Global analyses of biosynthetic gene clusters in phytobiomes reveal strong phylogenetic conservation of terpenes and aryl polyenes.

There are gaps in our understandings on how did the evolutionary relationships among members of the phytobiomes shape their ability to produce tremendously complex specialized metabolites under the influence of plant host. To determine these relationships, we investigated the phylogenetic conservation of biosynthetic gene clusters (BGCs) on a global collection of 4,519 high-quality and nonredundant (out of 12,181) bacterial isolates and metagenome-assembled genomes from 47 different plant hosts and soil, by adopting three independent phylogenomic approaches (D-test, Pagel's λ, and consenTRAIT). We report that the BGCs are phylogenetically conserved to varying strengths and depths in their different classes. We show that the ability to produce specialized metabolites qualifies as a complex trait, and the depth of conservation is equivalent to ecologically relevant complex microbial traits. Interestingly, terpene and aryl polyene BGCs had the strongest phylogenetic conservation in the phytobiomes, but not in the soil microbiomes. Furthermore, we showed that terpenes are largely uncharacterized in phytobiomes and pinpointed specific clades that harbor potentially novel terpenes. Taken together, this study sheds light on the evolution of specialized metabolites' biosynthesis potential in phytobiomes under the influence of plant hosts and presents strategies to rationally guide the discovery of potentially novel classes of metabolites. IMPORTANCE This study expands our understandings of the biosynthetic potential of phytobiomes by using such worldwide and extensive collection of microbiomes from plants and soil. Apart from providing such vital resource for the plant microbiome researchers, this study provides fundamental insights into the evolution of biosynthetic gene clusters (BGCs) in phytobiomes under the influence of plant host. Specifically, we report that the strength of phylogenetic conservation in microbiomes varies for different classes of BGCs and is influenced as a result of plant host association. Furthermore, our results indicate that biosynthetic potential of specialized metabolites is deeply conserved equivalent to other complex and ecologically relevant microbial traits. Finally, for the most conserved class of specialized metabolites (terpenes), we identified clades harboring potentially novel class of molecules. Future studies could focus on plant-microbe coevolution and interactions through specialized metabolites building upon these findings.

Pangenome-driven insights into nitrogen metabolic characteristics of Citrobacter portucalensis strain AAK_AS5 associated with wastewater nitrogen removal.

Nitrogen metabolism in the genus Citrobacter is very poorly studied despite its several implications in wastewater treatment. In the current study, Citrobacter portucalensis strain AAK_AS5 was assessed for remediation of simulated wastewater supplemented with different inorganic nitrogen sources. Combination of (NH4)2SO4 with KNO3 was the most preferred for achieving high growth density followed by (NH4)2SO4 and KNO3 alone. This was in agreement with highest ammonical nitrogen removal of 92.9% in the presence of combined nitrogen sources and the corresponding nitrate nitrogen removal of 93% in the presence of KNO3. Furthermore, these removal capacities were validated by investigating the uniqueness and the spread of metabolic features through pan-genomic approach that revealed the largest number of unique genes (2097) and accessory genes (705) in strain AAK_AS5. Of the total 44 different types of nitrogen metabolism-related genes, 39 genes were associated with the core genome, while 5 genes such as gltI, nasA, nasR, nrtA, and ntrC uniquely belonged to the accessory genome. Strain AAK_AS5 possessed three major nitrate removal pathways viz., assimilatory and dissimilatory nitrate reduction to ammonia (ANRA & DNRA), and denitrification; however, the absence of nitrification was compensated by ammonia assimilation catalyzed by gene products of the GDH and GS-GOGAT pathways. narGHIJ encoding the respiratory nitrate reductase was commonly identified in all the studied genomes, while genes such as nirK, norB, and nosZ were uniquely present in the strain AAK_AS5 only. A markedly different genetic content and metabolic diversity between the strains reflected their adaptive evolution in the environment thus highlighting the significance of C. portucalensis AAK_AS5 for potential application in nitrogen removal from wastewater.

Unique pool of carbohydrate-degrading enzymes in novel bacteria assembled from cow and buffalo rumen metagenomes.

