The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The Vmax and Km values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g–1 min–1 and 5.7 10–4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.
Microcystis aeruginosa was quantitatively surveyed in 88 freshwater environments across Japan within 3 weeks in 2011. In order to clarify the distribution pattern of M. aeruginosa at the intra-species level, three major genotypes, which were defined by 16S–23S rRNA inter-transcribed-spacer (ITS) regions, were selectively detected using quantitative real-time PCR assays. Of the 68 sites at which the Microcystis intergenic-spacer region of the phycocyanin (IGS-PC) gene was detected, the M. aeruginosa morphotype-related genotype (MG1) dominated in 41 sites, followed by the non-toxic M. wesenbergii-related genotype (MG3). A correlation analysis showed that total nitrogen and phosphate positively correlated with the abundance of IGS-PC, which positively correlated with microcystin synthetase gene abundance. A redundancy analysis of genotype compositions showed that pH positively correlated with the dominance of MG3 and negatively correlated with MG1, i.e., both toxic and non-toxic genotypes. Our survey of Microcystis populations over a wide area revealed that MG1 is a dominant genotype in Japan.
Bradyrhizobium elkanii, a rhizobium with a relatively wide host range, possesses a functional type III secretion system (T3SS) that is involved in symbiotic incompatibility against Rj4-genotype soybean (Glycine max) and some accessions of mung bean (Vigna radiata). To expand our knowledge on the T3SS-mediated partner selection mechanism in the symbiotic legume-rhizobia association, we inoculated three Lotus experimental accessions with wild-type and T3SS-mutant strains of B. elkanii USDA61. Different responses were induced by T3SS in a host genotype-dependent manner. Lotus japonicus Gifu inhibited infection; L. burttii allowed infection, but inhibited nodule maturation at the post-infection stage; and L. burttii and L. japonicus MG-20 both displayed a nodule early senescence-like response. By conducting inoculation tests with mutants of previously reported and newly identified effector protein genes of B. elkanii USDA61, we identified NopF as the effector protein triggering the inhibition of infection, and NopM as the effector protein triggering the nodule early senescence–like response. Consistent with these results, the B. elkanii USDA61 gene for NopF introduced into the Lotus symbiont Mesorhizobium japonicum induced infection inhibition in L. japonicus Gifu, but did not induce any response in L. burttii or L. japonicus MG-20. These results suggest that Lotus accessions possess at least three checkpoints to eliminate unfavorable symbionts, including the post-infection stage, by recognizing different T3SS effector proteins at each checkpoint.
Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.
The maximum ammonia oxidation potential (AOP) of a topsoil in Langhovde, East Antarctica was 22.1±2.4 ng N g–1 dry soil h–1 (2 mM ammonium, 10°C, n=3). This topsoil exhibited twin AOP peaks (1 and 2 mM ammonium) at 10°C, but not at 20°C. Six and ten operational taxonomic units (OTUs) were identified for ammonia-oxidizing bacteria (AOB) and archaea (AOA) amoA, respectively. AOB were classified into Nitrosospira; the two dominant OTUs corresponded to the Mount Everest cluster. AOA were classified into three clusters; Nitrososphaera and Nitrosocosmicus were the two dominant clusters.
We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the “PS protocol”), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.
In central Europe, soybean cultivation is gaining increasing importance to reduce protein imports from overseas and make cropping systems more sustainable. In the field, despite the inoculation of soybean with commercial rhizobia, its nodulation is low. In many parts of Europe, limited information is currently available on the genetic diversity of rhizobia and, thus, biological resources for selecting high nitrogen-fixing rhizobia are inadequate. These resources are urgently needed to improve soybean production in central Europe. The objective of the present study was to identify strains that have the potential to increase nitrogen fixation by and the yield of soybean in German soils. We isolated and characterized 77 soybean rhizobia from 18 different sampling sites. Based on a multilocus sequence analysis (MLSA), 71% of isolates were identified as Bradyrhizobium and 29% as Rhizobium. A comparative analysis of the nodD and nifH genes showed no significant differences, which indicated that the soybean rhizobia symbiotic genes in the present study belong to only one type. One isolate, GMF14 which was tolerant of a low temperature (4°C), exhibited higher nitrogen fixation in root nodules and a greater plant biomass than USDA 110 under cold conditions. These results strongly suggest that some indigenous rhizobia enhance biological nitrogen fixation and soybean yield due to their adaption to local conditions.
