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Background The advent of culture-independent, next-generation DNA sequencing has led to the discovery of distinct lung bacterial communities. Studies of lung microbiome taxonomy often reveal only subtle differences between health and disease, but host recognition and response may distinguish the members of similar bacterial communities in different populations. Magnetic-activated cell sorting has been applied to the gut microbiome to identify the numbers and types of bacteria eliciting a humoral response. We adapted this technique to examine the populations of immunoglobulin-bound bacteria in the lung. Methods Sixty-four individuals underwent bronchoalveolar lavage (BAL). We separated immunoglobulin G-bound bacteria using magnetic-activated cell sorting and sequenced the 16S rRNA gene on the Illumina MiSeq platform. We compared microbial sequencing data in IgG-bound bacterial communities compared to raw BAL then examined the differences in individuals with and without HIV as a representative disease state. Results Immunoglobulin G-bound bacteria were identified in all individuals. The community structure differed when compared to raw BAL, and there was a greater abundance of Pseudomonas and fewer oral bacteria in IgG-bound BAL. Examination of IgG-bound communities in individuals with HIV demonstrated the differences in Ig-bound bacteria by HIV status that were not seen in a comparison of raw BAL, and greater numbers of immunoglobulin-bound bacteria were associated with higher pulmonary cytokine levels. Conclusions We report a novel application of magnetic-activated cell sorting to identify immunoglobulin G-bound bacteria in the lung. This technique identified distinct bacterial communities which differed in composition from raw bronchoalveolar lavage, revealing the differences not detected by traditional analyses. Cytokine response was also associated with differential immunoglobulin binding of lung bacteria, suggesting the functional importance of these communities.

BackgroundChronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). There are currently no standard of care guidelines for the management of CF CRS, but treatment of upper airway disease may ameliorate lower airway disease. We sought to inform future treatment guidelines by determining whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation.MethodsWe performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS at the University of Pittsburgh’s CF Sinus Clinic. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation.FindingsSinus community diversity was low and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and Staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased IL-1β). Increased IL-1β was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics.InterpretationTo our knowledge, this is the largest longitudinal study of microbial communities and cytokine secretion in CF CRS. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease.FundingCystic Fibrosis Foundation and US National Institutes of Health.Research in ContextEvidence before this studyA search of the PubMed database on October 11, 2021 with the terms [cystic fibrosis sinus microbiome] yielded 16 results, and we have identified seven primary research articles on the CF CRS microbiome (including re-analyses of existing datasets). Most are cross-sectional cohort analyses, along with one prior longitudinal study of four adults at the University of Auckland, New Zealand. Together, these prior studies reveal similarities between CF CRS and CF sputum microbiomes, including low community diversity associated with sinus disease, the presence of common CF-associated microbes in the sinuses, and prevalence of sinus communities dominated by P. aeruginosa or Staphylococcus aureus. High levels of IL-1β are linked to the presence of nasal polyps in CF CRS, and polymorphisms in the IL-1 receptor antagonist gene are associated with risk of CRS outside of the context of CF. Two prior studies of this cohort have been performed by our laboratory. One describes clinical indicators of CF sinus disease and the other links sinus infection biogeography to P. aeruginosa evolutionary genomics.Added value of this studyOur study is the first to examine longitudinal relationships between the host immune response (through cytokine profiling) and microbiota dynamics in CF CRS, including linking elevated IL-1β to worse sinus disease through reduced sinus microbial community diversity. The longitudinal nature of our study also allowed us to uncover striking temporal instability of microbial communities in approximately half of our cohort’s sinus microbial communities over two years, including switching between communities dominated by P. aeruginosa and Staphylococcus spp. This instability could hinder attempts to link the relative abundance of taxa to clinical outcomes of interest in cross-sectional studies (e.g., markers of disease progression). We also identified patterns of synergy and antagonism between specific taxa, and impacts of the host immune response in the sinuses on community composition.Implications of all the available evidenceTogether with prior CF CRS microbiome studies, our study underscores similarities between sinus and lower respiratory tract microbial community structure in CF, and we show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1β in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1β. Finally, the widespread use of highly effective CFTR modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Imposition of social and health behavior mitigations are important control measures in response to the coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Although postulated that these measures may impact the human microbiota including losses in diversity from heightened hygiene and social distancing measures, this hypothesis remains to be tested. Other impacts on the microbiota and host mental and physical health status associations from these measures are also not well-studied. Here we examine changes in stool and oral microbiota by analyzing 16S rRNA gene sequence taxonomic profiles from the same individuals during pre-pandemic (before March 2020) and early pandemic (May-November 2020) phases. During the early pandemic phase, individuals were also surveyed using questionnaires to report health histories, anxiety, depression, sleep and other lifestyle behaviors in a cohort of predominantly Caucasian adults (mean age = 61.5 years) with the majority reporting at least one underlying co-morbidity. We identified changes in microbiota (stool n = 288; oral n = 89) between pre-pandemic and early pandemic time points from the same subject and associated these differences with questionnaire responses using linear statistical models and hierarchical clustering of microbiota composition coupled to logistic regression. While a trend in loss of diversity was identified between pre-pandemic and early pandemic time points it was not statistically significant. Paired difference analyses between individuals identified fewer significant changes between pre-pandemic and early pandemic microbiota in those who reported fewer comorbidities. Cluster transition analyses of stool and saliva microbiota determined most individuals remained in the same cluster assignments from the pre-pandemic to early pandemic period. Individuals with microbiota that shifted in composition, causing them to depart a pre-pandemic cluster, reported more health issues and pandemic-associated worries. Collectively, our study identified that stool and saliva microbiota from the pre-pandemic to early pandemic periods largely exhibited ecological stability (especially stool microbiota) with most associations in loss of diversity or changes in composition related to more reported health issues and pandemic-associated worries. Longitudinal observational cohorts are necessary to monitor the microbiome in response to pandemics and changes in public health measures.

