope probing to identify populations that incorporated (13)C from labeled hemicellulose, analyzing (13)C-enriched phospholipid fatty acids, bacterial 16 S rRNA genes and fungal ITS regions. In soil microcosms, we identified 104 bacterial and 52 fungal hemicellulolytic operational taxonomic units (OTUs). Several of these OTUs are affiliated with taxa not previously reported to degrade hemicellulose, including the bacterial genera Methylibium, Pelomonas and Rhodoferax, and the fungal genera Cladosporium, Pseudeurotiaceae, Capronia, Xenopolyscytalum and Venturia. The effect of harvesting on hemicellulolytic populations was evaluated based on in situ bacterial and fungal OTUs. Harvesting treatments had significant but modest long-term effects on relative abundances of hemicellulolytic populations, which differed in strength between two ecozones and between soil layers. For soils incubated in microcosms, prior harvesting treatments did not affect the rate of incorporation of hemicellulose carbon into microbial biomass. In six ecozones across North America, distributions of the bacterial hemicellulolytic OTUs were similar, whereas distributions of fungal ones differed. Our work demonstrates that diverse taxa in soil are hemicellulolytic, many of which are differentially affected by the impact of harvesting on environmental conditions. However, the hemicellulolytic capacity of soil communities appears resilient.

Soil is the key resource that must be managed to ensure sustainable forest productivity. Soil microbial communities mediate numerous essential ecosystem functions, and recent studies show that forest harvesting alters soil community composition. From a long-term soil productivity study site in a temperate coniferous forest in British Columbia, 21 forest soil shotgun metagenomes were generated, totaling 187 Gb. A method to analyze unassembled metagenome reads from the complex community was optimized and validated. The subsequent metagenome analysis revealed that, 12 years after forest harvesting, there were 16% and 8% reductions in relative abundances of biomass decomposition genes in the organic and mineral soil layers, respectively. Organic and mineral soil layers differed markedly in genetic potential for biomass degradation, with the organic layer having greater potential and being more strongly affected by harvesting. Gene families were disproportionately affected, and we identified 41 gene families consistently affected by harvesting, including families involved in lignin, cellulose, hemicellulose and pectin degradation. The results strongly suggest that harvesting profoundly altered below-ground cycling of carbon and other nutrients at this site, with potentially important consequences for forest regeneration. Thus, it is important to determine whether these changes foreshadow long-term changes in forest productivity or resilience and whether these changes are broadly characteristic of harvested forests.

The genus Bradyrhizobium has served as a model system for studying host-microbe symbiotic interactions and nitrogen fixation due to its importance in agricultural productivity and global nitrogen cycling. In this study, we identify a bacterial group affiliated with this genus that dominates the microbial communities of coniferous forest soils from six distinct ecozones across North America. Representative isolates from this group were obtained and characterized. Using quantitative population genomics, we show that forest soil populations of Bradyrhizobium represent ecotypes incapable of nodulating legume root hairs or fixing atmospheric nitrogen. Instead, these populations appear to be free living and have a greater potential for metabolizing aromatic carbon sources than their close symbiotic relatives. In addition, we identify fine-scaled differentiation between populations inhabiting neighboring soil layers that illustrate how diversity within Bradyrhizobium is structured by habitat similarity. These findings reconcile incongruent observations about this widely studied and important group of bacteria and highlight the value of ecological context to interpretations of microbial diversity and taxonomy. These results further suggest that the influence of this genus likely extends well beyond facilitating agriculture, especially as forest ecosystems are large and integral components of the biosphere. In addition, this study demonstrates how focusing research on economically important microorganisms can bias our understanding of the natural world.

Asthma is the most prevalent pediatric chronic disease and affects more than 300 million people worldwide. Recent evidence in mice has identified a "critical window" early in life where gut microbial changes (dysbiosis) are most influential in experimental asthma. However, current research has yet to establish whether these changes precede or are involved in human asthma. We compared the gut microbiota of 319 subjects enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study, and show that infants at risk of asthma exhibited transient gut microbial dysbiosis during the first 100 days of life. The relative abundance of the bacterial genera Lachnospira, Veillonella, Faecalibacterium, and Rothia was significantly decreased in children at risk of asthma. This reduction in bacterial taxa was accompanied by reduced levels of fecal acetate and dysregulation of enterohepatic metabolites. Inoculation of germ-free mice with these four bacterial taxa ameliorated airway inflammation in their adult progeny, demonstrating a causal role of these bacterial taxa in averting asthma development. These results enhance the potential for future microbe-based diagnostics and therapies, potentially in the form of probiotics, to prevent the development of asthma and other related allergic diseases in children., (Copyright © 2015, American Association for the Advancement of Science.)

