Nicotinic Acid Catabolism Modulates Bacterial Mycophagy in Burkholderia gladioli Strain NGJ1.

Burkholderia gladioli strain NGJ1 exhibits mycophagous activity on a broad range of fungi, including Rhizoctonia solani, a devastating plant pathogen. Here, we demonstrate that the nicotinic acid (NA) catabolic pathway in NGJ1 is required for mycophagy. NGJ1 is auxotrophic to NA and it potentially senses R. solani as a NA source. Mutation in the nicC and nicX genes involved in NA catabolism renders defects in mycophagy and the mutant bacteria are unable to utilize R. solani extract as the sole nutrient source. As supplementation of NA, but not FA (fumaric acid, the end product of NA catabolism) restores the mycophagous ability of ΔnicC/ΔnicX mutants, we anticipate that NA is not required as a carbon source for the bacterium during mycophagy. Notably, nicR, a MarR-type of transcriptional regulator that functions as a negative regulator of the NA catabolic pathway is upregulated in ΔnicC/ΔnicX mutant and upon NA supplementation the nicR expression is reduced to the basal level in both the mutants. The ΔnicR mutant produces excessive biofilm and is completely defective in swimming motility. On the other hand, ΔnicC/ΔnicX mutants are compromised in swimming motility as well as biofilm formation, potentially due to the upregulation of nicR. Our data suggest that a defect in NA catabolism alters the NA pool in the bacterium and upregulates nicR which in turn suppresses bacterial motility as well as biofilm formation, leading to mycophagy defects. IMPORTANCE Mycophagy is an important trait through which certain bacteria forage over fungal mycelia and utilize fungal biomass as a nutrient source to thrive in hostile environments. The present study emphasizes that nicotinic acid (NA) is important for bacterial motility and biofilm formation during mycophagy by Burkholderia gladioli strain NGJ1. Defects in NA catabolism potentially alter the cellular NA pool, upregulate the expression of nicR, a negative regulator of biofilm, and therefore suppress bacterial motility as well as biofilm formation, leading to mycophagy defects.

Environmental Selection and Biogeography Shape the Microbiome of Subsurface Petroleum Reservoirs.

Petroleum reservoirs within the deep biosphere are extreme environments inhabited by diverse microbial communities and represent biogeochemical hot spots in the subsurface. Despite the ecological and industrial importance of oil reservoir microbiomes, systematic study of core microbial taxa and their associated genomic attributes spanning different environmental conditions is limited. Here, we compile and compare 343 16S rRNA gene amplicon libraries and 25 shotgun metagenomic libraries from oil reservoirs in different parts of the world to test for the presence of core taxa and functions. These oil reservoir libraries do not share any core taxa at the species, genus, family, or order levels, and Gammaproteobacteria was the only taxonomic class detected in all samples. Instead, taxonomic composition varies among reservoirs with different physicochemical characteristics and with geographic distance highlighting environmental selection and biogeography in these deep biosphere habitats. Gene-centric metagenomic analysis reveals a functional core of metabolic pathways including carbon acquisition and energy-yielding strategies consistent with biogeochemical cycling in other subsurface environments. Genes for anaerobic hydrocarbon degradation are observed in a subset of the samples and are therefore not considered to represent core functions in oil reservoirs despite hydrocarbons representing an abundant source of carbon in these deep biosphere settings. Overall, this work reveals common and divergent features of oil reservoir microbiomes that are shaped by and responsive to environmental factors, highlighting controls on subsurface microbial community assembly. IMPORTANCE This comprehensive analysis showcases how environmental selection and geographic distance influence the microbiome of subsurface petroleum reservoirs. We reveal substantial differences in the taxonomy of the inhabiting microbes but shared metabolic function between reservoirs with different in situ temperatures and between reservoirs separated by large distances. The study helps understand and advance the field of deep biosphere science by providing an ecological framework and footing for geologists, chemists, and microbiologists studying these habitats to elucidate major controls on deep biosphere microbial ecology.

The Metabolic Potential of Endophytic Actinobacteria Associated with Medicinal Plant Thymus roseus as a Plant-Growth Stimulator.