Undernutrition is a public health challenge in sub-Saharan countries, including Uganda. In a previous randomized controlled trial (RCT) with a nutrition, hygiene and stimulation education intervention among mothers of 6 months’ old children, we found less caries in the intervention group when the children were 36 months of age. We now examined the effects of (i) the intervention on the microbiota, (ii) microbiota on caries, and (iii) the intervention and microbiota on caries. The original RCT comprised 511 mother/child pairs whereas in the current study we had access to data from 344/511 (67%) children aged 36 months. The saliva microbiota was determined using 16S rRNA gene sequencing. Carious lesions (a proxy for dental health) were identified using close-up intra-oral photographs of the upper front teeth. Statistical models were used to determine host-microbiota associations. The intervention had a significant effect on the microbiota, e.g. an increase in Streptococcus abundance and decreases in Alloprevotella and Tannerella. Significant associations between the microbiota and dental caries were identified: Positive associations of Capnocytophaga and Tannerella suggest that these taxa may be deleterious to dental health while negative associations of Granulicatella, Fusobacterium, and Abiotrophia suggest taxa potentially beneficial or benign contributors to dental health. Based on taxonomic profiles, the effects of the intervention and microbiota on dental health may be independent of one another. Educational interventions with emphasis on nutrition and oral hygiene may provide a feasible strategy to decrease progression of childhood caries in low-resource settings.

Background: The advent of culture-independent, next-generation DNA sequencing has led to discovery of distinct lung bacterial communities. Studies of lung microbiome taxonomy often reveal only subtle differences between health and disease, but microbial host response may distinguish members of similar communities in different populations. Magnetic-activated cell sorting has been applied to the gut microbiome to identify numbers and types of bacteria eliciting a humoral response. We adapted this technique to examine populations of immunoglobulin-bound bacteria and investigate the lung microbiota in Human Immunodeficiency Virus (HIV) as a representative disease.Methods: 42 people living with HIV and 22 HIV-uninfected individuals underwent bronchoalveolar lavage. We separated immunoglobulin G-bound bacteria using magnetic-activated cell sorting and sequenced the 16S rRNA gene on the Illumina MiSeq platform. We analyzed microbial sequencing data and quantified cytokines and bacterial rRNA copy numbers from bronchoalveolar lavage. Results: Immunoglobulin G-bound bacteria were detectable in the healthy lung microbiota. Comparison of raw bronchoalveolar lavage by HIV status showed no significant taxonomic differences, but the immunoglobulin-bound lung microbiota differed by HIV status with greater abundance of Pseudomonas in people living with HIV. Lung cytokine levels were also higher in people living with HIV, which correlated with increased quantity of immunoglobulin G-bound bacteria. Conclusions: We report a novel application of magnetic-activated cell sorting to identify immunoglobulin G-bound bacteria in the lung. This technique identified distinct bacterial communities which differed in composition from raw bronchoalveolar lavage, revealing differences in health and disease not detected by traditional analyses. Cytokine response was also associated with differential immunoglobulin binding of lung bacteria, suggesting functional importance of these communities.

