A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues.

Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol).

Impact of swapping soils on the endophytic bacterial communities of pre-domesticated, ancient and modern maize.

BACKGROUND: Endophytes are microbes that live within plants such as maize (corn, Zea mays L.) without causing disease. It is generally assumed that most endophytes originate from soil. If this is true, then as humans collected, domesticated, bred and migrated maize globally from its native Mexico, they moved the species away from its native population of endophyte donors. The migration of maize persists today, as breeders collect wild and exotic seed (as sources of diverse alleles) from sites of high genetic diversity in Mexico for breeding programs on distant soils. When transported to new lands, it is unclear whether maize permits only selective colonization of microbes from the Mexican soils on which it co-evolved, tolerates shifts in soil-derived endophytes, or prevents colonization of soil-based microbes in favour of seed-transmitted microbes. To test these hypotheses, non-sterilized seeds of three types of maize (pre-domesticated-Mexican, ancient-Mexican, modern-temperate) were planted side-by-side on indigenous Mexican soil, Canadian temperate soil or sterilized sand. The impact of these soil swaps on founder bacterial endophyte communities was tested using 16S-rDNA profiling, culturing and microbial trait phenotyping. RESULTS: Multivariate analysis showed that bacterial 16S-rDNA TRFLP profiles from young, surface-sterilized maize plants were more similar when the same host genotype was grown on the different soils than when different maize genotypes were grown on the same soil. There appeared to be two reasons for this result. First, the largest fraction of bacterial 16S-signals from soil-grown plants was shared with parental seeds and/or plants grown on sterilized sand, suggesting significant inheritance of candidate endophytes. The in vitro activities of soil-derived candidate endophytes could be provided by bacteria that were isolated from sterile sand grown plants. Second, many non-inherited 16S-signals from sibling plants grown on geographically-distant soils were shared with one another, suggesting maize can select microbes with similar TRFLP peak sizes from diverse soils. Wild, pre-domesticated maize did not possess more unique 16S-signals when grown on its native Mexican soil than on Canadian soil, pointing against long-term co-evolutionary selection. The modern hybrid did not reject more soil-derived 16S-signals than did ancestral maize, pointing against such rejection as a mechanism that contributes to yield stability across environments. A minor fraction of 16S-signals was uniquely associated with any one soil. CONCLUSION: Within the limits of TRFLP profiling, the candidate bacterial endophyte populations of pre-domesticated, ancient and modern maize are partially buffered against the effects of geographic migration --- from a Mexican soil associated with ancestral maize, to a Canadian soil associated with modern hybrid agriculture. These results have implications for understanding the effects of domestication, migration, ex situ seed conservation and modern breeding, on the microbiome of one of the world's most important food crops.

Stochastic Inoculum, Biotic Filtering and Species-Specific Seed Transmission Shape the Rare Microbiome of Plants.

A plant's health and productivity is influenced by its associated microbes. Although the common/core microbiome is often thought to be the most influential, significant numbers of rare or uncommon microbes (e.g., specialized endosymbionts) may also play an important role in the health and productivity of certain plants in certain environments. To help identify rare/specialized bacteria and fungi in the most important angiosperm plants, we contrasted microbiomes of the seeds, spermospheres, shoots, roots and rhizospheres of Arabidopsis, Brachypodium, maize, wheat, sugarcane, rice, tomato, coffee, common bean, cassava, soybean, switchgrass, sunflower, Brachiaria, barley, sorghum and pea. Plants were grown inside sealed jars on sterile sand or farm soil. Seeds and spermospheres contained some uncommon bacteria and many fungi, suggesting at least some of the rare microbiome is vertically transmitted. About 95% and 86% of fungal and bacterial diversity inside plants was uncommon; however, judging by read abundance, uncommon fungal cells are about half of the mycobiome, while uncommon bacterial cells make up less than 11% of the microbiome. Uncommon-seed-transmitted microbiomes consisted mostly of Proteobacteria, Firmicutes, Bacteriodetes, Ascomycetes and Basidiomycetes, which most heavily colonized shoots, to a lesser extent roots, and least of all, rhizospheres. Soil served as a more diverse source of rare microbes than seeds, replacing or excluding the majority of the uncommon-seed-transmitted microbiome. With the rarest microbes, their colonization pattern could either be the result of stringent biotic filtering by most plants, or uneven/stochastic inoculum distribution in seeds or soil. Several strong plant-microbe associations were observed, such as seed transmission to shoots, roots and/or rhizospheres of Sarocladium zeae (maize), Penicillium (pea and Phaseolus), and Curvularia (sugarcane), while robust bacterial colonization from cassava field soil occurred with the cyanobacteria Leptolyngbya into Arabidopsis and Panicum roots, and Streptomyces into cassava roots. Some abundant microbes such as Sakaguchia in rice shoots or Vermispora in Arabidopsis roots appeared in no other samples, suggesting that they were infrequent, stochastically deposited propagules from either soil or seed (impossible to know based on the available data). Future experiments with culturing and cross-inoculation of these microbes between plants may help us better understand host preferences and their role in plant productivity, perhaps leading to their use in crop microbiome engineering and enhancement of agricultural production.