Bio-fertilizer practice considers not only economical but also environmentally friendly, sustainable agriculture. Endophytes can play important beneficiary roles in plant development, directly, indirectly, or synergistically. In this study, the majority of our endophytic actinobacteria were able to possess direct plant growth-promoting (PGP) traits, including auxin (88%), ammonia (96%), siderophore production (94%), and phosphate solubilization (24%), along with cell-wall degrading enzymes such as protease (75%), cellulase (81%), lipase (81%), and chitinase (18%). About 45% of tested strains have an inhibitory effect on the phytopathogen Fusarium oxysporum, followed by 26% for Verticillium dahlia. Overall, our results showed that strains XIEG63 and XIEG55 were the potent strains with various PGP traits that caused a higher significant increase (p ≤ 0.05) in length and biomass in the aerial part and roots of tomato and cotton, compared to the uninoculated plants. Our data showed that the greatest inhibition percentages of two phytopathogens were achieved due to treatment with strains XIEG05, XIEG07, XIEG45, and XIEG51. The GC-MS analysis showed that most of the compounds were mainly alkanes, fatty acid esters, phenols, alkenes, and aromatic chemicals and have been reported to have antifungal activity. Our investigation emphasizes that endophytic actinobacteria associated with medicinal plants might help reduce the use of chemical fertilization and potentially lead to increased agricultural productivity and sustainability.

The Microbial Community and Functional Potential in the Midland Basin Reveal a Community Dominated by Both Thiosulfate and Sulfate-Reducing Microorganisms.

The Permian Basin is the highest producing oil and gas reservoir in the United States. Hydrocarbon resources in this region are often accessed by unconventional extraction methods, including horizontal drilling and hydraulic fracturing. Despite the importance of the Permian Basin, there is no publicly available microbiological data from this region. We completed an analysis of Permian produced water samples to understand the dynamics present in hydraulically fractured wells in this region. We analyzed produced water samples taken from 10 wells in the Permian region of the Midland Basin using geochemical measurements, 16S rRNA gene sequencing, and metagenomic sequencing. Compared to other regions, we found that Permian Basin produced water was characterized by higher sulfate and lower total dissolved solids (TDS) concentrations, with a median of 1,110 mg/L and 107,000 mg/L. Additionally, geochemical measurements revealed the presence of frac hits, or interwell communication events where an established well is affected by the pumping of fracturing fluid into a new well. The occurrence of frac hits was supported by correlations between the microbiome and the geochemical parameters. Our 16S rRNA gene sequencing identified a produced water microbiome characterized by anaerobic, halophilic, and sulfur reducing taxa. Interestingly, sulfate and thiosulfate reducing taxa including Halanaerobium, Orenia, Marinobacter, and Desulfohalobium were the most prevalent microbiota in most wells. We further investigated the metabolic potential of microorganisms in the Permian Basin with metagenomic sequencing. We recovered 15 metagenome assembled genomes (MAGs) from seven different samples representing 6 unique well sites. These MAGs corroborated the high presence of sulfate and thiosulfate reducing genes across all wells, especially from key taxa including Halanaerobium and Orenia. The observed microbiome composition and metabolic capabilities in conjunction with the high sulfate concentrations demonstrate a high potential for hydrogen sulfide production in the Permian Basin. Additionally, evidence of frac hits suggests the possibility for the exchange of microbial cells and/or genetic information between wells. This exchange would increase the likelihood of hydrogen sulfide production and has implications for the oil and gas industry. IMPORTANCE The Permian Basin is the largest producing oil and gas region in the United States and plays a critical role supplying national energy needs. Previous work in other basins has demonstrated that the geochemistry and microbiology of hydrocarbon regions can have a major impact on well infrastructure and production. Despite that, little work has been done to understand the complex dynamics present in the Permian Basin. This study characterizes and analyzes 10 unique wells and one groundwater sample in the Permian Basin using geochemical and microbial techniques. Across all wells we found a high number of classic and thiosulfate reducers, suggesting that hydrogen sulfide production may be especially prevalent in the Permian Basin. Additionally, our analysis revealed a biogeochemical signal impacted by the presence of frac hits, or interwell communication events where an established well is affected by the pumping of fracturing fluid into a new well. This information can be utilized by the oil and gas industry to improve oil recovery efforts and minimize commercial and environmental costs.

Synergistic Plant-Microbe Interactions between Endophytic Actinobacteria and Their Role in Plant Growth Promotion and Biological Control of Cotton under Salt Stress.

