Complex Environmental Communities Research Articles

Complementary effects of biochar, secondary metabolites, and bacteria biocontrol agents rejuvenate ratoon sugarcane traits and stimulate soil fertility

Bacteria biological control agents (BCAs) against diseases and pests are widely deemed a sustainable approach in agriculture. However, the extent to which the multifunctionality of plant secondary metabolites and BC interactions shape soil chemistry (e.g., C and N cycling enzymes) and BCAs communities in complex environments, especially under a cropping system (e.g., ratooning) prone to nutrient depletion, disease, etc., is not well established. Besides, the role metabolites-BCAs-BC interactions play in rejuvenating growth conditions necessary for ratoon sugarcane and suppressing plant pathogens/soil-borne diseases remains unclear. Here, we leveraged high-throughput sequencing and LC–MS/MS analyses to illuminate the regulatory effects of BC and plant secondary metabolites interactions on soil chemistry and BCAs communities. Additionally, we explored the roles metabolites-BCAs-BC interactions play in rejuvenating growth conditions necessary for ratoon sugarcane and suppressing plant pathogens/soil-borne diseases. Our finding revealed a pronounced rejuvenation of sugarcane ratoon traits and edaphic factors under the BC amendment soil, primarily due to the role metabolites-BCAs-BC interactions play in remediating soil fertility and health. We also proved that the BC-amended soils were worthy of enriching essential BCAs communities, including Pseudomonas, Bacillus, Streptomyces, Pantoea, Serratia, Weissella, and Leuconostoc. Abscisic aldehyde had a positive correlation with BCAs genera, including Bacillus, Pseudomonas, Serratia, Pantoea, and Ligilactobacillus. Whereas Pseudomonas, Pseudonocardia, Weissella, Bacillus, and Pantoea responded strongly and positively to jasmonic acid. Additionally, benzoate exhibited a strong positive association with BCAs genera, namely, Pseudomonas, Pseudonocardia, Weissella, Bacillus, Pantoea, and Ligilactobacillus in the different plant tissues. In the rhizosphere zone, BCAs genera such as Pseudomonas and Ligilactobacillus responded strongly to phenyllactic acid. Plant secondary metabolites, including apigenin and naringenin, demonstrated a strong positive relationship with N-cycling enzymes, including urease and phosphatase, respectively. Whereas palmitic acid, jasmonic acid, apiin, and abscisic aldehyde exhibited a significant positive correlation with soil TN, urease, pH, and AP, respectively. BugBase functional analysis confirmed that potentially pathogenic bacterial predicted phenotypes were low in the BC amendment. This work demonstrates how the multifunctionality of plant secondary metabolites-BCAs-BC interactions remediate soil fertility and suppress the presence of pathogens/parasites (e.g., Exserohilum, Aphis gossypii Glover, and Colletotrichum), thereby rejuvenating ratoon sugarcane growth and productivity.

VB12Path for Accurate Metagenomic Profiling of Microbially Driven Cobalamin Synthesis Pathways.

ABSTRACTCobalamin (vitamin B12; VB12) is an indispensable nutrient for all living entities in the Earth’s biosphere and plays a vital role in both natural and host environments. Currently in the metagenomic era, gene families of interest are extracted and analyzed based on functional profiles by searching shotgun metagenomes against public databases. However, critical issues exist in applying public databases for specific processes such as VB12 biosynthesis pathways. We developed a curated functional gene database termed VB12Path for accurate metagenomic profiling of VB12 biosynthesis gene families of microbial communities in complex environments. VB12Path contains a total of 60 VB12 synthesis gene families, 287,731 sequences, and 21,154 homology groups, and it aims to provide accurate functional and taxonomic profiles of VB12 synthesis pathways for shotgun metagenomes and minimize false-positive assignments. VB12Path was applied to characterize cobalamin biosynthesis gene families in human intestines and marine environments. The results demonstrated that ocean and human intestine had dramatically different VB12 synthesis processes and that gene families belonging to salvage and remodeling pathway dominated human intestine but were lowest in the ocean ecosystem. VB12Path is expected to be a useful tool to study cobalamin biosynthesis processes via shotgun metagenome sequencing in both environmental and human microbiome research.IMPORTANCE Vitamin B12 (VB12) is an indispensable nutrient for all living entities in the world but can only be synthesized by a small subset of prokaryotes. Therefore, this small subset of prokaryotes controls ecosystem stability and host health to some extent. However, critical accuracy and comprehensiveness issues exist in applying public databases to profile VB12 synthetic gene families and taxonomic groups in complex metagenomes. In this study, we developed a curated functional gene database termed VB12Path for accurate metagenomic profiling of VB12 communities in complex environments. VB12Path is expected to serve as a valuable tool to uncover the hidden microbial communities producing this precious nutrient on Earth.