Total body irradiation (TBI) has been demonstrated to alter the intestinal microbiome, but the effects of successful small molecule ionizing radiation mitigators on the intestinal microbiome are not well-known. Our survival experiments examined the effects of anti-cell death radiation mitigators on and in conjunction with the host’s microbiota. Mice received 9.25 Gy TBI and then were administered radiation mitigators 24 hours later. Passed stool were collected pre-irradiation, then on days 1, 3, 5, 7, 10, 14, 21, and 30 post-irradiation for 16S rRNA gene (V4 region) sequencing. The Cox proportional hazards (CPH) model was fit with taxonomic composition (time varying covariates) and treatment as predictors. In the first experiment, mice were administered drugs for “granulocyte stimulation and anti-apoptosis” in four protocol combinations: JP4-039 (anti-apoptosis), granulocyte colony-stimulating factor (G-CSF, granulopoietic precursor cell stimulator), both mitigators, and control. Survival improved relative to control (30.0%) for G-CSF (80%, p-value = 0.025), G-CSF/JP4-039 (70%, p-value = 0.084), but not for JP4-039 (50.0%). In the second experiment, mice were administered mitigation drugs “inhibiting programmed cell death” pathways: JP4-039 (anti-apoptosis), necrostatin-1 (anti-necroptosis), and baicalein (anti-ferroptosis), in eight combinations. The survival of JP4-039/baicalein (60.0%, p-value = 0.010) and JP4-039/baicalein/necrostatin-1 (60.0%, p-value = 0.06) treatment combinations were significantly different from the control (26.7%). The JP4-039/necrostatin-1 (46.7%) and baicalein/necrostatin-1 (40.0%) and singlet treatment combinations (26.7%) were not significantly different from the control. Despite differences between the baseline microbiota compositions of the two experiments, consistent changes in composition after irradiation were found: Lactobacillus decreased post-irradiation, relative to baseline. By day 7, microbiota perturbations had incompletely reversed, and no drug-specific differences were identifiable. The CPH model identified Lactobacillus and members of Ruminococcaceae, including Ruminococcus, as protective and Akkermansia as deleterious. By day 30, the microbiota of surviving mice had not returned to baseline, but the differences between experiments suggest the resultant microbiota composition of the survivors are stochastic or batch specific in nature, rather than a requirement for survival. In conclusion, the study determined that key taxa identified in fecal samples, when applied towards the prediction of TBI survival, improves the survival model relative to treatment information alone.

The microbiome has been proposed to play a role in the progression of idiopathic pulmonary fibrosis (IPF) based on bronchoalveolar lavage analyses, but the microbiome of lung tissue in IPF has not been explored. In a case–control study of lung explants analysed by 16S rRNA gene sequencing, we could not reliably detect bacterial DNA in basilar tissue samples from patients with either chronic or acute exacerbations of IPF, in contrast to control candidate-donor lungs or cystic fibrosis explants. Thus, our data do not indicate microbiome alterations in regions of IPF lung with advanced fibrosis.

Rationale: Mechanisms of HIV-associated chronic obstructive pulmonary disease (COPD) are poorly understood. The oral microbiome shapes the lung microbiome, and gut dysbiosis can affect lung diseases; however, relationships of the oral and gut microbiome to COPD in HIV have not been explored. Objectives: To examine alterations in the oral and gut microbiome associated with pulmonary disease in persons with HIV (PWH). Methods: Seventy-five PWH and 93 HIV-uninfected men from the MACS (Multicenter AIDS Cohort Study) performed pulmonary function testing. Sequencing of bacterial 16S ribosomal RNA in saliva and stool was performed. We used nonmetric multidimensional scaling, permutational multivariate ANOVA, and linear discriminant analysis to analyze communities by HIV and lung function. Measurements and Main Results: Oral microbiome composition differed by HIV and smoking status. Alterations of oral microbial communities were observed in PWH with abnormal lung function with increases in relative abundance of Veillonella, Streptococcus, and Lactobacillus. There were no significant associations between the oral microbiome and lung function in HIV-uninfected individuals. No associations with HIV status or lung function were seen with the gut microbiome. Conclusions: Alterations of oral microbiota in PWH were related to impaired pulmonary function and to systemic inflammation. These results suggest that the oral microbiome may serve as a biomarker of lung function in HIV and that its disruption may contribute to COPD pathogenesis.

IntroductionAs the population ages, there is interest in strategies to promote resiliency, especially for frail patients at risk of its complications. The physiological stress of surgery in high-risk individuals has been proposed both as an important cause of accelerated age-related decline in health and as a model testing the effectiveness of strategies to improve resiliency to age-related health decline. We describe a randomised, embedded, multifactorial, adaptative platform (REMAP) trial to investigate multiple perioperative interventions, the first of which is metformin and selected for its anti-inflammatory and anti-ageing properties beyond its traditional blood glucose control features.Methods and analysisWithin a multihospital, single healthcare system, the Core Protocol for Strategies to Promote ResiliencY (SPRY) will be embedded within both the electronic health record (EHR) and the healthcare culture generating a continuously self-learning healthcare system. Embedding reduces the administrative burden of a traditional trial while accessing and rapidly analysing routine patient care EHR data. SPRY-Metformin is a placebo-controlled trial and is the first SPRY domain evaluating the effectiveness of three metformin dosages across three preoperative durations within a heterogeneous set of major surgical procedures. The primary outcome is 90-day hospital-free days. Bayesian posterior probabilities guide interim decision-making with predefined rules to determine stopping for futility or superior dosing selection. Using response adaptative randomisation, a maximum of 2500 patients allows 77%–92% power, detecting >15% primary outcome improvement. Secondary outcomes include mortality, readmission and postoperative complications. A subset of patients will be selected for substudies evaluating the microbiome, cognition, postoperative delirium and strength.Ethics and disseminationThe Core Protocol of SPRY REMAP and associated SPRY-Metformin Domain-Specific Appendix have been ethically approved by the Institutional Review Board and are publicly registered. Results will be publicly available to healthcare providers, patients and trial participants following achieving predetermined platform conclusions.Trial registration numberNCT03861767.

