The Microbiome Protocols eBook initiative: Building a bridge to microbiome research.

The Microbiome Protocols eBook (MPB) serves as a crucial bridge, filling gaps in microbiome protocols for both wet experiments and data analysis. The first edition, launched in 2020, featured 152 meticulously curated protocols, garnering widespread acclaim. We now extend a sincere invitation to researchers to participate in the upcoming 2nd version of MPB, contributing their valuable protocols to advance microbiome research.

Community-level risk assessments on organophosphate esters in the sediments from the Bohai Sea of China based on multimodal species sensitivity distributions coupled with the equilibrium partitioning method.

Organophosphate esters (OPEs), increasingly used as alternatives to brominated flame retardants, are ubiquitous in the global aquatic environment. Despite their potential toxicological impact on ecosystems, community-level risk assessments for OPEs in sediments remain scarce. This study investigated OPE occurrences and composition characteristics in the Bohai Sea's sediments and appraised both individual and joint ecological risks posed by characteristic OPE homologs using ten commonly used species sensitivity distribution (SSD) models, integrating acute-to-chronic conversion and phase equilibrium partitioning. OPEs were detected across all sediment samples, with total concentrations ranging from 0.213 ng/g dry weight (dw) to 91.1 ng/g dw. The predominant congeners included tri-n-butyl phosphate (TnBP), triisobutyl phosphate (TiBP), tri(2-ethylhexyl) phosphate, tri(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), tris(1, 3-dichloro-2-propyl) phosphate (TDCIPP), and triphenylphosphine oxide. Best-fit SSD models varied among TnBP, TiBP, TCEP, TCPP, and TDCIPP, demonstrating Sigmoid, Burr III, Sigmoid, Burr III, and Burr III, respectively. The same parametric model demonstrated variability in the fitting process for different OPE congeners, which also happened to the fitting results of ten parametric models for the same specific characteristic congener, underscoring the necessity of employing multiple models for precise community-level risk assessments. Hazard concentrations for a 5% cumulative probability were 0.116 mg/L, 2.88 mg/L, 1.30 mg/L, 1.44 mg/L, and 1.85 mg/L for each respective congener. The resulting risk quotients (RQ) and overall hazard index (HI) were selected as criteria to assess the individual and joint ecological risks of OPEs in sediments from the Bohai Sea, respectively. RQ and HI were both below 0.1, indicating a low risk to the local ecosystems. Multi-model SSD analysis could provide refined data for community-level risk evaluation, offering valuable insights for the development of evidence-based environmental standards and pollution control strategies.

Drought-induced assembly of rhizosphere mycobiomes shows beneficial effects on plant growth.

Beneficial interactions between plants and rhizosphere fungi can enhance plant adaptability during drought stress. However, harnessing these interactions will require an in-depth understanding of the response of fungal community assembly to drought. Herein, by using different varieties of wheat plants, we analyzed the drought-induced changes in fungal community assembly in rhizosphere and bulk soil. We demonstrated that drought significantly altered the fungal communities, with the contribution of species richness to community beta diversity increased in both rhizosphere and bulk soil compartments during drought stress. The stochastic processes dominated fungal community assembly, but the relative importance of deterministic processes, mainly homogeneous selection, increased in the drought-stressed rhizosphere. Drought induced an increase in the relative abundance of generalists in the rhizosphere, as opposed to specialists, and the top 10 abundant taxa that enriched under drought conditions were predominantly generalists. Notably, the most abundant drought-enriched taxon in rhizosphere was a generalist, and the corresponding Chaetomium strain was found capable of improving root length and activating ABA signaling in wheat plants through culture-based experiment. Together, these findings provide evidence that host plants exert a strong influence on rhizospheric fungal community assembly during stress and suggest the fungal communities that have experienced drought have the potential to confer fitness advantages to the host plants. IMPORTANCE: We have presented a framework to integrate the shifts in community assembly processes with plant-soil feedback during drought stress. We found that environmental filtering and host plant selection exert influence on the rhizospheric fungal community assembly, and the re-assembled community has great potential to alleviate plant drought stress. Our study proposes that future research should incorporate ecology with plant, microbiome, and molecular approaches to effectively harness the rhizospheric microbiome for enhancing the resilience of crop production to drought.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar "Zhongzhimian No. 2." RESULTS: The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. CONCLUSIONS: This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

Biofilm formation promoted biodegradation of polyethylene in Gordonia polyisoprenivorans B251 isolated from bacterial enrichment acclimated by hexadecane for two years.