13C analysis of fatty acid fragments by gas chromatography mass spectrometry for metabolic flux analysis

When multiple metabolic pathways lead to the same product, compound-specific isotope analysis may not provide enough information to quantify the activities of the contributing pathways. Instead, identification of where in the molecule the 13C is incorporated is required. Here we show how knowledge of position-specific 13C incorporation in fatty acids (FA) and FA fragments can be used to quantitatively estimate the fluxes through the central C metabolic network.We developed a method to measure 13C enrichment of FA and FA fragments (ethanoate, propionate) using electron impact GC–MS. We tested the accuracy and repeatability of the measurements using natural abundance and position-specific 13C labelled standards and FA extracted from Bacillus licheniformis and Pseudomonas fluorescens grown with labelled and unlabelled glucose. The molecular ions of FA generally reflected theoretical predictions of mass isotopomer distributions for natural abundance values, but that of the associated FA fragments deviated from expected values, likely associated with McLafferty rearrangements of hydrogen. After correction for naturally occurring isotopes, 13C enrichments of FA and FA fragments showed good agreement with expected isotope composition of FA standards (root mean square error < 0.044 at%; δ13C of ∼ 40‰), natural abundance and labelled glucose. The unsaturated FA extracted from P. fluorescens deviated from expected values likely associated with problems of co-elution and ion suppression and were excluded from analysis.The ratio of glucose-1-13C to glucose-3-13C incorporation into FA fragments was high for B. licheniformis, but low for P. fluorescens. Metabolic flux modelling based on the 13C enrichment of ethanoate and propionate fragments showed that B. licheniformis used Embden-Meyerhof-Parnas and pentose phosphate pathway (66% and 30%, respectively), whereas P. fluorescens utilized Entner-Doudoroff and pentose phosphate pathway (72% and 27%, respectively). FA fragment analysis is therefore a promising tool to study central C metabolic network activities of co-occurring groups of microbes in intact and complex environmental communities.

Construction of Simplified Microbial Consortia to Degrade Recalcitrant Materials Based on Enrichment and Dilution-to-Extinction Cultures.

The capacity of microbes to degrade recalcitrant materials has been extensively explored for environmental remediation and industrial production. Significant achievements have been made with single strains, but focus is now going toward the use of microbial consortia owning to their functional stability and efficiency. However, assembly of simplified microbial consortia (SMC) from complex environmental communities is still far from trivial due to large diversity and the effect of biotic interactions. Here we propose a strategy, based on enrichment and dilution-to-extinction cultures, to construct SMC with reduced diversity for degradation of keratinous materials. Serial dilutions were performed on a keratinolytic microbial consortium pre-enriched from a soil sample, monitoring the dilution effect on community growth and enzymatic activities. An appropriate dilution regime (10–9) was selected to construct a SMC library from the enriched microbial consortium. Further sequencing analysis and keratinolytic activity assays demonstrated that obtained SMC displayed actual reduced microbial diversity, together with various taxonomic composition, and biodegradation capabilities. More importantly, several SMC possessed equivalent levels of keratinolytic efficiency compared to the initial consortium, showing that simplification can be achieved without loss of function and efficiency. This methodology is also applicable to other types of recalcitrant material degradation involving microbial consortia, thus considerably broadening its application scope.

Cytosine Methylation Within Marine Sediment Microbial Communities: Potential Epigenetic Adaptation to the Environment.