Infection with influenza can be aggravated by bacterial co-infections, which often results in disease exacerbation because of host responses and cellular damage. The native upper respiratory tract (URT) microbiome likely plays a role, yet the effects of influenza infection on the URT microbiome are largely unknown. We performed a longitudinal study to assess the temporal dynamics of the URT microbiomes of uninfected and influenza virus-infected humans and ferrets. Uninfected human patients and ferret URT microbiomes had stable “heathy ecostate” communities both within and between individuals. In contrast, infected patients and ferrets exhibited large changes in bacterial community composition over time and between individuals. The “unhealthy” ecostates of infected individuals progressed towards the “healthy ecostate” over time, coinciding with viral clearance and recovery. Blooms of Pseudomonas were a statistically associated constant in the disturbed microbiomes of infected individuals. The dynamic and resilient nature of the microbiome during influenza virus infection in multiple hosts provides a compelling rationale for the maintenance of the microbiome homeostasis as a potential therapeutic target to prevent IAV associated bacterial co-infections.One Sentence SummaryDynamics of the upper respiratory tract microbiome during influenza A virus infection

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.—Cox, L. M., Cho, I., Young, S. A., Kerr Anderson, W. H., Waters, B. J., Hung, S.-C., Gao, Z., Mahana, D., Bihan, M., Alekseyenko, A. V., Methé, B. A., Blaser, M. J. The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota.

Antibiotics administered in low doses have been widely used as growth promoters in the agricultural industry since the 1950s, yet the mechanisms for this effect are unclear. Because antimicrobial agents of different classes and varying activity are effective across several vertebrate species, we proposed that such subtherapeutic administration alters the population structure of the gut microbiome as well as its metabolic capabilities. We generated a model of adiposity by giving subtherapeutic antibiotic therapy to young mice and evaluated changes in the composition and capabilities of the gut microbiome. Administration of subtherapeutic antibiotic therapy increased adiposity in young mice and increased hormone levels related to metabolism. We observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol. In this model, we demonstrate the alteration of early-life murine metabolic homeostasis through antibiotic manipulation.

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies.

