Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields.

Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 ("Kv137-1036") retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Biological nitrogen fixation in maize: optimizing nitrogenase expression in a root-associated diazotroph.

Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume-rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strain exhibits repression of nitrogen fixation in conditions replete with bioavailable nitrogen, such as fertilized greenhouse and field experiments, remodeled strains show elevated levels in the rhizosphere of corn in the greenhouse and field even in the presence of exogenous nitrogen. Such strains could be used in commercial applications to supply fixed nitrogen to cereal crops.

Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest.

Bromeliads are a diverse group of plants that includes many species whose individuals are capable of retaining water, forming habitats called phytotelmata. These habitats harbor a diversity of organisms including prokaryotes, unicellular eukaryotes, metazoans, and fungi. Among single-celled eukaryotic organisms, ciliates are generally the most abundant. In the present study, we used Illumina DNA sequencing to survey the eukaryotic communities, especially ciliates, inhabiting the tanks of the bromeliads Aechmea gamosepala and Vriesea platynema in the Atlantic Forest of southern Brazil. Filtered sequences were clustered into distinct OTUs using a 99% identity threshold, and then assigned to phylum and genus using a BLAST-based approach (implemented in QIIME) and the SILVA reference database. Both bromeliad species harbored very diverse eukaryotic communities, with Arthropoda and Ciliophora showing the highest abundance (as estimated by the number of sequence reads). The ciliate genus Tetrahymena was the most abundant among single-celled organisms, followed by apicomplexan gregarines and the ciliate genus Glaucoma. Another interesting finding was the presence and high abundance of Trypanosoma in these bromeliad tanks, demonstrating their occurrence in this type of environment. The results presented here demonstrate a hidden diversity of eukaryotes in bromeliad tank waters, opening up new avenues for their in-depth characterization.

Corrigendum: Integrating DNA Methylation and Gene Expression Data in the Development of the Soybean-Bradyrhizobium N2-Fixing Symbiosis.

[This corrects the article on p. 518 in vol. 7, PMID: 27148207.].

Integrating DNA Methylation and Gene Expression Data in the Development of the Soybean-Bradyrhizobium N2-Fixing Symbiosis.

Very little is known about the role of epigenetics in the differentiation of a bacterium from the free-living to the symbiotic state. Here genome-wide analysis of DNA methylation changes between these states is described using the model of symbiosis between soybean and its root nodule-forming, nitrogen-fixing symbiont, Bradyrhizobium diazoefficiens. PacBio resequencing of the B. diazoefficiens genome from both states revealed 43,061 sites recognized by five motifs with the potential to be methylated genome-wide. Of those sites, 3276 changed methylation states in 2921 genes or 35.5% of all genes in the genome. Over 10% of the methylation changes occurred within the symbiosis island that comprises 7.4% of the genome. The CCTTGAG motif was methylated only during symbiosis with 1361 adenosines methylated among the 1700 possible sites. Another 89 genes within the symbiotic island and 768 genes throughout the genome were found to have methylation and significant expression changes during symbiotic development. Of those, nine known symbiosis genes involved in all phases of symbiotic development including early infection events, nodule development, and nitrogenase production. These associations between methylation and expression changes in many B. diazoefficiens genes suggest an important role of the epigenome in bacterial differentiation to the symbiotic state.

Identification of the Genes Required for the Culture of Liberibacter crescens, the Closest Cultured Relative of the Liberibacter Plant Pathogens.

Here Tn5 random transposon mutagenesis was used to identify the essential elements for culturing Liberibacter crescens BT-1 that can serve as antimicrobial targets for the closely related pathogens of citrus, Candidatus Liberibacter asiaticus (Las) and tomato and potato, Candidatus Liberibacter solanacearum (Lso). In order to gain insight on the virulence, metabolism, and culturability of the pathogens within the genus Liberibacter, a mini-Tn5 transposon derivative system consisting of a gene specifying resistance to kanamycin, flanked by a 19-base-pair terminal repeat sequence of Tn5, was used for the genome-wide mutagenesis of L. crescens BT-1 and created an insertion mutant library. By analyzing the location of insertions using Sanger and Illumina Mi-Seq sequencing, 314 genes are proposed as essential for the culture of L. crescens BT-1 on BM-7 medium. Of those genes, 76 are not present in the uncultured Liberibacter pathogens and, as a result, suggest molecules necessary for the culturing these pathogens. Those molecules include the aromatic amino acids, several vitamins, histidine, cysteine, lipopolysaccharides, and fatty acids. In addition, the 238 essential genes of L. crescens in common with L. asiaticus are potential targets for the development of therapeutics against the disease.

Towards a functional hypothesis relating anti-islet cell autoimmunity to the dietary impact on microbial communities and butyrate production.