Reconstruction of genomes from environmental metagenomes offers an excellent prospect for studying the metabolic potential of organisms resilient to isolation in laboratory conditions. Here, we assembled 12 high-quality metagenome-assembled genomes (MAGs) with an estimated completion of ≥ 90% from cow and buffalo rumen metagenomes. Average nucleotide identity (ANI) score-based screening with an existing database suggests the novelty of these genomes. Gene prediction led to the identification of 30,359 protein-encoding genes (PEGs) across 12 genomes, of which only 44.8% were annotated against a specific functional attribute. Further analysis revealed the presence of 985 carbohydrate-active enzymes (CAZymes) from more than 50 glycoside hydrolase families, of which 90% do not have a proper match in the CAZy database. Genome mining revealed the presence of a high frequency of plant biomass deconstructing genes in Bacteroidetes MAGs compared to Firmicutes. The results strongly indicate that the rumen chamber harbors high numbers of deeply branched and as-yet uncultured microbes that encode novel CAZymes, candidates for prospective usage in plant biomass-hydrolyzing and biofuels industries. KEY POINTS: • Genome binning plays a crucial role in revealing the metabolic potential of uncultivable microbes. • Assembled 12 novel genomes from cow and buffalo rumen metagenome datasets. • High frequency of plant biomass deconstructing genes identified in Bacteroidetes MAGs.

Hybrid Genome Assembly for Predicting Functional Potential of a Novel Streptomyces Strain as Plant Biomass Valorisation Agent.

Environmental bioremediation relies heavily on the realized potential of efficient bioremediation agents or microbial strains of interest. Identifying suitable microbial agents for plant biomass waste valorization requires (i) high-quality genome assemblies to predict the full metabolic and functional potential, (ii) accurate mapping of lignocellulose metabolizing enzymes. However, fragmented nature of the sequenced genomes often limits the prediction ability due to breaks occurring in coding sequences. To address these challenges and as part of our ongoing agri-culturomics efforts, we have performed a hybrid genome assembly using Illumina and Nanopore reads with modified assembly protocol, for a novel Streptomyces strain isolated from the rhizosphere niche of green leafy vegetables grown in a commercial urban farm. High-quality genome was assembled with the size of 8.6 Mb in just two contigs with N50 of 8,542,030 and coverage of 383X. This facilitated identification and complete arrangement of approximately 248 CAZymes and 38 biosynthetic gene clusters in the genome. Multiple gene clusters consisting of cellulases and hemicellulases associated with substrate recognition domain were identified in the genome. Genes for lignin, chitin, and even some aromatic compounds degradation were found in the Streptomyces sp. genome which makes it a promising candidate for lignocellulosic waste valorization. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00935-5.

Unfolding microbial community intelligence in aerobic and anaerobic biodegradation processes using metagenomics.

Environmental factors and available nutrients influence microbial communities, and with that, there exists a dynamic shift in community structure and hierarchy in wastewater treatment systems. Of the various factors, the availability and gradient of oxygen selectively enrich a typical microbial community and also form the community stratification which could be established through metagenomics studies. In recent years, metagenomics with various sets of bioinformatics tools has assisted in exploration and better insight into the organization and relation of the taxonomical and functional composition and associate physiological intelligence of the microbial communities. The microbial communities, under defined conditions acquire a typical hierarchy with flexible but active network of the metabolic route, which ensures the survival needs of every member residing in that community and their abundance. This knowledge of community functional organization defines the rule in designing and improving biodegradation processes in case of both aerobic and anaerobic systems.

Different Dimensions in Microbial Community Adaptation and Function.

With the omics tool, the challenges in understanding the microbial community functions are becoming more intriguing. It is the environment created scenario, which demands alignment of the different members of the community for the desired output leading to common condition for their survival. The resultant community pathways provide a broad umbrella of metabolic options giving the desired plasticity, which plays decision making role in the adaptation process. The initial step in community characterization must involve the discovery of key and core member of the community and monitoring the fluctuations in functional abundance over the space and time. The concept of entropy and metabolic fluxes must reflect the inner metabolic machinery of the taxon selection and route of functional operation in a community. The segregation of member based on their functional role and hierarchical level in the community must be an essential step to be followed by interaction mapping and measurement of metabolic fluxes to derive the flow of metabolites within the community. This conceptual framework and integrated omics tools with supported statistical modeling algorithm can help in bringing out finer details in the process of community functional adaptation in any given scenario.

Assembling a genome for novel nitrogen-fixing bacteria with capabilities for utilization of aromatic hydrocarbons.

Metagenome from refinery wastewater treatment plant running under nitrogen stress was analyzed for mining of novel aromatic hydrocarbon-degrading bacteria. The sequence data were assembled using metaspade followed by binning using the Metabat tool to assemble genome; where coverage and depth were calculated using bowtie and samtools. The analysis picked a novel genome belonging to family Bradyrhizobiaceae, identified based on 16S rDNA gene which was supported by CheckM and Kraken analysis. Using RAST, the assembled genome showed the capabilities for nitrogen fixation with the utilization of multiple hydrocarbon substrates with 14 different types of oxygenases as mapped by Minpath. An additional genetic feature like genes for stress and resistance towards heavy metals and antibiotic suggested that the genome has gone through the rigorous process of adaptation. If such bacteria could be cultivated then it will open the broad window of bioremediation strategies under nitrogen stress environment.