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

The modern ichthyofauna of the Southern Ocean is uniquely depauperate with respect to species diversity (Eastman, 2005). Although the Southern Ocean constitutes approximately 10% of our planet’s oceanic waters, the number of fish species is quite small (~300 species vs. a global diversity of ~25,000 species), and almost half belong to a single perciform suborder, the Notothenioidei. The origin of this taxonomic restriction results from the dramatic paleoclimatic and paleogeographic history of Antarctica (Eastman, ’93) during the Cenozoic Era. The opening of the Drake Passage, which separates Antarctica from South America, occurred via tectonic rifting ~40–34 million years (Myr) ago (Kennett, ’77; Livermore et al., 2005; Scher and Martin, 2006). The onset of widespread glaciation of Antarctica ~34 Myr ago (Zachos et al., 2001), which is attributable to declining atmospheric CO2 (DeConto and Pollard, 2003), the establishment of the Antarctic Circumpolar Current and the development of the Antarctic Polar Front (between ~50 and 60°S), decoupled the Southern Ocean from warmer, subtropical waters to the north (Kennett, ’77). A cooling trend that began in the late Paleocene/early Eocene (~55 Myr ago), when water temperatures were 15–20°C, accelerated during the Miocene until the Southern Ocean approached the freezing point of seawater (−1.9°C) ~14–5 Myr ago (Clarke, ’90). As the Southern Ocean cooled, the shallow water, cosmopolitan, and temperate fish fauna of late Eocene (38 Myr ago) became largely extinct owing to the destruction of inshore habitat and changes in trophic structure caused by repeated ice sheet scouring of the continental margin (Eastman, 2005). Thus, fish diversity was reduced and new ecological niches became available to taxa, such as the notothenioid fishes, which were diversifying in situ. Today, species of the notothenioid suborder constitute 46% of all the fish species of the Southern Ocean. At the highest latitudes, notothenioid species dominance is particularly clear, encompassing 77% of species diversity and 90% of biomass (Eastman, 2005). The ancestral notothenioid stock probably arose as a sluggish, bottom-dwelling perciform species some 40–60 Myr ago in the cool temperate shelf waters of the Antarctic continent (DeWitt, ’71; Eastman, ’93; Eastman and Clarke, ’98). With the elimination of most of the Eocene ichthyofauna, the notothenioids diversified to fill the many vacated niches. Lacking swim bladders, the Notothenioidei evolved toward pelagic or partially pelagic zooplanktivory and piscivory by reduction of skeletal mineralization and enhancement of lipid deposition. Termed pelagization, the tailoring of morphology for life in the water column is the hallmark of the notothenioid radiation, has arisen independently several times in different clades (Eastman, ’97, ’99; Near et al., 2007), and reflects the retention of larval characteristics in the adult (pedomorphism) (Eastman, ’97). In the dominant family Nototheniidae, ~50% of the Antarctic species are semipelagic, epibenthic, cryopelagic, or pelagic (Eastman, 2005). On the basis of their rapid speciation (average time for speciation of 0.76–2.1 Myr), geographical restriction, and high endemism, the Notothenioidei are the best described example of a marine species flock (Eastman, 2000; Eastman and McCune, 2000). Absence of niche competition alone would not have guaranteed the radiation of the notothenioids had they not evolved biochemical and physiological adaptations to their frigid environment. One key evolutionary adaptation of the notothenioids was the de novo acquisition of antifreeze glycoproteins, which bind to the surfaces of ice crystals that form in these fishes to inhibit ice propagation in their hyposmotic fluids (Cheng, ’98; Cheng and Detrich, 2007). The evolution of the antifreeze glycoprotein genes from a pancreatic, trypsinogen-like gene (Chen et al., ’97; Cheng and Chen, ’99) is a remarkable exemplar of a critical genetic innovation that fostered an extensive species radiation (Montgomery and Clements, 2000). The notothenioids are notable not only for their resistance and compensatory adaptations to the extreme Antarctic marine environment, but also for their regressive evolutionary changes or losses of function [the “disaptations” of Montgomery and Clements (2000) and references therein]. Antarctic notothenioids are cold stenotherms—these high latitude species typically live at temperatures between −1.9 and +2°C and have incipient lethal temperatures near 5°C that cannot be raised by acclimation in the laboratory (Somero and DeVries, ’67). This stenothermality results in part from adaptive alterations to the biochemical, cellular, and physiological systems of these fishes. Major examples of compensatory adaptation include efficient microtubule assembly (Williams et al., ’85; Detrich et al., ’89, ’92, 2000; Paluh et al., 2004) and protein translocation at cold temperatures (Romisch et al., 2003), homeoviscous adaptation of membrane lipids to preserve membrane fluidity (Logue et al., 2000), and cold-stable lens crystallins that prevent cataract formation (Kiss et al., 2004). Consistent with their long isolation in a constantly cold environment, the Antarctic notothenioids have lost the inducible heat shock response (Hofmann et al., 2000; Buckley et al., 2004), which seems to have been recruited to an adaptive, constitutive status to deal with elevated denaturation of proteins caused by cold stress (Place et al., 2004; Place and Hofmann, 2005; Todgham et al., 2007). Other striking regressive changes include the loss of erythrocytes (Ruud, ’54) and the respiratory transport protein hemoglobin (Cocca et al., ’95, 2000; Zhao et al., ’98; Near et al., 2006) by all species of the icefish family and independent losses of cardiac myoglobin in a subset of icefish species (Sidell et al., ’97; O’Brien and Sidell, 2000; Sidell and O’Brien, 2006). Recently, Chen et al. (2008) reported a large-scale transcriptomic up-regulation of 177 gene families in the Antarctic toothfish, Dissostichus mawsoni (Nototheniodei: Nototheniidae), with respect to their orthologs in temperate and tropical fish. At the genomic level, they found that 118 gene families in several notothenioid species have undergone substantial gene duplication relative to the temperate/tropical species. Overlap of the duplicated gene set with the transcriptionally up-regulated gene set suggested that gene family expansion may contribute to cold adaptation of cellular and physiological function, a conclusion supported by the augmentation of tubulin genes in the bullhead notothen, Notothenia coriiceps (Parker and Detrich, ’98). It is notable that 17 families of LINEs showed Antarctic-specific duplication, which suggests that LINE expansion may have facilitated gene duplication in Antarctic notothenioids by retrotransposition-mediated gene transduction (Xing et al., 2006). The above findings suggest that the genomes of notothenioids are evolutionarily dynamic and that this plasticity may have contributed to the overall success of the group. The development of genomic resources from these difficult-to-obtain specimens is thus important, well justified, and valuable to a wide community of genomic, evolutionary, and ecological biologists and biomedical scientists. In 2003, we collected high molecular weight (HMW) DNA from a wide assortment of notothenioid fishes, with the aim of enabling genomic studies in this group, and subsequently submitted a white paper to the BAC library network at the National Human Genome Research Institute (NHGRI) to request construction of high-quality and high-representation BAC genomic libraries from selected notothenioid species. We were informed that BAC libraries would be generated from two of the species: Chaenocephalus aceratus (blackfin or Scotia Arc icefish) and N. coriiceps (bullhead notothen or yellowbelly rockcod). The selection of these two species was largely strategic in that C. aceratus represents a lineage (icefishes) devoid of erythrocytes and lacking hemoglobin genes, whereas N. coriiceps represents an erythrocyte-producing species in which hemoglobin genes are intact and functional. Thus, these two species, although not representative of the entire gamut of notothenioids, are extremely useful for genomic comparisons of processes that involve differences in their respective physiologies and anatomies as a result of hemoglobin loss by the icefishes. In this article, we describe the generation of BAC libraries from these 2 representative notothenioid species and further provide genome size estimates for 11 species of notothenioid fishes. Quality assurance data are provided for both BAC libraries that validate the utility of these resources for genomic analyses.

Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified by microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high-affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium-bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve because of the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.

The development of new technologies to measure gene expression has been calling for statistical methods to integrate findings across multiple-platform studies. A common goal of microarray analysis is to identify genes with differential expression between two conditions, such as treatment versus control. Here, we introduce a hierarchical Bayesian meta-analysis model to pool gene expression studies from different microarray platforms: spotted DNA arrays and short oligonucleotide arrays. The studies have different array design layouts, each with multiple sources of data replication, including repeated experiments, slides and probes. Our model produces the gene-specific posterior probability of differential expression, which is the basis for inference. In simulations combining two and five independent studies, our meta-analysis model outperformed separate analyses for three commonly used comparison measures; it also showed improved receiver operating characteristic curves. When combining spotted DNA and Combi...

Insertion sequences (ISs) are mobile genetic elements widely distributed among bacteria. Their impact on the bacterial genome is multifold, including transfer of genetic information, shuttle of adaptive traits and influence on the genomic content. As a result, ISs play an important role in the organization, plasticity and evolution of bacterial genomes. In this study, four new IS elements: ISMbov7; ISMbov4 and ISMbov5; and ISMbov6, related, respectively, to the IS3, IS4 and IS30 gene families, were identified and characterized with respect to inverted repeat (IR) and directly repeated (DR) sequences, putative target specificity and motifs related to transposase function. For instance, IS30-related ISMbov6 isoform elements were shown to (1) contain an alpha-helix-turn-alpha-helix homeodomain (HTH), (2) generate long DR and (3) possess target specificity for a palindromic sequence derived from putative rho-independent transcription terminators. Members of the IS3 family, which had not been documented previously in Mycoplasma bovis, contain HTH, leucine zipper and AT-hook motifs, which may be involved in DNA binding. In addition, the availability of the M. bovis PG45 genome sequence allowed us to elucidate the genomic organization of 54 intact or truncated IS elements and their possible effect on the expression of adjacent genes.

Geobacteraceae are a family of metal reducing bacteria with important applications in bioremediation and electricity generation. G. sulfurreducens is a representative of Geobacteraceae that has been extensively studied with the goal of extending the understanding of this family of organisms for optimizing their practical applications. Here, we have analyzed gene expression data from 10 experiments involving environmental and genetic perturbations and have identified putative transcription factor binding sites (TFBS) involved in regulating key aspects of metabolism. Specifically, we considered data from both a subset of 10 microarray experiments (7 of 10) and all 10 experiments. The expression data from these two sets were independently clustered, and the upstream regions of genes and operons from the clusters in both sets were used to identify TFBS using the AlignACE program. This analysis resulted in the identification of motifs upstream of several genes involved in central metabolism, sulfate assimilation, and energy metabolism, as well as genes potentially encoding acetate permease. Further, similar TFBS were identified from the analysis of both sets, suggesting that these TFBS are significant in the regulation of metabolism in G. sulfurreducens. In addition, we have utilized microarray data to derive condition specific constraints on the capacity of key enzymes in central metabolism. We have incorporated these constraints into the metabolic model of G. sulfurreducens and simulated Fe(II)-limited growth. The resulting prediction was consistent with data, suggesting that regulatory constraints are important for simulating growth phenotypes in nonoptimal environments.

Growth using Fe(III) as a terminal electron acceptor is a critical physiological process in Geobacter sulfurreducens. However, the mechanisms of electron transfer during Fe(III) reduction are only now being understood. It has been demonstrated that the pili in G. sulfurreducens function as microbial nanowires conducting electrons onto Fe(III) oxides. A number of c-type cytochromes have also been shown to play important roles in Fe(III) reduction. However, the regulatory networks controlling the expression of the genes involved in such processes are not well known. Here we report that the expression of pilA, which encodes the pilistructural protein, is directly regulated by a two-component regulatory system in which PilR functions as an RpoN-dependent enhancer binding protein. Surprisingly, a deletion of the pilR gene affected not only insoluble Fe(III) reduction, which requires pili, but also soluble Fe(III) reduction, which, in contrast, does not require pili. Gene expression profiling using whole-genome DNA microarray and quantitative RT-PCR analyses obtained with a PilR-deficient mutant revealed that the expression of pilA and other pilin-related genes are downregulated, while many c-type cytochromes involved in Fe(III) reduction were differentially regulated. This is the first instance of an enhancer binding protein implicated in regulating genes involved in Fe(III) respiratory functions.