Bacterial endophytes are well-acknowledged inoculants to promote plant growth and enhance their resistance toward various pathogens and environmental stresses. In the present study, 71 endophytic strains associated with the medicinal plant Thymus roseus were screened for their plant growth promotion (PGP), and the applicability of potent strains as bioinoculant has been evaluated. Regarding PGP traits, the percentage of strains were positive for the siderophore production (84%), auxin synthesis (69%), diazotrophs (76%), phosphate solubilization (79%), and production of lytic enzymes (i.e., cellulase (64%), lipase (62%), protease (61%), chitinase (34%), and displayed antagonistic activity against Verticillium dahliae (74%) in vitro. The inoculation of strain XIEG05 and XIEG12 enhanced plant tolerance to salt stress significantly (p < 0.05) through the promotion of shoot, root development, and reduced the activities of antioxidant enzymes (SOD, POD, and CAT), compared with uninoculated controls in vivo. Furthermore, inoculation of strain XIEG57 was capable of reducing cotton disease incidence (DI) symptoms caused by V. dahliae at all tested salt concentrations. The GC-MS analysis showed that many compounds are known to have antimicrobial and antifungal activity. Our findings provide valuable information for applying strains XIEG05 and XIEG12 as bioinoculant fertilizers and biological control agent of cotton under saline soil conditions.

Potassium and Sodium Salt Stress Characterization in the Yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, and Rhodotorula toruloides.

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is a vital cell factory, but more diverse cell factories are essential for the sustainable use of natural resources. Here, we benchmarked nonconventional yeasts Kluyveromyces marxianus and Rhodotorula toruloides against S. cerevisiae strains CEN.PK and W303 for their responses to potassium and sodium salt stress. We found an inverse relationship between the maximum growth rate and the median cell volume that was responsive to salt stress. The supplementation of K+ to CEN.PK cultures reduced Na+ toxicity and increased the specific growth rate 4-fold. The higher K+ and Na+ concentrations impaired ethanol and acetate metabolism in CEN.PK and acetate metabolism in W303. In R. toruloides cultures, these salt supplementations induced a trade-off between glucose utilization and cellular aggregate formation. Their combined use increased the beta-carotene yield by 60% compared with that of the reference. Neural network-based image analysis of exponential-phase cultures showed that the vacuole-to-cell volume ratio increased with increased cell volume for W303 and K. marxianus but not for CEN.PK and R. toruloides in response to salt stress. Our results provide insights into common salt stress responses in yeasts and will help design efficient bioprocesses. IMPORTANCE Characterization of microbial cell factories under industrially relevant conditions is crucial for designing efficient bioprocesses. Salt stress, typical in industrial bioprocesses, impinges upon cell volume and affects productivity. This study presents an open-source neural network-based analysis method to evaluate volumetric changes using yeast optical microscopy images. It allows quantification of cell and vacuole volumes relevant to cellular physiology. On applying salt stress in yeasts, we found that the combined use of K+ and Na+ improves the cellular fitness of Saccharomyces cerevisiae strain CEN.PK and increases the beta-carotene productivity in Rhodotorula toruloides, a commercially important antioxidant and a valuable additive in foods.

Diversity and Antimicrobial Potential of Cultivable Endophytic Actinobacteria Associated With the Medicinal Plant Thymus roseus.