Polyethylene (PE) mulch films have been widely used in agriculture and led to a significant pollution in cultivated soils. It is desirable to develop the sustainable method for the degradation of PE. As an environment friendly approach, microbial or enzymatic degradation of PE could meet this demanding. Thus, more microbial strains are required for illustrating biodegrading pathway and developing efficient biological method. In this study, Gordonia polyisoprenivorans B251 capable of degrading PE was isolated from bacterial enrichment with hexadecane as a sole carbon source for two years, in which genus Gordonia had dominated. As revealed by microbial growth curve, the strain B251 had the highest growth rate than other tested strains in the mediums either with hexadecane or PE particles as sole carbon source. The formation of biofilms in both enriched culture and G. polyisoprenivorans B251 pure culture attached to PE film was observed. The capability for PE degradation of individual strain was screened by 30-day incubation with PE film and confirmed by the presence of hydroxyl, carbonyl, carbon-carbon double bond and ether groups in FT-IR analysis and cracks on the surface of PE film observed by scanning electron microscopy (SEM). Therefore, Gordonia polyisoprenivorans, reported as their degradation of environmental contaminants in previous study, were also identified in current study as a candidate for polyethylene biodegradation.

Unlocking antagonistic potential of Bacillus amyloliquefaciens KRS005 to control gray mold.

To establish a safe, efficient, and simple biocontrol measure for gray mold disease caused by Botrytis cinerea, the basic characteristics and antifungal activity of KRS005 were studied from multiple aspects including morphological observation, multilocus sequence analysis and typing (MLSA-MLST), physical-biochemical assays, broad-spectrum inhibitory activities, control efficiency of gray mold, and determination of plant immunity. The strain KRS005, identified as Bacillus amyloliquefaciens, demonstrated broad-spectrum inhibitory activities against various pathogenic fungi by dual confrontation culture assays, of which the inhibition rate of B. cinerea was up to 90.3%. Notably, through the evaluation of control efficiency, it was found that KRS005 fermentation broth could effectively control the occurrence of tobacco leaves gray mold by determining the lesion diameter and biomass of B. cinerea on tobacco leaves still had a high control effect after dilution of 100 folds. Meanwhile, KRS005 fermentation broth had no impact on the mesophyll tissue of tobacco leaves. Further studies showed that plant defense-related genes involved in reactive oxygen species (ROS), salicylic acid (SA), and jasmonic acid (JA)-related signal pathways were significantly upregulated when tobacco leaves were sprayed with KRS005 cell-free supernatant. In addition, KRS005 could inhibit cell membrane damage and increase the permeability of B. cinerea. Overall, KRS005, as a promising biocontrol agent, would likely serve as an alternative to chemical fungicides to control gray mold.

Soil physiochemical properties and bacterial community changes under long-term polycyclic aromatic hydrocarbon stress insitu steel plant soils.

In situ soils were collected at two depths in Jinan and Hangzhou steel plants, which both have a long history of operation and polycyclic aromatic hydrocarbons (PAHs) contamination. The richness of 16 S rRNA gene and bacterial community of the soil were determined by real-time PCR and high-throughput sequencing. Soil physicochemical properties, PAHs contamination characteristics, and their interrelationships were also analyzed. In general, the PAHs contamination decreased with increasing soil depths. The physicochemical properties and PAH concentration of soil had synergistic impacts on the composition of the bacterial community. The long-term higher PAHs stress in Hangzhou contaminated soil (982 mg kg-1) increased the bacterial abundance and diversity, while that of Jinan contaminated soil (63 mg kg-1) decreased bacterial abundance and diversity. The pH value, sand content of the soil were positively correlated (P < 0.05) with the bacterial diversity including Simpson, Shannon, Observed_species and Chao1 indexes., and the other soil properties exhibited negative correlations with different strengths. The abundances of Curvibacter, Pseudomonas, Thiobacillus, Lysobacter, and Limnobacter were positively correlated with the PAHs concentration (P < 0.01). Additionally, the network structure of the PAHs-contaminated soils was more complex compared to that of uncontaminated soils, with stronger linkages and correlations between the different bacteria. These findings provide a theoretical basis for microbial remediation of PAHs-polluted soil.

Structure and Function Analysis of Cultivated Meconopsis integrifolia Soil Microbial Community Based on High-Throughput Sequencing and Culturability.