Rationale: The relatively sparse but diverse microbiome in human lungs may become less diverse in chronic obstructive pulmonary disease (COPD). This article examines the relationship of this microbiome to emphysematous tissue destruction, number of terminal bronchioles, infiltrating inflammatory cells, and host gene expression., Methods: Culture-independent pyrosequencing microbiome analysis was used to examine the V3-V5 regions of bacterial 16S ribosomal DNA in 40 samples of lung from 5 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 4) and 28 samples from 4 donors (controls). A second protocol based on the V1-V3 regions was used to verify the bacterial microbiome results. Within lung tissue samples the microbiome was compared with results of micro-computed tomography, infiltrating inflammatory cells measured by quantitative histology, and host gene expression., Measurements and Main Results: Ten operational taxonomic units (OTUs) was found sufficient to discriminate between control and GOLD stage 4 lung tissue, which included known pathogens such as Haemophilus influenzae. We also observed a decline in microbial diversity that was associated with emphysematous destruction, remodeling of the bronchiolar and alveolar tissue, and the infiltration of the tissue by CD4(+) T cells. Specific OTUs were also associated with neutrophils, eosinophils, and B-cell infiltration (P < 0.05). The expression profiles of 859 genes and 235 genes were associated with either enrichment or reductions of Firmicutes and Proteobacteria, respectively, at a false discovery rate cutoff of less than 0.1., Conclusions: These results support the hypothesis that there is a host immune response to microorganisms within the lung microbiome that appears to contribute to the pathogenesis of COPD.

Separating edaphic impacts on tree distributions from those of climate and geography is notoriously difficult. Aboveground and belowground factors play important roles, and determining their relative contribution to tree success will greatly assist in refining predictive models and forestry strategies in a changing climate. In a common glasshouse, seedlings of interior Douglas-fir (Pseudotsuga menziesii var. glauca) from multiple populations were grown in multiple forest soils. Fungicide was applied to half of the seedlings to separate soil fungal and nonfungal impacts on seedling performance. Soils of varying geographic and climatic distance from seed origin were compared, using a transfer function approach. Seedling height and biomass were optimized following seed transfer into drier soils, whereas survival was optimized when elevation transfer was minimised. Fungicide application reduced ectomycorrhizal root colonization by c. 50%, with treated seedlings exhibiting greater survival but reduced biomass. Local adaptation of Douglas-fir populations to soils was mediated by soil fungi to some extent in 56% of soil origin by response variable combinations. Mediation by edaphic factors in general occurred in 81% of combinations. Soil biota, hitherto unaccounted for in climate models, interacts with biogeography to influence plant ranges in a changing climate., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Extensive regions of interior Douglas-fir (Pseudotsuga menziesii var. glauca, IDF) forests in North America are being damaged by drought and western spruce budworm (Choristoneura occidentalis). This damage is resulting from warmer and drier summers associated with climate change. To test whether defoliated IDF can directly transfer resources to ponderosa pine (Pinus ponderosae) regenerating nearby, thus aiding in forest recovery, we examined photosynthetic carbon transfer and defense enzyme response. We grew pairs of ectomycorrhizal IDF 'donor' and ponderosa pine 'receiver' seedlings in pots and isolated transfer pathways by comparing 35 μm, 0.5 μm and no mesh treatments; we then stressed IDF donors either through manual defoliation or infestation by the budworm. We found that manual defoliation of IDF donors led to transfer of photosynthetic carbon to neighboring receivers through mycorrhizal networks, but not through soil or root pathways. Both manual and insect defoliation of donors led to increased activity of peroxidase, polyphenol oxidase and superoxide dismutase in the ponderosa pine receivers, via a mechanism primarily dependent on the mycorrhizal network. These findings indicate that IDF can transfer resources and stress signals to interspecific neighbors, suggesting ectomycorrhizal networks can serve as agents of interspecific communication facilitating recovery and succession of forests after disturbance.