We report for the first time the isolation of endophytic actinobacteria associated with wild populations of the Chinese medicinal herb Thymus roseus Schipcz obtained from the arid land in Ili and Tacheng of the Xinjiang Province, China. Strains were isolated by special pretreatment of plant tissues and identified based on their 16S rRNA gene sequences, and their antimicrobial activities in vitro were evaluated. A total of 126 endophytic actinobacteria belonging to two classes, eight orders, 14 families, and 24 genera were isolated from different organs at the Ili and Tacheng sites. In addition, the diversity of culturable endophytic actinobacteria genera was higher at Tacheng site (n = 71, 56.35%) than the Ili site (n = 55, 43.65%). A neighbor-joining tree of 126 isolated actinobacteria showing the phylogenetic relationships based on 16S rRNA gene sequences and the genus Streptomyces was the most dominant isolate. The number of endophytic actinobacteria genera obtained from root tissues (n = 54, 42.86%) was higher compared to stem (n = 35, 27.78%) and leaf tissue (n = 37, 29.36%). Among 126 endophytic actinobacteria, 54 strains were antagonistic against at least one or more indicator organisms in vitro. Notably, most strains of Streptomyces proved antagonistic activities. For example, strain T4SB028, namely Streptomyces polyantibioticus, showed the highest inhibition ratio reached 67.06, 64.20, and 70.55% against Alternaria solani, Valsa malicola, and Valsa mali, respectively. The results demonstrate that about 30.95%, 23.01% of the tested endophytic actinobacteria were capable of producing siderophores and chitinase, respectively. Additionally, the results of the amplification of biosynthetic genes polyketide synthetase (PKS-I) and non-ribosomal peptide synthetase (NRPS) indicated that at least one antibiotic biosynthetic gene was detected in 27 (50%) of the tested strains. Our result emphasizes that the endophytic actinobacteria communities are different based on the plant tissues and the geographical environment of the sampled area. Thus, we conclude that T. roseus Schipcz. provided a rich source of endophytic actinobacteria that exhibited a broad-spectrum antimicrobial agent.

Beneficial Endophytic Bacterial Populations Associated With Medicinal Plant Thymus vulgaris Alleviate Salt Stress and Confer Resistance to Fusarium oxysporum.

As a result of climate change, salinity has become a major abiotic stress that reduces plant growth and crop productivity worldwide. A variety of endophytic bacteria alleviate salt stress; however, their ecology and biotechnological potential has not been fully realized. To address this gap, a collection of 117 endophytic bacteria were isolated from wild populations of the herb Thymus vulgaris in Sheikh Zuweid and Rafah of North Sinai Province, Egypt, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 17 genera and 30 species. The number of bacterial species obtained from root tissues was higher (n = 18) compared to stem (n = 14) and leaf (n = 11) tissue. The endophytic bacteria exhibited several plant growth-promoting activities in vitro, including auxin synthesis, diazotrophy, phosphate solubilization, siderophore production, and production of lytic enzymes (i.e., chitinase, cellulase, protease, and lipase). Three endophytes representing Bacillus species associated with T. vulgaris such as EGY05, EGY21, and EGY25 were selected based on their ex-situ activities for growth chamber assays to test for their ability to promote the growth of tomato (Solanum lycopersicum L.) under various NaCl concentrations (50-200 mM). All three strains significantly (P < 0.05) promoted the growth of tomato plants under salt stress, compared to uninoculated controls. In addition, inoculated tomato plants by all tested strains decreased (P < 0.05) the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase). Six strains, representing Bacillus and Enterobacter species EGY01, EGY05, EGY16, EGY21, EGY25, and EGY31 were selected based on in vitro antagonistic activity to F. oxysporum for pot experiments under salt stress. All tested strains reduced the disease severity index (DSI) of tomato plants at all tested salt concentrations. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of B. subtilis (EGY16) showed at least ten compounds were known to have antimicrobial activity, with the major peaks being benzene, 1,3-dimethyl-, p-xylene, dibutyl phthalate, bis (2-ethylhexyl) phthalate, and tetracosane. This study demonstrates that diverse endophytes grow in wild thyme populations and that some are able to alleviate salinity stress and inhibit F. oxysporum pathogenesis, making them promising candidates for biofertilizers and biocontrol agents.

Evaluation of the Antimicrobial Activity of Endophytic Bacterial Populations From Chinese Traditional Medicinal Plant Licorice and Characterization of the Bioactive Secondary Metabolites Produced by Bacillus atrophaeus Against Verticillium dahliae.

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant Glycyrrhiza uralensis (licorice) were screened for their in vitro antimicrobial activities against common fungal pathogens of tomato (Fusarium oxysporum f. sp., Fulvia fulva, Alternaria solani), cotton (Fusarium oxysporum f. sp. Vesinfectum, Verticillium dahliae), pomegranite (Ceratocystis fimbriata), Cymbidinium (Colletotrichum gloeosporioides), and Tsao-ko (Pestalotiopsis microspora and Fusarium graminearum) and the common bacteria Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, and Escherichia coli. Several Bacillus strains, particularly Bacillus atrophaeus and Bacillus mojavensis, had a broad spectrum of antifungal and antibacterial activity. A total of 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing Bacillus atrophaeus and Bacillus mojavensis, were selected for plant growth chamber experiments based on strong in vitro antifungal activities. All five strains significantly reduced disease severity in Arabidopsis thaliana plants challenged with V. dahlia infection. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of Bacillus atrophaeus strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with V. dahlia, including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the first to report that bacteria isolated from G. uralensis have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly B. atrophaeus, and suggest this species may a promising candidate as a biocontrol agent to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops.