(1) Background: The structure, function, and community interactions of soil microbial communities of cultivated Meconopsis integrifolia were characterized by studying this alpine flower and traditional endangered Tibetan medicine. (2) Methods: Soil bacteria and fungi were studied based on high-throughput sequencing technology. Bacteria were isolated using culturomics and functionally identified as IAA-producing, organic phosphorus-dissolving, inorganic phosphorus-dissolving, and iron-producing carriers. (3) Results: The dominant bacterial phyla were found to be Proteobacteria and Acidobacteria, and unclassified_Rhizobiales was the most abundant genus. Ascomycota and Mortierellomycota were the dominant fungal phyla. The bacteria were mainly carbon and nitrogen metabolizers, and the fungi were predominantly Saprotroph-Symbiotroph. The identified network was completely dominated by positive correlations, but the fungi were more complex than the bacteria, and the bacterial keystones were unclassified_Caulobacteraceae and Pedobacter. Most of the keystones of fungi belonged to the phyla Ascomycetes and Basidiomycota. The highest number of different species of culturable bacteria belonged to the genus Streptomyces, with three strains producing IAA, 12 strains solubilizing organic phosphorus, one strain solubilizing inorganic phosphorus, and nine strains producing iron carriers. (4) Conclusions: At the cost of reduced ecological stability, microbial communities increase cooperation toward promoting overall metabolic efficiency and enabling their survival in the extreme environment of the Tibetan Plateau. These pioneering results have value for the protection of endangered Meconopsis integrifolia under global warming and the sustainable utilization of its medicinal value.

Reduction, evolutionary pattern and positive selection of genes encoding formate dehydrogenase in Wood-Ljungdahl pathway of gastrointestinal acetogens suggests their adaptation to formate-rich habitats.

Acetogens are anaerobes using Wood-Ljungdahl pathway (WLP) as the terminal electron acceptor for both assimilation and dissimilation of CO2 and widely distributed in diverse habitats. However, their habitat adaptation is often unclear. Given that bacterial genome evolution is often the result of environmental selective pressure, hereby we analysed gene copy number, phylogeny and selective pressure of genes involved in WLP within known genomes of 43 species to study the habitat adaption of gastrointestinal acetogens. The gene copy number of formate dehydrogenase (FDH) in gastrointestinal acetogens was much lower than that of non-gastrointestinal acetogens, and in five cases, no FDH genes were found in the genomes of five gastrointestinal acetogens, but that of the other WLP genes showed no difference. The evolutionary pattern of FDH genes was significantly different from that of the other enzymes. Additionally, seven positively selected sites were only identified in the fdhF genes, which means fdhF mutations favoured their adaptation. Collectively, reduction or loss of FDH genes and their evolutionary pattern as well as positive selection in gastrointestinal acetogens indicated their adaptation to formate-rich habitats, implying that FDH genes catalysing CO2 reduction to formate as the first step of methyl branch of WLP may have evolved independently.

Role of key-stone microbial taxa in algae amended soil for mediating nitrogen transformation.

Although the plant-growth promotion by algae have been studied comprehensively, their impacts on indigenous soil microbiome remain largely unexplored. Herein we conducted a greenhouse experiment to investigate the changes in soil properties and corresponding microbial communities (bacterial, fungal and protists) after 2-year application of algae and their dynamic variation within 60 days immediately after algae addition. In comparison with Control treatment, the impact of algae on soil properties and microbial communities was huge, especially the content of nitrate was decreased however soluble organic nitrogen (SON) was increased. The increased copies of nifH gene suggested the improved potential of nitrogen fixation in algae treated soil. By constructing multitrophic ecological network, soil microorganisms were divided into several modules, and two key-stone microbial taxa (module 1 and 2) showed strong associations with the content of nitrate and SON. With addition of algae, the abundance of most microbial taxa was decreased and increased in module 1 and module 2, respectively. Particularly, module 1 and module 2 were proved to be taxonomically and functionally comprised of different microbes. Moreover, random forest analysis and structural equation model indicated that the key-stone microbial taxa were more important factors affecting the content of nitrate and SON than algae, bacterial, fungal and protistan communities and the influence of algae on soil nitrogen cycling mostly depended on their indirect effects via module 1 and 2.

Genome-Resolved Metagenomics Reveals Distinct Phosphorus Acquisition Strategies between Soil Microbiomes.