Cholesterol catabolism is thought to be a key factor contributing to the pathogenesis of Mycobacterium tuberculosis. Previous epistasis and mutant screening studies predicted that the P55 efflux pump (Rv1410c) positively interacts with the Mce4 transporter, a major cholesterol import system of M. tuberculosis and is needed for optimal growth in vitro, in macrophages, and in vivo. Using a combination of cell growth kinetic techniques, cholesterol consumption, and [4-(14)C]cholesterol uptake studies, we demonstrated that the Mycobacterium bovis BCG rv1410c gene indeed is needed for optimal in vitro growth on cholesterol and other carbon sources. Our data, together with previous predictions, support hypotheses that the P55 efflux pump functions in maintaining general metabolism or as a subunit of the Mce4 transport apparatus (catalyzing its assembly or providing cell wall integrity) to allow more efficient cholesterol uptake.

Background: Resident gut microbiota are now recognized as potent modifiers of host immune responses in various scenarios. Recently, we demonstrated that perinatal exposure to vancomycin, but not streptomycin, profoundly alters gut microbiota and enhances susceptibility to a TH2 model of allergic asthma., Objective: Here we sought to further clarify the etiology of these changes by determining whether perinatal antibiotic treatment has a similar effect on the TH1/TH17-mediated lung disease, hypersensitivity pneumonitis., Methods: Hypersensitivity pneumonitis was induced in C57BL/6 wild-type or recombination-activating gene 1-deficient mice treated perinatally with vancomycin or streptomycin by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Disease severity was assessed by measuring lung inflammation, pathology, cytokine responses, and serum antibodies. Microbial community analyses were performed on stool samples via 16S ribosomal RNA pyrosequencing and correlations between disease severity and specific bacterial taxa were identified., Results: Surprisingly, in contrast to our findings in an allergic asthma model, we found that the severity of hypersensitivity pneumonitis was unaffected by vancomycin, but increased dramatically after streptomycin treatment. This likely reflects an effect on the adaptive, rather than innate, immune response because the effects of streptomycin were not observed during the early phases of disease and were abrogated in recombination-activating gene 1-deficient mice. Interestingly, Bacteroidetes dominated the intestinal microbiota of streptomycin-treated animals, while vancomycin promoted the expansion of the Firmicutes., Conclusions: Perinatal antibiotics exert highly selective effects on resident gut flora, which, in turn, lead to very specific alterations in susceptibility to TH2- or TH1/TH17-driven lung inflammatory disease., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a major soil and groundwater contaminant. Organisms such as Gordonia sp. KTR9, capable of degrading RDX and using it as an N source, may prove useful for bioremediation of contaminated sites. XplA is a cytochrome P450 monooxygenase responsible for RDX degradation. Expression of xplA in KTR9 was not induced by RDX but was strongly induced (50-fold) during N-limited growth. When glnR, encoding a regulatory protein affecting N assimilation in diverse Actinobacteria, was deleted from KTR9, the bacterium lost the ability to use nitrate, nitrite, and RDX as N sources. Deletion of glnR also abolished the inhibition of xplA expression by nitrite. Our results confirm the essential role of GlnR in regulating assimilation of nitrite, but there was no evidence for a direct role of GlnR in regulating XplA expression. Rather, the general availability of nitrogen repressed XplA expression. We conclude that the inability of the glnR mutant to use RDX as an N source was due to its inability to assimilate nitrite, an intermediate in the assimilation of nitrogen from RDX. Regulation of XplA does not seem adaptive for KTR9, but it is important for RDX bioremediation with KTR9 or similar bacteria.

Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating (13)C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography-tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% (13)C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Engineering the microbial transformation of lignocellulosic biomass is essential to developing modern biorefining processes that alleviate reliance on petroleum-derived energy and chemicals. Many current bioprocess streams depend on the genetic tractability of Escherichia coli with a primary emphasis on engineering cellulose/hemicellulose catabolism, small molecule production, and resistance to product inhibition. Conversely, bioprocess streams for lignin transformation remain embryonic, with relatively few environmental strains or enzymes implicated. Here we develop a biosensor responsive to monoaromatic lignin transformation products compatible with functional screening in E. coli. We use this biosensor to retrieve metagenomic scaffolds sourced from coal bed bacterial communities conferring an array of lignin transformation phenotypes that synergize in combination. Transposon mutagenesis and comparative sequence analysis of active clones identified genes encoding six functional classes mediating lignin transformation phenotypes that appear to be rearrayed in nature via horizontal gene transfer. Lignin transformation activity was then demonstrated for one of the predicted gene products encoding a multicopper oxidase to validate the screen. These results illuminate cellular and community-wide networks acting on aromatic polymers and expand the toolkit for engineering recombinant lignin transformation based on ecological design principles.