BACKGROUND: The development of anti-islet cell autoimmunity precedes clinical type 1 diabetes and occurs very early in life. During this early period, dietary factors strongly impact on the composition of the gut microbiome. At the same time, the gut microbiome plays a central role in the development of the infant immune system. A functional model of the association between diet, microbial communities, and the development of anti-islet cell autoimmunity can provide important new insights regarding the role of the gut microbiome in the pathogenesis of type 1 diabetes. RESULTS: A novel approach was developed to enable the analysis of the microbiome on an aggregation level between a single microbial taxon and classical ecological measures analyzing the whole microbial population. Microbial co-occurrence networks were estimated at age 6 months to identify candidates for functional microbial communities prior to islet autoantibody development. Stratification of children based on these communities revealed functional associations between diet, gut microbiome, and islet autoantibody development. Two communities were strongly associated with breast-feeding and solid food introduction, respectively. The third community revealed a subgroup of children that was dominated by Bacteroides abundances compared to two subgroups with low Bacteroides and increased Akkermansia abundances. The Bacteroides-dominated subgroup was characterized by early introduction of non-milk diet, increased risk for early autoantibody development, and by lower abundances of genes for the production of butyrate via co-fermentation of acetate. By combining our results with information from the literature, we provide a refined functional hypothesis for a protective role of butyrate in the pathogenesis of type 1 diabetes. CONCLUSIONS: Based on functional traits of microbial communities estimated from co-occurrence networks, we provide evidence that alterations in the composition of mucin degrading bacteria associate with early development of anti-islet cell autoimmunity. We hypothesize that lower levels of Bacteroides in favor of increased levels of Akkermansia lead to a competitive advantage of acetogens compared to sulfate reducing bacteria, resulting in increased butyrate production via co-fermentation of acetate. This hypothesis suggests that butyrate has a protective effect on the development of anti-islet cell autoantibodies.

A model for the role of gut bacteria in the development of autoimmunity for type 1 diabetes.

Several lines of evidence suggest a role for the gut microbiome in type 1 diabetes. Treating diabetes-prone rodents with probiotics or antibiotics prevents the development of the disorder. Diabetes-prone rodents also have a distinctly different gut microbiome compared with healthy rodents. Recent studies in children with a high genetic risk for type 1 diabetes demonstrate significant differences in the gut microbiome between children who develop autoimmunity for the disease and those who remain healthy. However, the differences in microbiome composition between autoimmune and healthy children are not consistent across all studies because of the strong environmental influences on microbiome composition, particularly diet and geography. Controlling confounding factors of microbiome composition uncovers bacterial associations with disease. For example, in a human cohort from a single Finnish city where geography is confined, a strong association between one dominant bacterial species, Bacteroides dorei, and type 1 diabetes was discovered (Davis-Richardson et al. Front Microbiol 2014;5:678). Beyond this, recent DNA methylation analyses suggest that a thorough epigenetic analysis of the gut microbiome may be warranted. These studies suggest a testable model whereby a diet high in fat and gluten and low in resistant starch may be the primary driver of gut dysbiosis. This dysbiosis may cause a lack of butyrate production by gut bacteria, which, in turn, leads to the development of a permeable gut followed by autoimmunity. The bacterial community responsible for these changes in butyrate production may vary around the world, but bacteria of the genus Bacteroides are thought to play a key role.

Early childhood gut microbiomes show strong geographic differences among subjects at high risk for type 1 diabetes.

OBJECTIVE: Gut microbiome dysbiosis is associated with numerous diseases, including type 1 diabetes. This pilot study determines how geographical location affects the microbiome of infants at high risk for type 1 diabetes in a population of homogenous HLA class II genotypes. RESEARCH DESIGN AND METHODS: High-throughput 16S rRNA sequencing was performed on stool samples collected from 90 high-risk, nonautoimmune infants participating in The Environmental Determinants of Diabetes in the Young (TEDDY) study in the U.S., Germany, Sweden, and Finland. RESULTS: Study site-specific patterns of gut colonization share characteristics across continents. Finland and Colorado have a significantly lower bacterial diversity, while Sweden and Washington state are dominated by Bifidobacterium in early life. Bacterial community diversity over time is significantly different by geographical location. CONCLUSIONS: The microbiome of high-risk infants is associated with geographical location. Future studies aiming to identify the microbiome disease phenotype need to carefully consider the geographical origin of subjects.

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei.