Enhancing soil phosphate solubilization is a promising strategy for agricultural sustainability, while little is known about the mechanisms of how microorganisms cope with differing phosphorus availability. Using a combination of genome-resolved metagenomics and amplicon sequencing, we investigated the microbial mechanisms involved in phosphorus cycling under three agricultural treatments in a wheat-maize rotation system and two natural reforestation treatments. Available soil phosphorus was the key factor shaping bacterial and fungal community composition and function across our agricultural and reforestation sites. Membrane-bound quinoprotein glucose dehydrogenase (PQQGDH) and exopolyphosphatases (PPX) governed microbial phosphate solubilization in agroecosystems. In contrast, genes encoding glycerol-3-phosphate transporters (ugpB, ugpC, and ugpQ) displayed a significantly greater abundance in the reforestation soils. The gcd gene encoding PQQGDH was found to be the best determinant for bioavailable soil phosphorus. Metagenome-assembled genomes (MAGs) affiliated with Cyclobacteriaceae and Vicinamibacterales were obtained from agricultural soils. Their MAGs harbored not only gcd but also the pit gene encoding low-affinity phosphate transporters. MAGs obtained from reforestation soils were affiliated with Microtrichales and Burkholderiales. These contain ugp genes but no gcd, and thereby are indicative of a phosphate transporter strategy. Our study demonstrates that knowledge of distinct microbial phosphorus acquisition strategies between agricultural and reforestation soils could help in linking microbial processes with phosphorus cycling. IMPORTANCE The soil microbiome is the key player regulating phosphorus cycling processes. Identifying phosphate-solubilizing bacteria and utilizing them for release of recalcitrant phosphate that is bound to rocks or minerals have implications for improving crop nutrient acquisition and crop productivity. In this study, we combined functional metagenomics and amplicon sequencing to analyze microbial phosphorus cycling processes in natural reforestation and agricultural soils. We found that the phosphorus acquisition strategies significantly differed between these two ecosystems. A microbial phosphorus solubilization strategy dominated in the agricultural soils, while a microbial phosphate transporter strategy was observed in the reforestation soils. We further identified microbial taxa that contributed to enhanced phosphate solubilization in the agroecosystem. These microbes are predicted to be beneficial for the increase in phosphate bioavailability through agricultural practices.

Insights Into Mechanism of the Naphthalene-Enhanced Biodegradation of Phenanthrene by Pseudomonas sp. SL-6 Based on Omics Analysis.

The existence of polycyclic aromatic hydrocarbons (PAHs) in contaminated environment is multifarious. At present, studies of metabolic regulation focus on the degradation process of single PAH. The global metabolic regulatory mechanisms of microorganisms facing coexisting PAHs are poorly understood, which is the major bottleneck for efficient bioremediation of PAHs pollution. Naphthalene (NAP) significantly enhanced the biodegradation of phenanthrene (PHE) by Pseudomonas sp. SL-6. To explore the underlying mechanism, isobaric tags for relative and absolute quantification (iTRAQ) labeled quantitative proteomics was used to characterize the differentially expressed proteins of SL-6 cultured with PHE or NAP + PHE as carbon source. Through joint analysis of proteome and genome, unique proteins were identified and quantified. The up-regulated proteins mainly concentrated in PAH catabolism, Transporters and Electron transfer carriers. In the process, the regulator NahR, activated by salicylate (intermediate of NAP-biodegradation), up-regulates degradation enzymes (NahABCDE and SalABCDEFGH), which enhances the biodegradation of PHE and accumulation of toxic intermediate-1-hydroxy-2-naphthoic acid (1H2Na); 1H2Na stimulates the expression of ABC transporter, which maintains intracellular physiological activity by excreting 1H2Na; the up-regulation of cytochrome C promotes the above process running smoothly. Salicylate works as a trigger that stimulates cell to respond globally. The conjecture was verified at transcriptional and metabolic levels. These new insights contribute to improving the overall understanding of PAHs-biodegradation processes under complex natural conditions, and promoting the application of microbial remediation technology for PAHs pollution.

Root Morphogenesis of Arabidopsis thaliana Tuned by Plant Growth-Promoting Streptomyces Isolated From Root-Associated Soil of Artemisia annua.

In this study, the capacity to tune root morphogenesis by a plant growth-promoting rhizobacterium, Streptomyces lincolnensis L4, was investigated from various aspects including microbial physiology, root development, and root endophytic microbial community. Strain L4 was isolated from the root-associated soil of 7-year plantation of Artemisia annua. Aiming at revealing the promotion mechanism of Streptomyces on root growth and development, this study first evaluated the growth promotion characters of S. lincolnensis L4, followed by investigation in the effect of L4 inoculation on root morphology, endophytic microbiota of root system, and expression of genes involved in root development in Arabidopsis thaliana. Streptomyces lincolnensis L4 is able to hydrolyze organic and inorganic phosphorus, fix nitrogen, and produce IAA, ACC deaminase, and siderophore, which shaped specific structure of endophytic bacterial community with dominant Streptomyces in roots and promoted the development of roots. From the observation of root development characteristics, root length, root diameter, and the number of root hairs were increased by inoculation of strain L4, which were verified by the differential expression of root development-related genes in A. thaliana. Genomic traits of S. lincolnensis L4 which further revealed its capacity for plant growth promotion in which genes involved in phosphorus solubilization, ACC deamination, iron transportation, and IAA production were identified. This root growth-promoting strain has the potential to develop green method for regulating plant development. These findings provide us ecological knowledge of microenvironment around root system and a new approach for regulating root development.

Formate-Dependent Acetogenic Utilization of Glucose by the Fecal Acetogen Clostridium bovifaecis.

Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO2 fixation and energy conservation. The conversion of 2 mol CO2 into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO2 to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens.IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO2 fixation, which converts 2 mol of CO2 into acetyl-CoA. Autotrophic growth with H2 and CO2 via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

Complete Genome Sequence of a Plant-Derived Phenylpropanoid-Degrading Bacterium, Pseudomonas putida JYR-1.

Pseudomonas putida JYR-1 was isolated from soil contaminated with industrial oil because of its ability to utilize trans-anethole as a carbon and energy source. The complete genome is 5.41 Mb with 4,834 protein-coding genes. Study of this isolate will provide insight into biotransformation pathways for phenylpropanoids.

Unusual Genomic Traits Suggest Methylocystis bryophila S285 to Be Well Adapted for Life in Peatlands.

The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum-iron and vanadium-iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands.

Response of Methylocystis sp. Strain SC2 to Salt Stress: Physiology, Global Transcriptome, and Amino Acid Profiles.

Soil microorganisms have to rapidly respond to salt-induced osmotic stress. Type II methanotrophs of the genus Methylocystis are widely distributed in upland soils but are known to have a low salt tolerance. Here, we tested the ability of Methylocystis sp. strain SC2 to adapt to increased salinity. When exposed to 0.75% NaCl, methane oxidation was completely inhibited for 2.25 h and fully recovered within 6 h. Growth was inhibited for 23.5 h and then fully recovered. Its transcriptome was profiled after 0 min (control), 45 min (early response), and 14 h (late response) of stress exposure. Physiological and transcriptomic stress responses corresponded well. Salt stress induced the differential expression of 301 genes, with sigma factor σ32 being a major controller of the transcriptional stress response. The transcript levels of nearly all the genes involved in oxidizing CH4 to CO2 remained unaffected, while gene expression involved in energy-yielding reactions (nuoA-N) recovered concomitantly with methane oxidation from salt stress shock. Glutamate acted as an osmoprotectant. Its accumulation in late stress response corresponded to increased production of glutamate dehydrogenase 1. Chromosomal genes whose products (stress-induced protein, DNA-binding protein from starved cells, and CsbD family protein) are known to confer stress tolerance showed increased expression. On plasmid pBSC2-1, genes encoding type IV secretion system and single-strand DNA-binding protein were upregulated in late response, suggesting stress-induced activation of the plasmid-borne conjugation machinery. Collectively, our results show that Methylocystis sp. strain SC2 is able to adapt to salt stress, but only within a narrow range of salinities.IMPORTANCE Besides the oxic interface of methanogenic environments, Methylocystis spp. are widely distributed in upland soils, where they may contribute to the oxidation of atmospheric methane. However, little is known about their ability to cope with changes in soil salinity. Growth and methane oxidation of Methylocystis sp. strain SC2 were not affected by the presence of 0.5% NaCl, while 1% NaCl completely inhibited its activity. This places strain SC2 into the low-salt-tolerance range reported for other Methylocystis species. Our results show that, albeit in a narrow range, strain SC2 is able to respond and adapt to salinity changes. It possesses various stress response mechanisms, which allow resumption of growth within 24 h when exposed to 0.75% NaCl. Presumably, these mechanisms allow Methylocystis spp., such as strain SC2, to thrive in upland soils and to adapt to certain fluctuations in soil salinity.

In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins.

The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-L-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-L-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.