Synergistic plant-microbe interactions between endophytic bacterial communities and the medicinal plant Glycyrrhiza uralensis F.

Little is known about the composition, diversity, and geographical distribution of bacterial communities associated with medicinal plants in arid lands. To address this, a collection of 116 endophytic bacteria were isolated from wild populations of the herb Glycyrrhiza uralensis Fisch (licorice) in Xinyuan, Gongliu, and Tekesi of Xinjiang Province, China, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 20 genera and 35 species. The number of distinct bacterial genera obtained from root tissues was higher (n = 14) compared to stem (n = 9) and leaf (n = 6) tissue. Geographically, the diversity of culturable endophytic genera was higher at the Tekesi (n = 14) and Xinyuan (n = 12) sites than the Gongliu site (n = 4), reflecting the extremely low organic carbon content, high salinity, and low nutrient status of Gongliu soils. The endophytic bacteria exhibited a number of plant growth-promoting activities ex situ, including diazotrophy, phosphate and potassium solubilization, siderophore production, auxin synthesis, and production of hydrolytic enzymes. Twelve endophytes were selected based on their ex situ plant growth-promoting activities for growth chamber assays to test for their ability to promote growth of G. uralensis F. and Triticum aestivum (wheat) plants. Several strains belonging to the genera Bacillus (n = 6) and Achromobacter (n = 1) stimulated total biomass production in both G. uralensis and T. aestivum under low-nutrient conditions. This work is the first report on the isolation and characterization of endophytes associated with G. uralensis F. in arid lands. The results demonstrate the broad diversity of endophytes associated with wild licorice and suggest that some Bacillus strains may be promising candidates for biofertilizers to promote enhanced survival and growth of licorice and other valuable crops in arid environments.

Biogeographical distribution analysis of hydrocarbon degrading and biosurfactant producing genes suggests that near-equatorial biomes have higher abundance of genes with potential for bioremediation.

BACKGROUND: Bacterial and Archaeal communities have a complex, symbiotic role in crude oil bioremediation. Their biosurfactants and degradation enzymes have been in the spotlight, mainly due to the awareness of ecosystem pollution caused by crude oil accidents and their use. Initially, the scientific community studied the role of individual microbial species by characterizing and optimizing their biosurfactant and oil degradation genes, studying their individual distribution. However, with the advances in genomics, in particular with the use of New-Generation-Sequencing and Metagenomics, it is now possible to have a macro view of the complex pathways related to the symbiotic degradation of hydrocarbons and surfactant production. It is now possible, although more challenging, to obtain the DNA information of an entire microbial community before automatically characterizing it. By characterizing and understanding the interconnected role of microorganisms and the role of degradation and biosurfactant genes in an ecosystem, it becomes possible to develop new biotechnological approaches for bioremediation use. This paper analyzes 46 different metagenome samples, spanning 20 biomes from different geographies obtained from different research projects. RESULTS: A metagenomics bioinformatics pipeline, focused on the biodegradation and biosurfactant-production pathways, genes and organisms, was applied. Our main results show that: (1) surfactation and degradation are correlated events, and therefore should be studied together; (2) terrestrial biomes present more degradation genes, especially cyclic compounds, and less surfactation genes, when compared to water biomes; and (3) latitude has a significant influence on the diversity of genes involved in biodegradation and biosurfactant production. This suggests that microbiomes found near the equator are richer in genes that have a role in these processes and thus have a higher biotechnological potential. CONCLUSION: In this work we have focused on the biogeographical distribution of hydrocarbon degrading and biosurfactant producing genes. Our principle results can be seen as an important step forward in the application of bioremediation techniques, by considering the biostimulation, optimization or manipulation of a starting microbial consortia from the areas with higher degradation and biosurfactant producing genetic diversity.