Genomically Defined Paenibacillus polymyxa ND24 for Efficient Cellulase Production Utilizing Sugarcane Bagasse as a Substrate.

Cellulolytic bacteria from cattle rumen with ability to hydrolyze cellulose rich biomass were explored. The study selected Paenibacillus polymyxa ND24 from 847 isolates as the most potent strain, which can efficiently produce cellulase by utilizing sugarcane bagasse, rice straw, corn starch, CMC, and avicel as a sole carbon source. On annotation of P. polymyxa ND24 genome, 116 members of glycoside hydrolase (GH) family from CAZy clusters were identified and the presence of 10 potential cellulases was validated using protein folding information. Cellulase production was further demonstrated at lab-scale 5-L bioreactor exhibiting maximum endoglucanase activity up to 0.72 U/mL when cultivated in the medium containing bagasse (2% w/v) after 72 h. The bagasse hydrolysate so produced was further utilized for efficient biogas production. The presence of diverse hydrolytic enzymes and formidable cellulase activity supports the use of P. polymyxa ND24 for cost-effective bioprocessing of cellulosic biomass.

Exploring the rearrangement of sensory intelligence in proteobacteria: insight of Pho regulon.

Pho regulon is a highly evolved and conserved mechanism across the microbes to fulfil their phosphate need. In this study, 52 proteobacteria genomes were analyzed for the presence of phosphorus acquisition genes, their pattern of arrangement and copy numbers. The diverse genetic architecture of the Pho regulon genes indicates the evolutionary challenge of nutrient limitation, particularly phosphorus, faced by bacteria in their environment. The incongruence between the Pho regulon proteins phylogeny and species phylogeny along with the presence of additional copies of pstS and pstB genes, having cross similarity with other genera, suggest the possibility of horizontal gene transfer event. The substitution rate analysis and multiple sequence alignment of the Pho regulon proteins were analyzed to gain additional insight into the evolution of the Pho regulon system. This comprehensive study confirms that genes perform the regulatory function (phoBR) were vertically inherited, whereas interestingly, genes whose product involved in direct interaction with the environment (pstS) acquired by horizontal gene transfer. The substantial amino acid substitutions in PstS most likely contribute to the successful adaptation of bacteria in different ecological condition dealing with different phosphorus availability. The findings decipher the intelligence of the bacteria which enable them to carry out the targeted alteration of genes to cope up with the environmental condition.

An Approach to In Silico Dissection of Bacterial Intelligence Through Selective Genomic Tools.

All the genetic potential and the intelligence a bacteria can showcase in a given environment are embedded in its genome. In this study, we have presented systematic guidelines to understand a bacterial genome with the relevant set of in silico tools using a novel bacteria as an example. This study presents a multi-dimensional approach from genome annotation to tracing genes and their network of metabolism operating in an organism. It also shows how the sequence can be used to mine the enzymes and construction of its 3-dimensional structure so that its functional behavior can be predicted and compared. The discriminating algorithm allows analysis of the promoter region and provides the insight in the regulation of genes in spite of the similarity in its sequences. The ecological niche specific bacterial behavior and adapted altered physiology can be understood through the presence of secondary metabolite, antibiotic resistance genes, and viral genes; and it helps in the valorization of genetic information for developing new biological application/processes. This study provides an in silico work plan and necessary steps for genome analysis of novel bacteria without any rigorous wet lab experiments.

Dissolved oxygen-mediated enrichment of quorum-sensing phenomenon in the bacterial community to combat oxidative stress.

Microbial community with their plasticity follows a course of changes that allow adaptation and survival in a particular habitat. In this study perturbations in microbial flora dwelling in two reactors with phenol as a carbon source under the limiting nitrogen and phosphorus conditions were monitored for 3 months with alterations of dissolved oxygen (DO). With the time, the shift in diversity and abundance of bacteria were observed with simultaneous increase in biofilm-forming bacteria like Pseudomonas, Escherichia, etc. Functional level screening revealed that the abundance of core metabolic genes were not much altered, however, the regulated level of increase in quorum sensing genes (acyl-homoserine lactone), biofilm-forming genes, catalase and ferroxidase enzymes at high DO suggest the survival mechanism of the community. This study sheds light on survival route followed by the bacterial community with abiotic stress, such as an increase in DO.

Genomic characterization of key bacteriophages to formulate the potential biocontrol agent to combat enteric pathogenic bacteria.

Combating bacterial pathogens has become a global concern especially when the antibiotics and chemical agents are failing to control the spread due to its resistance. Bacteriophages act as a safe biocontrol agent by selectively lysing the bacterial pathogens without affecting the natural beneficial microflora. The present study describes the screening of prominent enteric pathogens NDK1, NDK2, NDK3, and NDK4 (Escherichia, Klebsiella, Enterobacter, and Serratia) mostly observed in domestic wastewater; against which KNP1, KNP2, KNP3, and KNP4 phages were isolated. To analyze their potential role in eradicating enteric pathogens and toxicity issue, these bacteriophages were sequenced using next-generation sequencing and characterized based on its genomic content. The isolated bacteriophages were homologous to Escherichia phage (KNP1), Klebsiella phage (KNP2), Enterobacter phage (KNP3), Serratia phage (KNP4), and belonged to Myoviridae family of Caudovirales except for the unclassified KNP4 phage. Draft genome analysis revealed the presence of lytic enzymes such as holing and lysozyme in KNP1 phage, endolysin in KNP2 phage, and endopeptidase with holin in KNP3 phage. The absence of any lysogenic and virulent genes makes this bacteriophage suitable candidate for preparation of phage cocktail to combat the pathogens present in wastewater. However, KNP4 contained a virulent gene rendering it unsuitable to be used as a biocontrol agent. These findings make the phages (KNP1-KNP3) as a promising alternative for the biocontrol of pathogens in wastewater which is the main culprit to spread these dominated pathogens in different natural water bodies. This study also necessitates for genomic screening of bacteriophages for lysogenic and virulence genes prior to its use as a biocontrol agent.

An Insight into Phage Diversity at Environmental Habitats using Comparative Metagenomics Approach.

Bacteriophages play significant role in driving microbial diversity; however, little is known about the diversity of phages in different ecosystems. A dynamic predator-prey mechanism called "kill the winner" suggests the elimination of most active bacterial populations through phages. Thus, interaction between phage and host has an effect on the composition of microbial communities in ecosystems. In this study, secondary phage metagenome data from aquatic habitats: wastewater treatment plant (WWTP), fresh, marine, and hot water spring habitat were analyzed using MG-RAST and STAMP tools to explore the diversity of the viruses. Differential relative abundance of phage families-Siphoviridae (34%) and Myoviridae (26%) in WWTP, Myoviridae (30%) and Podoviridae (23%) in fresh water, and Myoviridae (41%) and Podoviridae (8%) in marine-was found to be a discriminating factor among four habitats while Rudiviridae (9%), Globuloviridae (8%), and Lipothrixviridae (1%) were exclusively observed in hot water spring. Subsequently, at genera level, Bpp-1-like virus, Chlorovirus, and T4-like virus were found abundant in WWTP, fresh, and marine habitat, respectively. PCA analysis revealed completely disparate composition of phage in hot water spring from other three ecosystems. Similar analysis of relative abundance of functional features corroborated observations from taxa analysis. Functional features corresponding to phage packaging machinery, replication, integration and excision, and gene transfer discriminated among four habitats. The comparative metagenomics approach exhibited genetically distinct phage communities among four habitats. Results revealed that selective distribution of phage communities would help in understanding the role of phages in food chains, nutrient cycling, and microbial ecology. Study of specific phages would also help in controlling environmental pathogens including MDR bacterial populations using phage therapy approach by selective mining and isolation of phages against specific pathogens persisting in a given environment.

In silico analysis for prediction of degradative capacity of Pseudomonas putida SF1.

The study employs draft genome sequence data to explore p-nitrophenol (PNP) degradation activity of Pseudomonas putida strain SF-1 at a genomic scale. Annotation analysis proposes that the strain SF1 not only possesses the gene cluster for PNP utilization but also for the utilization of benzoate, catechol, hydroxybenzoate, protocatechuate, and homogentisate. Further, the analysis was carried out to understand more details of PNP 4-monooxygenase and its regulator. A comparative analysis of PNP 4-monooxygenase from SF1 was carried out for prediction of its tertiary structure; and also its binding affinity with PNP, FAD, NADH and NADPH using FlexX docking. The tertiary structure of regulator was also predicted along with its conserved DNA binding residues. Regulator binding site (RBS) and promoter region were mapped for the PNP degradation gene cluster. Based on genome sequence analysis, the study unveiled the genomic attributes for a versatile catabolic potential of Pseudomonas putida strain SF-1 for different aromatic compounds.