Despite the large interest in the human microbiome in recent years, there are no reports of bacterial DNA methylation in the microbiome. Here metagenomic sequencing using the Pacific Biosciences platform allowed for rapid identification of bacterial GATC methylation status of a bacterial species in human stool samples. For this work, two stool samples were chosen that were dominated by a single species, Bacteroides dorei. Based on 16S rRNA analysis, this species represented over 45% of the bacteria present in these two samples. The B. dorei genome sequence from these samples was determined and the GATC methylation sites mapped. The Bacteroides dorei genome from one subject lacked any GATC methylation and lacked the DNA adenine methyltransferase genes. In contrast, B. dorei from another subject contained 20,551 methylated GATC sites. Of the 4970 open reading frames identified in the GATC methylated B. dorei genome, 3184 genes were methylated as well as 1735 GATC methylations in intergenic regions. These results suggest that DNA methylation patterns are important to consider in multi-omic analyses of microbiome samples seeking to discover the diversity of bacterial functions and may differ between disease states.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter.

The Gram-stain-negative, rod-shaped bacterial isolate BT-1(T) is the closest relative to the genus 'Candidatus Liberibacter' cultured to date. BT-1(T) was recovered from the phloem sap of a defoliating mountain papaya in Puerto Rico. The BT-1(T) 16S rRNA gene sequence showed that strain BT-1(T) is most closely related to members of the genus 'Ca. Liberibacter' sharing 94.7% 16S rRNA gene sequence similarity with 'Ca. Liberibacter americanus' and 'Ca. Liberibacter asiaticus'. Additionally, average nucleotide identity, 16S rRNA gene sequences and conserved protein sequences supported inclusion of the previously described species of the genus 'Ca. Liberibacter' in a genus with BT-1(T). The prominent fatty acids of isolate BT-1(T) were C18 : 1ω7c (77.2%), C16 : 0 OH (4.8%), C18 : 0 (4.4%) and C16 : 0 (3.5%). Both physiological and genomic characteristics support the creation of the genus Liberibacter, as well as the novel species Liberibacter crescens gen. nov., sp. nov. with type strain BT-1(T) ( = ATCC BAA-2481(T) = DSM 26877(T)).

Compromised gut microbiota networks in children with anti-islet cell autoimmunity.

The gut microbiome is suggested to play a role in the pathogenesis of autoimmune disorders such as type 1 diabetes. Evidence of anti-islet cell autoimmunity in type 1 diabetes appears in the first years of life; however, little is known regarding the establishment of the gut microbiome in early infancy. Here, we sought to determine whether differences were present in early composition of the gut microbiome in children in whom anti-islet cell autoimmunity developed. We investigated the microbiome of 298 stool samples prospectively taken up to age 3 years from 22 case children in whom anti-islet cell autoantibodies developed, and 22 matched control children who remained islet cell autoantibody-negative in follow-up. The microbiome changed markedly during the first year of life, and was further affected by breast-feeding, food introduction, and birth delivery mode. No differences between anti-islet cell autoantibody-positive and -negative children were found in bacterial diversity, microbial composition, or single-genus abundances. However, substantial alterations in microbial interaction networks were observed at age 0.5 and 2 years in the children in whom anti-islet cell autoantibodies developed. The findings underscore a role of the microbiome in the pathogenesis of anti-islet cell autoimmunity and type 1 diabetes.

Meconium microbiome analysis identifies bacteria correlated with premature birth.

BACKGROUND: Preterm birth is the second leading cause of death in children under the age of five years worldwide, but the etiology of many cases remains enigmatic. The dogma that the fetus resides in a sterile environment is being challenged by recent findings and the question has arisen whether microbes that colonize the fetus may be related to preterm birth. It has been posited that meconium reflects the in-utero microbial environment. In this study, correlations between fetal intestinal bacteria from meconium and gestational age were examined in order to suggest underlying mechanisms that may contribute to preterm birth. METHODS: Meconium from 52 infants ranging in gestational age from 23 to 41 weeks was collected, the DNA extracted, and 16S rRNA analysis performed. Resulting taxa of microbes were correlated to clinical variables and also compared to previous studies of amniotic fluid and other human microbiome niches. FINDINGS: Increased detection of bacterial 16S rRNA in meconium of infants of <33 weeks gestational age was observed. Approximately 61·1% of reads sequenced were classified to genera that have been reported in amniotic fluid. Gestational age had the largest influence on microbial community structure (R = 0·161; p = 0·029), while mode of delivery (C-section versus vaginal delivery) had an effect as well (R = 0·100; p = 0·044). Enterobacter, Enterococcus, Lactobacillus, Photorhabdus, and Tannerella, were negatively correlated with gestational age and have been reported to incite inflammatory responses, suggesting a causative role in premature birth. INTERPRETATION: This provides the first evidence to support the hypothesis that the fetal intestinal microbiome derived from swallowed amniotic fluid may be involved in the inflammatory response that leads to premature birth.

Interactions between specific phytoplankton and bacteria affect lake bacterial community succession.