Histochemical Evidence for Nitrogen-Transfer Endosymbiosis in Non-Photosynthetic Cells of Leaves and Inflorescence Bracts of Angiosperms.

We used light and confocal microscopy to visualize bacteria in leaf and bract cells of more than 30 species in 18 families of seed plants. Through histochemical analysis, we detected hormones (including ethylene and nitric oxide), superoxide, and nitrogenous chemicals (including nitric oxide and nitrate) around bacteria within plant cells. Bacteria were observed in epidermal cells, various filamentous and glandular trichomes, and other non-photosynthetic cells. Most notably, bacteria showing nitrate formation based on histochemical staining were present in glandular trichomes of some dicots (e.g., Humulus lupulus and Cannabis sativa). Glandular trichome chemistry is hypothesized to function to scavenge oxygen around bacteria and reduce oxidative damage to intracellular bacterial cells. Experiments to assess the differential absorption of isotopic nitrogen into plants suggest the assimilation of nitrogen into actively growing tissues of plants, where bacteria are most active and carbohydrates are more available. The leaf and bract cell endosymbiosis types outlined in this paper have not been previously reported and may be important in facilitating plant growth, development, oxidative stress resistance, and nutrient absorption into plants. It is unknown whether leaf and bract cell endosymbioses are significant in increasing the nitrogen content of plants. From the experiments that we conducted, it is impossible to know whether plant trichomes evolved specifically as organs for nitrogen fixation or if, instead, trichomes are structures in which bacteria easily colonize and where some casual nitrogen transfer may occur between bacteria and plant cells. It is likely that the endosymbioses seen in leaves and bracts are less efficient than those of root nodules of legumes in similar plants. However, the presence of endosymbioses that yield nitrate in plants could confer a reduced need for soil nitrogen and constitute increased nitrogen-use efficiency, even if the actual amount of nitrogen transferred to plant cells is small. More research is needed to evaluate the importance of nitrogen transfer within leaf and bract cells of plants.

Loss of gut microbial diversity in the cultured, agastric fish, Mexican pike silverside (Chirostoma estor: Atherinopsidae).

Teleost fish are the most diverse group of extant vertebrates and have varied digestive anatomical structures and strategies, suggesting they also possess an array of different host-microbiota interactions. Differences in fish gut microbiota have been shown to affect host development, the process of gut colonization, and the outcomes of gene-environment or immune system-microbiota interactions. There is generally a lack of studies on the digestive mechanisms and microbiota of agastric short-intestine fish however, meaning that we do not understand how changes in gut microbial diversity might influence the health of these types of fish. To help fill these gaps in knowledge, we decided to study the Mexican pike silverside (Chirostoma estor) which has a simplified alimentary canal (agastric, short-intestine, 0.7 gut relative length) to observe the diversity and metabolic potential of its intestinal microbiota. We characterized gut microbial populations using high-throughput sequencing of the V3 region in bacterial 16S rRNA genes while searching for population shifts resulting associated with fish development in different environments and cultivation methods. Microbiota samples were taken from the digesta, anterior and posterior intestine (the three different intestinal components) of fish that grew wild in a lake, that were cultivated in indoor tanks, or that were raised in outdoor ponds. Gut microbial diversity was significantly higher in wild fish than in cultivated fish, suggesting a loss of diversity when fish are raised in controlled environments. The most abundant phyla observed in these experiments were Firmicutes and Proteobacteria, particularly of the genera Mycoplasma, Staphylococcus, Spiroplasma, and Aeromonas. Of the 14,161 OTUs observed in this experiment, 133 were found in all groups, and 17 of these, belonging to Acinetobacter, Aeromonas, Pseudomonas, and Spiroplasma genera, were found in all samples suggesting the existence of a core C. estor microbiome. Functional metagenomic prediction of bacterial ecological functions using PICRUSt2 suggested that different intestinal components select for functionally distinct microbial populations with variation in pathways related to the metabolism of amino acids, vitamins, cofactors, and energy. Our results provide, for the first time, information on the bacterial populations present in an agastric, short-gut teleost with commercial potential and show that controlled cultivation of this fish reduces the diversity of its intestinal microbiota.

Seed-Transmitted Bacteria and Fungi Dominate Juvenile Plant Microbiomes.