Marine sediments harbor a vast amount of Earth’s microbial biomass, yet little is understood regarding how cells subsist in this low-energy, presumably slow-growth environment. Cells in marine sediments may require additional methods for genetic regulation, such as epigenetic modification via DNA methylation. We investigated this potential phenomenon within a shallow estuary sediment core spanning 100 years of age. Here, we provide evidence of dynamic community m5-cytosine methylation within estuarine sediment metagenomes. The methylation states of individual CpG sites were reconstructed and quantified across three depths within the sediment core. A total of 6,254 CpG sites were aligned for direct comparison of methylation states between samples, and 4,235 of these sites mapped to taxa and genes. Our results demonstrate the presence of differential methylation within environmental CpG sites across an age gradient of sediment. We show that epigenetic modification can be detected via Illumina sequencing within complex environmental communities. The change in methylation state of environmentally relevant genes across depths may indicate a dynamic role of DNA methylation in regulation of biogeochemical processes.

Stable Isotope-Labeled Single-Cell Raman Spectroscopy Revealing Function and Activity of Environmental Microbes.

Microorganisms play a key role in driving the global element (C, N, H, P, and S) cycling. However, the function and activity of environmental microbes remain largely elusive because the vast majority of them are yet uncultured. Recent achievements in single cell stable isotope-labeled Raman spectroscopy enable direct investigation of function and activity of individual microbes in complex environmental communities. Here, this protocol describes a workflow to investigate environmental microbes in soil and water by combining 15N, 2D, and 13C stable isotope labeling with different single-cell Raman techniques, including normal Raman, resonance Raman (RR), and surface-enhanced Raman spectroscopy (SERS). Their applications in investigating functional bacteria driving the N and C cycles, and metabolically active cells are described.

Does universal 16S rRNA gene amplicon sequencing of environmental communities provide an accurate description of nitrifying guilds?

Universal (i.e., targeting most bacteria/prokaryotes) 16S rRNA gene based amplicon sequencing is widely used for assessing microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community. However, it is currently unclear if it can yield reliable information on specific microbial guilds, as obtained by using primer sets targeting functional genes or specific16S rRNA gene sequences. Here, we compared the relative abundance, diversity, richness, and composition of selected guilds (nitrifiers), obtained from universal 16S rRNA gene based amplicon sequencing and from guild targeted approaches. The universal amplicon sequencing provided 1) accurate estimates of nitrifier composition, 2) clustering of the samples based on these compositions consistent with sample origin, 3) estimates of the relative abundance of the guilds correlated with those obtained from the targeted approaches and within ~1.2 orders of magnitude of them, but with measurable bias that should be considered when comparing estimates from both approaches. In contrast, the diversity and richness estimations using the universal 16S rRNA based amplicon sequencing were likely limited by the sequencing depth; therefore, we suggest preferring targeted approaches for assessing nitrifiers diversity and richness or using sequencing depth larger than those currently typically practiced. Overvall, we conclude that universal amplicon sequencing provides, in a single analysis, useful information on the abundance and composition of diverse guilds in complex environmental communities.

Inconsistent Denoising and Clustering Algorithms for Amplicon Sequence Data.

Natural microbial communities have been studied for decades using the 16S rRNA gene as a marker. In recent years, the application of second-generation sequencing technologies has revolutionized our understanding of the structure and function of microbial communities in complex environments. Using these highly parallel techniques, a detailed description of community characteristics are constructed, and even the rare biosphere can be detected. The new approaches carry numerous advantages and lack many features that skewed the results using traditional techniques, but we are still facing serious bias, and the lack of reliable comparability of produced results. Here, we contrasted publicly available amplicon sequence data analysis algorithms by using two different data sets, one with defined clone-based structure, and one with food spoilage community with well-studied communities. We aimed to assess which software and parameters produce results that resemble the benchmark community best, how large differences can be detected between methods, and whether these differences are statistically significant. The results suggest that commonly accepted denoising and clustering methods used in different combinations produce significantly different outcome: clustering method impacts greatly on the number of operational taxonomic units (OTUs) and denoising algorithm influences more on taxonomic affiliations. The magnitude of the OTU number difference was up to 40-fold and the disparity between results seemed highly dependent on the community structure and diversity. Statistically significant differences in taxonomies between methods were seen even at phylum level. However, the application of effective denoising method seemed to even out the differences produced by clustering.

Strain/species identification in metagenomes using genome-specific markers

Shotgun metagenome sequencing has become a fast, cheap and high-throughput technology for characterizing microbial communities in complex environments and human body sites. However, accurate identification of microorganisms at the strain/species level remains extremely challenging. We present a novel k-mer-based approach, termed GSMer, that identifies genome-specific markers (GSMs) from currently sequenced microbial genomes, which were then used for strain/species-level identification in metagenomes. Using 5390 sequenced microbial genomes, 8 770 321 50-mer strain-specific and 11 736 360 species-specific GSMs were identified for 4088 strains and 2005 species (4933 strains), respectively. The GSMs were first evaluated against mock community metagenomes, recently sequenced genomes and real metagenomes from different body sites, suggesting that the identified GSMs were specific to their targeting genomes. Sensitivity evaluation against synthetic metagenomes with different coverage suggested that 50 GSMs per strain were sufficient to identify most microbial strains with ≥0.25× coverage, and 10% of selected GSMs in a database should be detected for confident positive callings. Application of GSMs identified 45 and 74 microbial strains/species significantly associated with type 2 diabetes patients and obese/lean individuals from corresponding gastrointestinal tract metagenomes, respectively. Our result agreed with previous studies but provided strain-level information. The approach can be directly applied to identify microbial strains/species from raw metagenomes, without the effort of complex data pre-processing.

Respiration Response Imaging for Real-Time Detection of Microbial Function at the Single-Cell Level

The ability to detect specific functions of uncultured microbial cells in complex natural communities remains one of the most difficult tasks of environmental microbiology. Here we present respiration response imaging (RRI) as a novel fluorescence microscopy-based approach for the identification of microbial function, such as the ability to use C(1) substrates, at a single-cell level. We demonstrate that RRI could be used for the investigation of heterogeneity of a single microbial population or for functional profiling of microbial cells from complex environmental communities, such as freshwater lake sediment.

Microbial dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid bacteria ecology

The dynamics of dominant microflora throughout the manufacture and ripening processes were evaluated in three batches of traditional Castelmagno PDO cheese. Milk, curd and cheese samples, at different stages during cheesemaking, were collected and subjected to culture-dependent and -independent analysis. Traditional plating and genetic identification of lactic acid bacteria (LAB) isolates, and PCR-DGGE analysis of V1 region of 16S rRNA gene were carried out. The collected samples were also monitored by HPLC for the presence of organic acids, sugars and ketones. LAB resulted to be the prevailing microflora in all production stages although enterococci, coagulase-negative cocci and yeasts also showed considerable viable counts probably related to the presence, in the dairy samples analysed, of free short-chain fatty acids detected by HPLC. Lactococcus lactis subsp. lactis was the species most frequently isolated during Castelmagno PDO manufacture, while Lactobacillus plantarum and Lactobacillus paracasei were isolated with the highest frequencies from ripened Castelmagno PDO cheese samples. Occasionally strains of Lactobacillus delbrueckii subsp. lactis, Lactobacillus coryniformis subsp. torquens and Lactobacillus casei were isolated. The results obtained on Castelmagno PDO microflora underlines a partial correspondence between culture-dependent method and DGGE analysis. Thus, in this study, it is highlighted once more the importance to combine molecular culture-independent approaches with classical microbiological methods for the study of complex environmental communities occurring in food matrices.

An efficient strategy for screening large cloned libraries of amplified 16S rDNA sequences from complex environmental communities

We propose a strategy for the efficient screening of large libraries of amplified 16S rRNA genes from complex environmental samples. It consists of processing sets of multiple clones simultaneously. This strategy saves up to 90% of the costs and labor spent in the process of screening a 16S rDNA library.