Time-series observations and a phytoplankton manipulation experiment were combined to test the hypothesis that phytoplankton succession effects changes in bacterial community composition. Three humic lakes were sampled weekly May-August and correlations between relative abundances of specific phytoplankton and bacterial operational taxonomic units (OTUs) in each time series were determined. To experimentally characterize the influence of phytoplankton, bacteria from each lake were incubated with phytoplankton from one of the three lakes or no phytoplankton. Following incubation, variation in bacterial community composition explained by phytoplankton treatment increased 65%, while the variation explained by bacterial source decreased 64%. Free-living bacteria explained, on average, over 60% of the difference between phytoplankton and corresponding no-phytoplankton control treatments. Fourteen out of the 101 bacterial OTUs that exhibited positively correlated patterns of abundance with specific algal populations in time-series observations were enriched in mesocosms following incubation with phytoplankton, and one out of 59 negatively correlated bacterial OTUs was depleted in phytoplankton treatments. Bacterial genera enriched in mesocosms containing specific phytoplankton assemblages included Limnohabitans (clade betI-A), Bdellovibrio and Mitsuaria. These results suggest that effects of phytoplankton on certain bacterial populations, including bacteria tracking seasonal changes in algal-derived organic matter, result in correlations between algal and bacterial community dynamics.

Microbial hitchhikers on intercontinental dust: catching a lift in Chad.

Ancient mariners knew that dust whipped up from deserts by strong winds travelled long distances, including over oceans. Satellite remote sensing revealed major dust sources across the Sahara. Indeed, the Bodélé Depression in the Republic of Chad has been called the dustiest place on earth. We analysed desert sand from various locations in Chad and dust that had blown to the Cape Verde Islands. High throughput sequencing techniques combined with classical microbiological methods showed that the samples contained a large variety of microbes well adapted to the harsh desert conditions. The most abundant bacterial groupings in four different phyla included: (a) Firmicutes-Bacillaceae, (b) Actinobacteria-Geodermatophilaceae, Nocardiodaceae and Solirubrobacteraceae, (c) Proteobacteria-Oxalobacteraceae, Rhizobiales and Sphingomonadaceae, and (d) Bacteroidetes-Cytophagaceae. Ascomycota was the overwhelmingly dominant fungal group followed by Basidiomycota and traces of Chytridiomycota, Microsporidia and Glomeromycota. Two freshwater algae (Trebouxiophyceae) were isolated. Most predominant taxa are widely distributed land inhabitants that are common in soil and on the surfaces of plants. Examples include Bradyrhizobium spp. that nodulate and fix nitrogen in Acacia species, the predominant trees of the Sahara as well as Herbaspirillum (Oxalobacteraceae), a group of chemoorganotrophic free-living soil inhabitants that fix nitrogen in association with Gramineae roots. Few pathogenic strains were found, suggesting that African dust is not a large threat to public health.

Characterization of the Relative Abundance of the Citrus Pathogen Ca. Liberibacter Asiaticus in the Microbiome of Its Insect Vector, Diaphorina citri, using High Throughput 16S rRNA Sequencing.

The relationship between the causal agent of Huanglongbing (HLB), Ca. Liberibacter asiaticus(Las), and the naturally occurring endosymbiotic community of its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri, was studied. Variation was observed in the titer of Las within an ACP population feeding on the same material. The cause of this disparity is unknown, and has implications for Las transmission and the spread of HLB. This study utilizes culture independent methods to establish the relationship between the ACP's microbial community and Las acquisition. DNA from 21 psyllids was amplified using universal 16S rRNA primers with Illumina adaptor regions and a sample-specific 7- base identifier. These amplicons were then batch-sequenced on the Illumina platform. The resulting sequences were separated by the identifier, and compared to known sequences in a 16S rRNA database. The microbial communities of each psyllid were compared to determine whether a correlation exists between the ACP's endosymbionts and level of Las acquisition.ACPs were dominated by the same four bacterialgenera regardless of the abundance of Ca.Liberibacter. A combination of qPCR and Illumina sequencing was used to establish an infection gradient among the sampled ACPs. The Ca. Liberibacter titer within the insect was found to have a strong negative relationship with an endosymbiont residing in the syncytium of the mycetocyte and a positive relationship with Wolbachia. These correlations have implications in the acquisition of Las by the ACP as well as the activities of Las within this vector.

Complete genome sequence of Liberibacter crescens BT-1.

Liberibacter crescens BT-1, a Gram-negative, rod-shaped bacterial isolate, was previously recovered from mountain papaya to gain insight on Huanglongbing (HLB) and Zebra Chip (ZC) diseases. The genome of BT-1 was sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. A finished assembly and annotation yielded one chromosome with a length of 1,504,659 bp and a G+C content of 35.4%. Comparison to other species in the Liberibacter genus, L. crescens has many more genes in thiamine and essential amino acid biosynthesis. This likely explains why L. crescens BT-1 is culturable while the known Liberibacter strains have not yet been cultured. Similar to CandidatusL. asiaticus psy62, the L. crescens BT-1 genome contains two prophage regions.