A soluble ferric reductase, SfrAB, which catalysed the NADPH-dependent reduction of chelated Fe(III), was previously purified from the dissimilatory Fe(III)-reducing micro-organism Geobacter sulfurreducens, suggesting that reduction of chelated forms of Fe(III) might be cytoplasmic. However, metabolically active spheroplast suspensions could not catalyse acetate-dependent Fe(III) citrate reduction, indicating that periplasmic and/or outer-membrane components were required for Fe(III) citrate reduction. Furthermore, phenotypic analysis of an SfrAB knockout mutant suggested that SfrAB was involved in acetate metabolism rather than respiration-linked Fe(III) reduction. The mutant could not grow via the reduction of either Fe(III) citrate or fumarate when acetate was the electron donor but could grow with either acceptor if either hydrogen or formate served as the electron donor. Following prolonged incubation in acetate : fumarate medium in the absence of hydrogen and formate, an 'acetate-adapted' SfrAB-null strain was isolated that was capable of growth on acetate : fumarate medium but not acetate : Fe(III) citrate medium. Comparison of gene expression in this strain with that of the wild-type revealed upregulation of a potential NADPH-dependent ferredoxin oxidoreductase as well as genes involved in energy generation and amino acid uptake, suggesting that NADPH homeostasis and the tricarboxylic acid (TCA) cycle were perturbed in the 'acetate-adapted' SfrAB-null strain. Membrane and soluble fractions prepared from the 'acetate-adapted' strain were depleted of NADPH-dependent Fe(III), viologen and quinone reductase activities. These results indicate that cytoplasmic, respiration-linked reduction of Fe(III) by SfrAB in vivo is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.

Background Viral infections such as influenza have been shown to predispose hosts to increased colonization of the respiratory tract by pathogenic bacteria and secondary bacterial pneumonia. To examine how viral infections and host antiviral immune responses alter the upper respiratory microbiota, we analyzed nasal bacterial composition by 16S ribosomal RNA (rRNA) gene sequencing in healthy adults at baseline and at 1 to 2 weeks and 4 to 6 weeks following instillation of live attenuated influenza vaccine or intranasal sterile saline. A subset of these samples was submitted for microarray host gene expression profiling. Results We found that live attenuated influenza vaccination led to significant changes in microbial community structure, diversity, and core taxonomic membership as well as increases in the relative abundances of Staphylococcus and Bacteroides genera (both p

Severe deficiency of alpha-1 antitrypsin has a highly variable clinical presentation. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis α1 Study is a prospective, multicenter, cross-sectional study of adults older than age 35 years with PiZZ or PiMZ alpha-1 antitrypsin genotypes. It is designed to better understand if microbial factors influence this heterogeneity. Clinical symptoms, pulmonary function testing, computed chest tomography, exercise capacity, and bronchoalveolar lavage (BAL) will be used to define chronic obstructive pulmonary disease (COPD) phenotypes that can be studied with an integrated systems biology approach that includes plasma proteomics; mouth, BAL, and stool microbiome and virome analysis; and blood microRNA and blood mononuclear cell RNA and DNA profiling. We will rely on global genome, transcriptome, proteome, and metabolome datasets. Matched cohorts of PiZZ participants on or off alpha-1 antitrypsin augmentation therapy, PiMZ participants not on augmentation therapy, and control participants from the Subpopulations and Intermediate Outcome Measures in COPD Study who match on FEV1 and age will be compared. In the primary analysis, we will determine if the PiZZ individuals on augmentation therapy have a difference in lower respiratory tract microbes identified compared with matched PiZZ individuals who are not on augmentation therapy. By characterizing the microbiome in alpha-1 antitrypsin deficiency (AATD), we hope to define new phenotypes of COPD that explain some of the diversity of clinical presentations. As a unique genetic cause of COPD, AATD may inform typical COPD pathogenesis, and better understanding of it may illuminate the complex interplay between environment and genetics. Although the biologic approaches are hypothesis generating, the results may lead to development of novel biomarkers, better understanding of COPD phenotypes, and development of novel diagnostic and therapeutic trials in AATD and COPD. Clinical trial registered with www.clinicaltrials.gov (NCT01832220).