Plant microbiomes play an important role in agricultural productivity, but there is still much to learn about their provenance, diversity, and organization. In order to study the role of vertical transmission in establishing the bacterial and fungal populations of juvenile plants, we used high-throughput sequencing to survey the microbiomes of seeds, spermospheres, rhizospheres, roots, and shoots of the monocot crops maize (B73), rice (Nipponbare), switchgrass (Alamo), Brachiaria decumbens, wheat, sugarcane, barley, and sorghum; the dicot crops tomato (Heinz 1706), coffee (Geisha), common bean (G19833), cassava, soybean, pea, and sunflower; and the model plants Arabidopsis thaliana (Columbia-0) and Brachypodium distachyon (Bd21). Unsterilized seeds were planted in either sterile sand or farm soil inside hermetically sealed jars, and after as much as 60 days of growth, DNA was extracted to allow for amplicon sequence-based profiling of the bacterial and fungal populations that developed. Seeds of most plants were dominated by Proteobacteria and Ascomycetes, with all containing operational taxonomic units (OTUs) belonging to Pantoea and Enterobacter. All spermospheres also contained DNA belonging to Pseudomonas, Bacillus, and Fusarium. Despite having only seeds as a source of inoculum, all plants grown on sterile sand in sealed jars nevertheless developed rhizospheres, endospheres, and phyllospheres dominated by shared Proteobacteria and diverse fungi. Compared to sterile sand-grown seedlings, growth on soil added new microbial diversity to the plant, especially to rhizospheres; however, all 63 seed-transmitted bacterial OTUs were still present, and the most abundant bacteria (Pantoea, Enterobacter, Pseudomonas, Klebsiella, and Massilia) were the same dominant seed-transmitted microbes observed in sterile sand-grown plants. While most plant mycobiome diversity was observed to come from soil, judging by read abundance, the dominant fungi (Fusarium and Alternaria) were also vertically transmitted. Seed-transmitted fungi and bacteria appear to make up the majority of juvenile crop plant microbial populations by abundance, and based on occupancy, there seems to be a pan-angiosperm seed-transmitted core bacterial microbiome. Further study of these seed-transmitted microbes will be important to understand their role in plant growth and health, as well as their fate during the plant life cycle and may lead to innovations for agricultural inoculant development.

Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study plant-associated communities of bacteria and fungi.

High-throughput sequencing technologies have revolutionized the study of plant-associated microbial populations, but they are relatively expensive. Molecular fingerprinting techniques are more affordable, yet yield considerably less information about the microbial community. Does this mean they are no longer useful for plant microbiome research? In this paper, we review the past 10 years of studies on plant-associated microbiomes using molecular fingerprinting methodologies, including single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), amplicon length heterogeneity PCR (LH-PCR), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA), and terminal restriction fragment length polymorphism (TRFLP). We also present data juxtaposing results from TRFLP methods with those generated using Illumina sequencing in the comparison of rhizobacterial populations of Brazilian maize and fungal surveys in Canadian tomato roots. In both cases, the TRFLP approach yielded the desired results at a level of resolution comparable to that of the MiSeq method, but at a fraction of the cost. Community fingerprinting methods (especially TRFLP) remain relevant for the identification of dominant microbes in a population, the observation of shifts in plant microbiome community diversity, and for screening samples before their use in more sensitive and expensive approaches.

Draft Genome Sequence of Burkholderia gladioli Strain UCD-UG_CHAPALOTE (Phylum Proteobacteria).

Here, we present the draft genome of Burkholderia gladioli strain UCD-UG_CHAPALOTE. This strain is an endophyte isolated from surface sterilized seeds of an ancient Mexican landrace of corn, Chapalote. The genome contains 8,527,129 bp in 109 scaffolds.

Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology.

Endophytes are non-pathogenic microbes living inside plants. We asked whether endophytic species were conserved in the agriculturally important plant genus Zea as it became domesticated from its wild ancestors (teosinte) to modern maize (corn) and moved from Mexico to Canada. Kernels from populations of four different teosintes and 10 different maize varieties were screened for endophytic bacteria by culturing, cloning and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. Principle component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny. However, there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology. The majority of seed endophytes in the wild ancestor persist today in domesticated maize, though ancient selection against the hard fruitcase surrounding seeds may have altered the abundance of endophytes. Four TRFLP signals including two predicted to represent Clostridium and Paenibacillus species were conserved across all Zea genotypes, while culturing showed that Enterobacter, Methylobacteria, Pantoea and Pseudomonas species were widespread, with γ-proteobacteria being the prevalent class. Twenty-six different genera were cultured, and these were evaluated for their ability to stimulate plant growth, grow on nitrogen-free media, solubilize phosphate, sequester iron, secrete RNAse, antagonize pathogens, catabolize the precursor of ethylene, produce auxin and acetoin/butanediol. Of these traits, phosphate solubilization and production of acetoin/butanediol were the most commonly observed. An isolate from the giant Mexican landrace Mixteco, with 100% identity to Burkholderia phytofirmans, significantly promoted shoot potato biomass. GFP tagging and maize stem injection confirmed that several seed endophytes could spread systemically through the plant. One seed isolate, Enterobacter asburiae, was able to exit the root and colonize the rhizosphere. Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration.