Functional analyses of bacterial genomes found in Symbiodiniaceae genome assemblies.

Bacterial-algal interactions strongly influence marine ecosystems. Bacterial communities in cultured dinoflagellates of the family Symbiodiniaceae have been characterized by metagenomics. However, little is known about whole-genome analysis of marine bacteria associated with these dinoflagellates. We performed in silico analysis of four bacterial genomes from cultures of four dinoflagellates of the genera Symbiodinium, Breviolum, Cladocopium and Durusdinium. Comparative analysis showed that the former three contain the alphaproteobacterial family Parvibaculaceae and that the Durusdinium culture includes the family Sphingomonadaceae. There were no large genomic reductions in the alphaproteobacteria with genome sizes of 2.9-3.9 Mb, implying they are not obligate intracellular bacteria. Genomic annotations of three Parvibaculaceae detected the gene for diacetylchitobiose deacetylase (Dac), which may be involved in the degradation of dinoflagellate cell surfaces. They also had metabolic genes for dissimilatory nitrate reduction to ammonium (DNRA) in the nitrogen (N) cycle and cobalamin (vitamin B12 ) biosynthetic genes in the salvage pathway. Those three characters were not found in the Sphingomonadaceae genome. Predicted biosynthetic gene clusters for secondary metabolites indicated that the Parvibaculaceae likely produce the same secondary metabolites. Our study suggests that the Parvibaculaceae is a major resident of Symbiodiniaceae cultures with antibiotics.

Erythrobacter oryzae sp. nov., isolated from rice paddy soil.

Two novel bacterial strains, designated as COR-2T and CR-8, were isolated from paddy soil. These isolates were aerobic, Gram-stain-negative, non-spore-forming, non-motile, rod-shaped, and formed orange-coloured colonies. Phylogenetic analysis based on 16S rRNA gene sequences showed that two strains formed a clear phylogenetic lineage with the genus Erythrobacter. Strains COR-2T and CR-8 showed 99.9 % 16S rRNA gene sequence similarity. Both strains had the highest 16S rRNA gene similarity of 99.1-99.7 % to Erythrobacter colymbi TPW-24T, Erythrobacter donghaensis SW-132T and Erythrobacter tepidarius DSM 10594T, respectively. The genome of strain COR-2T comprised 3 559 918 bp and the genomic DNA G + C content was 67.7 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain COR-2T and its closely related species of the genus Erythrobacter were 79.3-85.5% and 24.1-29.1 %, respectively. The major respiratory quinone was Q-10, while the major fatty acids were C18 : 1 ω7c and C17 : 1 ω6c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids and eight unidentified lipids. Based on phylogenetic and phenotypic considerations, the two strains [COR-2T (type strain; = KACC 22941T=JCM 35529T) and CR-8 (= KACC 22945=JCM 35530)] are considered to represent novel species of the genus Erythrobacter, for which the name Erythrobacter oryzae sp. nov. is proposed.

Revealing the biological significance of multiple metabolic pathways of chloramphenicol by Sphingobium sp. WTD-1.

Chloramphenicol (CAP) is an antibiotic that commonly pollutes the environment, and microorganisms primarily drive its degradation and transformation. Although several pathways for CAP degradation have been documented in different bacteria, multiple metabolic pathways in the same strain and their potential biological significance have not been revealed. In this study, Sphingobium WTD-1, which was isolated from activated sludge, can completely degrade 100 mg/L CAP within 60 h as the sole energy source. UPLC-HRMS and HPLC analyses showed that three different pathways, including acetylation, hydroxyl oxidation, and oxidation (C1-C2 bond cleavage), are responsible for the metabolism of CAP. Importantly, acetylation and C3 hydroxyl oxidation reduced the cytotoxicity of the substrate to strain WTD-1, and the C1-C2 bond fracture of CAP generated the metabolite p-nitrobenzoic acid (PNBA) to provide energy for its growth. This indicated that the synergistic action of three metabolic pathways caused WTD-1 to be adaptable and able to degrade high concentrations of CAP in the environment. This study deepens our understanding of the microbial degradation pathway of CAP and highlights the biological significance of the synergistic metabolism of antibiotic pollutants by multiple pathways in the same strain.

MlrA, an Essential Enzyme for Microcystins and Nodularin on First Step Biodegradation in Microcystin-Degrading Bacteria.

Microcystin-degrading bacteria first degrade microcystins by microcystinase A (MlrA) to cleave the cyclic structure of microcystins at the Adda-Arg site of microcystin-LR, microcystin-RR, and microcystin-YR, but the cleavage of the other microcystins was not clear. In our study, the microcystin-degrading bacterium Sphingopyxis sp. C-1 as wild type and that of mlrA-disrupting mutant, Sphingopyxis sp. CMS01 were used for microcystins biodegradation. The results showed MlrA degraded microcystin-LA, microcystin-LW, microcystin-LY, microcystin-LF, and nodularin. MlrA could cleave the Adda-L-amino acid site.

Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments, and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske-type dioxygenases and flavin-dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth-supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde; Group 2: p-coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre-growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor (Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost-efficient approach that reveals mechanistic insights into OMP-DOM biodegradation.

Comparative genomic analysis and functional investigations for MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria in ecology.

Microcystins (MCs) significantly threaten the ecosystem and public health. Biodegradation has emerged as a promising technology for removing MCs. Many MCs-degrading bacteria have been identified, including an indigenous bacterium Sphingopyxis sp. YF1 that could degrade MC-LR and Adda completely. Herein, we gained insight into the MCs biodegradation mechanisms and evolutionary dynamics of MCs-degrading bacteria, and revealed the toxic risks of the MCs degradation products. The biochemical characteristics and genetic repertoires of strain YF1 were explored. A comparative genomic analysis was performed on strain YF1 and six other MCs-degrading bacteria to investigate their functions. The degradation products were investigated, and the toxicity of the intermediates was analyzed through rigorous theoretical calculation. Strain YF1 might be a novel species that exhibited versatile substrate utilization capabilities. Many common genes and metabolic pathways were identified, shedding light on shared functions and catabolism in the MCs-degrading bacteria. The crucial genes involved in MCs catabolism mechanisms, including mlr and paa gene clusters, were identified successfully. These functional genes might experience horizontal gene transfer events, suggesting the evolutionary dynamics of these MCs-degrading bacteria in ecology. Moreover, the degradation products for MCs and Adda were summarized, and we found most of the intermediates exhibited lower toxicity to different organisms than the parent compound. These findings systematically revealed the MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria. Consequently, this research contributed to the advancement of green biodegradation technology in aquatic ecology, which might protect human health from MCs.

The etherase system of Novosphingobium sp. MBES04 functions as a sensor of lignin fragments through phenylpropanone production to induce specific transcriptional responses.

The MBES04 strain of Novosphingobium accumulates phenylpropanone monomers as end-products of the etherase system, which specifically and reductively cleaves the ß-O-4 ether bond (a major bond in lignin molecules). However, it does not utilise phenylpropanone monomers as an energy source. Here, we studied the response to the lignin-related perturbation to clarify the physiological significance of its etherase system. Transcriptome analysis revealed two gene clusters, each consisting of four tandemly linked genes, specifically induced by a lignin preparation extracted from hardwood (Eucalyptus globulus) and a ß-O-4-type lignin model biaryl compound, but not by vanillin. The most strongly induced gene was a 2,4'-dihydroxyacetophenone dioxygenase-like protein, which leads to energy production through oxidative degradation. The other cluster was related to multidrug resistance. The former cluster was transcriptionally regulated by a common promoter, where a phenylpropanone monomer acted as one of the effectors responsible for gene induction. These results indicate that the physiological significance of the etherase system of the strain lies in its function as a sensor for lignin fragments. This may be a survival strategy to detect nutrients and gain tolerance to recalcitrant toxic compounds, while the strain preferentially utilises easily degradable aromatic compounds with lower energy demands for catabolism.

Development of a rapid assay for ß-etherase activity using a novel chromogenic substrate.

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as ß-etherases, capable of breaking ß-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying ß-etherase activity was developed based on the ß-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate ß-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.

Engineering Novosphingobium aromaticivorans to produce cis,cis-muconic acid from biomass aromatics.

The platform chemical cis,cis-muconic acid (ccMA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the ß-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of ccMA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to ccMA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in ccMA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of ccMA from PCA and produced greater than 100% yield of ccMA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of ccMA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce ccMA from deconstructed biomass.IMPORTANCEThe production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis-muconic acid from renewable and abundant biomass aromatics.

Catabolic System of 5-Formylferulic Acid, a Downstream Metabolite of a ß-5-Type Lignin-Derived Dimer, in Sphingobium lignivorans SYK-6.

Sphingobium lignivorans SYK-6 can assimilate various lignin-derived aromatic compounds, including a ß-5-type (phenylcoumaran-type) dimer, dehydrodiconiferyl alcohol (DCA). SYK-6 converts DCA to a stilbene-type intermediate via multiple reaction steps and then to vanillin and 5-formylferulic acid (FFA). Here, we first elucidated the catabolic pathway of FFA, which is the only unknown pathway in DCA catabolism. Then, we identified and characterized the enzyme-encoding genes responsible for this pathway. Analysis of the metabolites revealed that FFA was converted to 5-carboxyferulic acid (CFA) through oxidation of the formyl group, followed by conversion to ferulic acid by decarboxylation. A comprehensive analysis of the aldehyde dehydrogenase genes in SYK-6 indicated that NAD+-dependent FerD (SLG_12800) is crucial for the conversion of FFA to CFA. LigW and LigW2, which are 5-carboxyvanillic acid decarboxylases involved in the catabolism of a 5,5-type dimer, were found to be involved in the conversion of CFA to ferulic acid, and LigW2 played a significant role. The ligW2 gene forms an operon with ferD, and their transcription was induced during growth in DCA.

Biosorption of U(VI) and mechanisms by live and dead cells of Sphingopyxis sp. YF1.

Heavy metal pollution seriously threatens the environment and human health. The biosorption of heavy metals has attracted worldwide attention due to its cost-effectiveness and environmental friendliness. It is significant to develop biosorbents with excellent adsorption performance. Sphingopyxis is widely used in the removal of various organic pollutants, but its potential application in heavy metal adsorption has been largely overlooked. This study investigates the biosorption of U(VI) onto live and dead cells of a Sphingopyxis strain YF1. The effects of pH, contact time and initial ion concentration on U(VI) adsorption investigated, and kinetic and isothermal models were used to fit the adsorption results. The results show that under pH 3-6, the adsorption of U(VI) by YF1 live cells increased with the increase of the pH. Both the pseudo-first order and pseudo-second order models can satisfactorily interpret the adsorption by live and dead cells. Three isothermal adsorption models (Langmuir, Freundlich, and Sips) were used to fit the adsorption process. The adsorption of uranium by live and dead cells was best fitted by the Sips model. The maximal adsorption capacities of U(VI) by live and dead cells were 140.7 mg g-1 and 205.7 mg g-1, respectively. The mechanisms of U(VI) adsorption by Sphingopyxis sp. YF1 were revealed. Scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS) show that U(VI) was deposited on the surface of the bacterial cells. Fourier-transform infrared spectroscopy (FTIR) shows that amine, hydroxyl, alkyl, amide I, amide II, phosphate, carboxylates and carboxylic acids were the major functional groups that are involved in U(VI) adsorption by live and dead cells. X-ray photoelectron spectroscopy (XPS) suggests that the main functional groups of live cells involved in adsorption were O = C-O, C-OH/C-O-C and N-C = O. This study indicates Sphingopyxis sp. YF1 is a high-efficiency U(VI)-adsorbing strain, promising to remove U(VI) from aquatic environment.

Alteromonas gilva sp. nov. and Erythrobacter fulvus sp. nov., isolated from a tidal mudflat.

Strains chi3T and sf7T were collected from a tidal mudflat around Dongmak beach in Ganghwa, Republic of Korea. Both strains were Gram-stain-negative, aerobic or facultatively anaerobic, and rod-shaped. Results of phylogenetic tree analysis based on 16S rRNA and whole-genome sequences suggested that strains chi3T and sf7T belong to the genera Alteromonas and Erythrobacter, respectively. The cells of strain chi3T were non-motile and grew at 15-45 °C (optimum, 38 °C), at pH 6.0-10.0 (optimum, pH 8.0) and in the presence of 0-9.0 % (w/v) NaCl (optimum, 2.0 %). The cells of strain sf7T were motile as they had flagella and grew at 20-48 °C (optimum, 38 °C), at pH 6.0-10.0 (optimum, pH 9.0) and in the presence of 0-5.0 % (w/v) NaCl (optimum, 1.0 %). Strains chi3T and sf7T have average nucleotide identity values (70.0-70.4% and 78.9-81.7 %) and digital DNA-DNA hybridization values (21.8-22.3% and 21.0-25.6 %) with reference strains in the genera Alteromonas and Erythrobacter, respectively. Data from digital DNA-DNA hybridization, as well as phylogenetic, biochemical and physiological analyses, indicated the distinction of the two strains from the genera Alteromonas and Erythrobacter, respectively, and we thus propose the names Alteromonas gilva sp. nov. (type strain chi3T=KACC 22866T=TBRC 16612T) and Erythrobacter fulvus sp. nov. (type strain sf7T=KACC 22865T=TBRC 16611T).

Marine Bacterioplankton Community Dynamics and Potentially Pathogenic Bacteria in Seawater around Jeju Island, South Korea, via Metabarcoding.

Understanding marine bacterioplankton composition and distribution is necessary for improving predictions of ecosystem responses to environmental change. Here, we used 16S rRNA metabarcoding to investigate marine bacterioplankton diversity and identify potential pathogenic bacteria in seawater samples collected in March, May, September, and December 2013 from two sites near Jeju Island, South Korea. We identified 1343 operational taxonomic units (OTUs) and observed that community diversity varied between months. Alpha- and Gamma-proteobacteria were the most abundant classes, and in all months, the predominant genera were Candidatus Pelagibacter, Leisingera, and Citromicrobium. The highest number of OTUs was observed in September, and Vibrio (7.80%), Pseudoalteromonas (6.53%), and Citromicrobium (6.16%) showed higher relative abundances or were detected only in this month. Water temperature and salinity significantly affected bacterial distribution, and these conditions, characteristic of September, were adverse for Aestuariibacter but favored Citromicrobium. Potentially pathogenic bacteria, among which Vibrio (28 OTUs) and Pseudoalteromonas (six OTUs) were the most abundant in September, were detected in 49 OTUs, and their abundances were significantly correlated with water temperature, increasing rapidly in September, the warmest month. These findings suggest that monthly temperature and salinity variations affect marine bacterioplankton diversity and potential pathogen abundance.

Novosphingobium beihaiensis sp. nov., a novel pesticide-tolerant bacterium isolated from mangrove sediments.

A novel Novosphingobium species, designated strain B2638T, was isolated from mangrove sediments which was collected from Beibu Gulf, Beihai, P. R. China. The isolate could grow in the presence of chlorpyrifos. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the isolate belonged to the genus Novosphingobium, showing 99.9% sequence similarity with N. decloroationis 502str22T and less than 98% similarity with other type strain of species of this genus. Molecular typing by BOX-PCR divided strain B2638T and N. declorationis 502str22T into two clusters and indicated that they were not identical. Genomic comparison referenced by values of the DNA-DNA hybridization (dDDH) and the average nucleotide identity (ANI) between strain B2638T and its close phylogenetic neighbors were 20.0-29.5% and 75.3-85.3%, respectively, that were lower than proposed thresholds for bacterial species delineation. The major fatty acids (> 10%) were identified as C18:1 ω7c, C17:1 iso ω9c and C16:0. The main polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, phosphatidyl glycerol, unidentified lipid and unidentified aminolipid. Results from phenotypic, chemotaxonomic and genotypic analyses proposed that strain B2638T (= MCCC 1K07406T = KCTC 72968 T) is represented a novel species in the genus Novosphingobium, for which the names Novosphingobium beihaiensis sp. nov. is proposed.

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05.

Hepatotoxic microcystins (MCs) are produced and released by the harmful bloom-forming cyanobacteria, which severely threaten drinking water safety and human health due to their high toxicity, widespread distribution, and structural stability. The linearized microcystinase (MlrB) further hydrolyses the poisonous linearized MCs produced by the microcystinase-catalysed MCs to form tetrapeptides. Here, the purification and activity of MlrB were investigated. The results showed that the linearized products generated by 12.5 mg/L MC-LR and MC-RR were removed by purified recombinant MlrB at a protein concentration of 1 mg/L within 30 min. The high catalytic activity of MlrB can be obtained via heterologous expression and affinity purification, which lays the foundation for further studies on the properties and mechanism of MCs biodegradation enzymes.

Metagenomic insights into the mechanisms of triphenyl phosphate degradation by bioaugmentation with Sphingopyxis sp. GY.

Biodegradation of triphenyl phosphate (TPHP) by Sphingopyxis sp. GY was investigated, and results demonstrated that TPHP could be completely degraded in 36 h with intracellular enzymes playing a leading role. This study, for the first time, systematically explores the effects of the typical brominated flame retardants, organophosphorus flame retardants, and heavy metals on TPHP degradation. Our findings reveal that TCPs, BDE-47, HBCD, Cd and Cu exhibit inhibitory effects on TPHP degradation. The hydrolysis-, hydroxylated-, monoglucosylated-, methylated products and glutathione (GSH) conjugated derivative were identified and new degradation pathway of TPHP mediated by microorganism was proposed. Moreover, toxicity evaluation experiments indicate a significant reduction in toxicity following treatment with Sphingopyxis sp. GY. To evaluate its potential for environmental remediation, we conducted bioaugmentation experiments using Sphingopyxis sp. GY in a TPHP contaminated water-sediment system, which resulted in excellent remediation efficacy. Twelve intermediate products were detected in the water-sediment system, including the observation of the glutathione (GSH) conjugated derivative, monoglucosylated product, (OH)2-DPHP and CH3-O-DPHP for the first time in microorganism-mediated TPHP transformation. We further identify the active microbial members involved in TPHP degradation within the water-sediment system using metagenomic analysis. Notably, most of these members were found to possess genes related to TPHP degradation. These findings highlight the significant reduction of TPHP achieved through beneficial interactions and cooperation established between the introduced Sphingopyxis sp. GY and the indigenous microbial populations stimulated by the introduced bacteria. Thus, our study provides valuable insights into the mechanisms, co-existed pollutants, transformation pathways, and remediation potential associated with TPHP biodegradation, paving the way for future research and applications in environmental remediation strategies.

Identification of key degraders for controlling toxicity risks of disguised toxic pollutants with division of labor mechanisms in activated sludge microbiomes: Using nonylphenol ethoxylate as an example.

Efficient management of disguised toxic pollutants (DTPs), which can undergo microbial degradation and convert into more toxic substances, necessitates the collaboration of diverse microbial populations in wastewater treatment plants. However, the identification of key bacterial degraders capable of controlling the toxicity risks of DTPs through division of labor mechanisms in activated sludge microbiomes has received limited attention. In this study, we investigated the key degraders capable of controlling the risk of estrogenicity associated with nonylphenol ethoxylate (NPEO), a representative DTP, in textile activated sludge microbiomes. The results of our batch experiments revealed that the transformation of NPEO into NP and subsequent NP degradation were the rate-limiting processes for controlling the risk of estrogenicity, resulting in an inverted V-shaped curve of estrogenicity in water samples during the biodegradation of NPEO by textile activated sludge. By utilizing enrichment sludge microbiomes treated with NPEO or NP as the sole carbon and energy source, a total of 15 bacterial degraders, including Sphingbium, Pseudomonas, Dokdonella, Comamonas, and Hyphomicrobium, were identified as capable of participating in these processes, Among them, Sphingobium and Pseudomonas were the two key degraders that could cooperatively interact in the degradation of NPEO with division of labor mechanisms. Co-culturing Sphingobium and Pseudomonas isolates exhibited a synergistic effect in degrading NPEO and reducing estrogenicity. Our study underscores the potential of the identified functional bacteria for controlling estrogenicity associated with NPEO and provides a methodological framework for identifying key cooperators engaged in labor division, contributing to the management of risks associated with DTPs by leveraging intrinsic microbial metabolic interactions.

Fine-scale mapping of the microbiome on phylloplane and spermoplane of aromatic and non-aromatic rice genotypes.

Adaxial, abaxial phylloplane (leaf), and spermoplane (seed) are proximal yet contrasting habitats for a microbiota that needs to be adequately explored. Here, we proposed novel methods to decipher the adaxial/abaxial-phylloplane and spermoplane-microbiomes. Comparison of 22 meta barcoded-NGS datasets (size of total data set-1980.48 Mb) enabled us to fine-map the microbiome of the rice foliar niche, which encompasses the lower, middle, top leaf as well panicle. Here, the total- and the cultivable-microbiome profiling revealed 157 genera representing ten phyla and 87 genera from 4 bacterial phyla, respectively, with a predominance of Proteobacteria and Actinobacteria. Interestingly, more bacterial communities (124-genera) preferred the abaxial than the adaxial phylloplane (104-genera) and spermoplane (67-genera) for colonization. The microbiome profiles were nearly identical on the aromatic (125-genera) and non-aromatic rice (116-genera) with high representation of Pantoea, Methylobacterium, Curtobacterium, Sphingopyxis, and Microbacterium. The culturomics investigation confirmed the abundance of Pantoea, Chryseobacterium, Pseudomonas, Acinetobacter, Sphingobacterium, and Exiguobacterium. One hundred bacterial isolates characterized and identified by polyphasic-taxonomic tools revealed the dominance of Acinetobacter, Chryseobacterium, Enterobacter, Massilia, Pantoea, Pseudomonas, and Stenotrophomonas on adaxial/abaxial-phylloplane and spermoplane. The study culminated in identifying hitherto unexplored bacterial communities on the adaxial/abaxial phylloplane and spermoplane of rice that can be harnessed for microbiome-assisted rice cultivation in the future.

Microgravity stress alters bacterial community assembly and co-occurrence networks during wheat seed germination.

Bacterial interactions occurring on and around seeds are integral to plant fitness, health and productivity. Although seed- and plant-associated bacteria are sensitive to environmental stress, the effects of microgravity, as present during plant cultivation in space, on microbial assembly during seed germination are not clear. Here, we characterized the bacterial microbiome assembly process and mechanisms during seed germination of two wheat varieties under simulated microgravity by 16S rRNA gene amplicon sequencing and metabolome analysis. We found that the bacterial community diversity, and network complexity and stability were significantly decreased under simulated microgravity. In addition, the effects of simulated microgravity on the plant bacteriome of the two wheat varieties tended to be consistent in seedlings. At this stage, the relative abundance of Oxalobacteraceae, Paenibacillaceae, Xanthomonadaceae, Lachnospiraceae, Sphingomonadaceae and Ruminococcaceae decreased, while the relative abundance of Enterobacteriales increased under simulated microgravity. Analysis of predicted microbial function revealed that simulated microgravity exposure leads to lower sphingolipid signaling and calcium signaling pathways. We also found that simulated microgravity drove the strengthening of deterministic processes in microbial community assembly. Importantly, some specific metabolites exhibited significant changes under simulated microgravity, suggesting that bacteriome assembly is mediated, at least in part, by metabolites altered by microgravity. The data we present here moves us closer to a holistic understanding of the plant bacteriome under microgravity stress at plant emergence, and provides a theoretical basis for the precise utilization of microorganisms in microgravity to improve plant adaptation to the challenge of cultivation in space.

How Single Amino Acid Substitutions Can Disrupt a Protein Hetero-Dimer Interface: Computational and Experimental Studies of the LigAB Dioxygenase from Sphingobium sp. Strain SYK-6.

Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous research has indicated that the phenylalanine at the 103 position of the alpha subunit (F103α) controls selectivity for the C5 position of the aromatic substrates, and mutations of this residue can enhance the rate of catalysis for substrates with larger functional groups at this position. While several of the mutations to this position (Valine, V; Threonine, T; Leucine, L; and Histidine, H) were catalytically active, other mutations (Alanine, A; and Serine, S) were found to have reduced dimer interface affinity, leading to challenges in copurifing the catalytically active enzyme complex under high salt conditions. In this study, we aimed to experimentally and computationally interrogate residues at the dimer interface to discern the importance of position 103α for maintaining the integrity of the heterodimer. Molecular dynamic simulations and electrophoretic mobility assays revealed a preference for nonpolar/aromatic amino acids in this position, suggesting that while substitutions to polar amino acids may produce a dioxygenase with a useful substrate utilization profile, those considerations may be off-set by potential destabilization of the catalytically active oligomer. Understanding the dimerization of LigAB provides insight into the multimeric proteins within the largely uncharacterized superfamily and characteristics to consider when engineering proteins that can degrade lignin efficiently. These results shed light on the challenges associated with engineering proteins for broader substrate specificity.