A deep sleep in coal beds Deep below the ocean floor, microorganisms from forest soils continue to thrive. Inagaki et al. analyzed the microbial communities in several drill cores off the coast of Japan, some sampling more than 2 km below the seafloor (see the Perspective by Huber). Although cell counts decreased with depth, deep coal beds harbored active communities of methanogenic bacteria. These communities were more similar to those found in forest soils than in other deep marine sediments. Science , this issue p. 420 ; see also p. 376

Geobacterspecies are key members of the microbial community in many subsurface environments in which dissimilatory metal reduction is an important process. The genome ofGeobacter sulfurreducenscontains a gene designatedrelGsu, which encodes a RelA homolog predicted to catalyze both the synthesis and the degradation of guanosine 3′,5′-bispyrophosphate (ppGpp), a regulatory molecule that signals slow growth in response to nutrient limitation in bacteria. To evaluate the physiological role of RelGsuinG. sulfurreducens, arelGsumutant was constructed and characterized, and ppGpp levels were monitored under various conditions in both the wild-type andrelGsumutant strains. In the wild-type strain, ppGpp and ppGp were produced in response to acetate and nitrogen deprivation, whereas exposure to oxygen resulted in an accumulation of ppGpp alone. Neither ppGpp nor ppGp could be detected in therelGsumutant. TherelGsumutant consistently grew to a higher cell density than the wild type in acetate-fumarate medium and was less tolerant of oxidative stress than the wild type. The capacity for Fe(III) reduction was substantially diminished in the mutant. Microarray and quantitative reverse transcription-PCR analyses indicated that during stationary-phase growth, protein synthesis genes were up-regulated in therelGsumutant and genes involved in stress responses and electron transport, including several implicated in Fe(III) reduction, were down-regulated in the mutant. The results are consistent with a role for RelGsuin regulating growth, stress responses, and Fe(III) reduction inG. sulfurreducensunder conditions likely to be prevalent in subsurface environments.

Previous studies demonstrated that an outer membrane c -type cytochrome, OmcB, was involved in Fe(III) reduction in Geobacter sulfurreducens . An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c -type cytochromes in G. sulfurreducens , increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens , which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides.

The completion of the 5,373,180-bp genome sequence of the marine psychrophilic bacterium Colwellia psychrerythraea 34H, a model for the study of life in permanently cold environments, reveals capabilities important to carbon and nutrient cycling, bioremediation, production of secondary metabolites, and cold-adapted enzymes. From a genomic perspective, cold adaptation is suggested in several broad categories involving changes to the cell membrane fluidity, uptake and synthesis of compounds conferring cryotolerance, and strategies to overcome temperature-dependent barriers to carbon uptake. Modeling of three-dimensional protein homology from bacteria representing a range of optimal growth temperatures suggests changes to proteome composition that may enhance enzyme effectiveness at low temperatures. Comparative genome analyses suggest that the psychrophilic lifestyle is most likely conferred not by a unique set of genes but by a collection of synergistic changes in overall genome content and amino acid composition.

A DNA microarray representing the genome of Geobacter sulfurreducens was constructed for use in global gene expression profiling of cells under steady-state conditions with acetate as the electron donor and Fe(III) or fumarate as the electron acceptor. Reproducible differences in transcript levels were also observed in comparisons between cells grown with ammonia and those fixing atmospheric nitrogen. There was a high correlation between changes in transcript levels determined with microarray analyses and an evaluation of a subset of the genome with quantitative PCR. As expected, cells required to fix nitrogen had higher levels of transcripts of genes associated with nitrogen fixation, further demonstrating that the microarray approach could reliably detect important physiological changes. Cells grown with Fe(III) as the electron acceptor had higher levels of transcripts for omcB , a gene coding for an outer membrane c -type cytochrome that is essential for Fe(III) reduction. Several other c -type cytochrome genes also appeared to be up-regulated. An unexpected result was significantly higher levels of transcripts for genes which have a role in metal efflux, potentially suggesting the importance of maintaining metal homeostasis during release of soluble metals when reducing Fe(III). A substantial proportion (30%) of significantly expressed genes during Fe(III) reduction were genes of unknown function or hypothetical proteins, suggesting differences in Fe(III) reduction physiology among microorganisms which perform this metabolic process.

Dehalococcoides ethenogenes is the only bacterium known to reductively dechlorinate the groundwater pollutants, tetrachloroethene (PCE) and trichloroethene, to ethene. Its 1,469,720–base pair chromosome contains large dynamic duplicated regions and integrated elements. Genes encoding 17 putative reductive dehalogenases, nearly all of which were adjacent to genes for transcription regulators, and five hydrogenase complexes were identified. These findings, plus a limited repertoire of other metabolic modes, indicate that D. ethenogenes is highly evolved to utilize halogenated organic compounds and H 2 . Diversification of reductive dehalogenase functions appears to have been mediated by recent genetic exchange and amplification. Genome analysis provides insights into the organism's complex nutrient requirements and suggests that an ancestor was a nitrogen-fixing autotroph.