Recent studies demonstrated that phylogenetically more diverse and abundant bacteria and fungi than previously considered are responsible for denitrification in terrestrial environments. We herein examined the effects of land-use types on the community composition of those denitrifying microbes based on their nitrite reductase gene (nirK and nirS) sequences. These genes can be phylogenetically grouped into several clusters. We used cluster-specific PCR primers to amplify nirK and nirS belonging to each cluster because the most widely used primers only amplify genes belonging to a single cluster. We found that the dominant taxa as well as overall community composition of denitrifying bacteria and fungi, regardless of the cluster they belonged to, differed according to the land-use type. We also identified distinguishing taxa based on individual land-use types, the distribution of which has not previously been characterized, such as denitrifying bacteria or fungi dominant in forest soils, Rhodanobacter having nirK, Penicillium having nirK, and Bradyrhizobium having nirS. These results suggest that land-use management affects the ecological constraints and consequences of denitrification in terrestrial environments through the assembly of distinct communities of denitrifiers.
The pmoA gene, encoding particulate methane monooxygenase in methanotrophs, and nirS and nirK genes, encoding bacterial nitrite reductases, were examined in the root and rhizosphere sediment of three common emergent macrophytes (Phragmites australis, Typha angustifolia, and Scirpus triqueter) and unvegetated sediment from eutrophic Wuliangsuhai Lake in China. Sequencing analyses indicated that 334 out of 351 cloned pmoA sequences were phylogenetically the most closely related to type I methanotrophs (Gammaproteobacteria), and Methylomonas denitrificans-like organisms accounted for 44.4% of the total community. In addition, 244 out of 250 cloned nirS gene sequences belonged to type I methanotrophs, and 31.2% of nirS genes were the most closely related to paddy rice soil clone SP-2-12 in Methylomonas of the total community. Three genera of type I methanotrophs, Methylomonas, Methylobacter, and Methylovulum, were common in both pmoA and nirS clone libraries in each sample. A quantitative PCR (qPCR) analysis demonstrated that the copy numbers of the nirS and nirK genes were significantly higher in rhizosphere sediments than in unvegetated sediments in P. australis and T. angustifolia plants. In the same sample, the nirS gene copy number was significantly higher than that of nirK. Furthermore, type I methanotrophs were localized in the root tissues according to catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Thus, nirS-carrying type I methanotrophs were enriched in macrophyte root and rhizosphere sediment and are expected to play important roles in carbon/nitrogen cycles in a eutrophic wetland.
The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial effects on plant growth and health. Sixty-one Methylobacterium strains belonging to seven species were predominantly isolated from the phyllosphere of rice cultivated in six Mekong delta provinces. Inoculation tests revealed that some strains exhibited plant growth-promoting activity. Moreover, three strains possessed the novel characteristics of inducing leaf bleaching and killing rice seedlings. These results revealed the complex diversity of Methylobacterium in Mekong delta rice and that healthy and productive rice cultivation requires a proper balance of Methylobacterium.
MicroRNAs (miRNAs) are a group of small non-coding RNAs that suppress the expression of target mRNAs. The seed sequence of miRNA plays a crucial role in recognizing the 3′-untranslated region of the target mRNA. Cells infected with a simian foamy virus (SFV) isolated from an African green monkey (Chlorocebus aethiops) (SFVcae) showed high expression levels of viral miRNAs encoded in the long terminal repeat of SFVcae. In the present study, we investigated the roles and expression of miRNAs derived from an SFV isolated from a Japanese macaque (Macaca fuscata) (SFVmfu) using next-generation sequencing technologies. The results obtained showed that SFVmfu also expressed viral miRNAs; however, the seed sequences of most miRNAs derived from SFVmfu differed from those reported previously from SFVcae. Cells persistently infected with SFVmfu strongly expressed an miRNA with the same seed sequence as the miR-1 microRNA precursor family. Luciferase reporter assays indicated that this miRNA down-regulates the expression of adenylyl cyclase-associated protein 1, which is up-regulated in several solid tumors. The present results suggest that SFVmfu utilizes viral miRNAs to establish long-term co-existence with the Japanese macaque.
Vigna is a genus of legumes cultivated in specific areas of tropical countries. Species in this genus are important crops worldwide. Vigna species are of great agronomic interest in Venezuela because Vigna beans are an excellent alternative to other legumes. However, this type of crop has some cultivation issues due to sensitivity to acidic soils, high temperatures, and salinity stress, which are common in Venezuela. Vigna species establish symbioses mainly with Bradyrhizobium and Ensifer, and Vigna-rhizobia interactions have been examined in Asia, Africa, and America. However, the identities of the rhizobia associated with V. radiata and V. unguiculata in Venezuela remain unknown. In the present study, we isolated Venezuelan symbiotic rhizobia associated with Vigna species from soils with contrasting agroecosystems or from fields in Venezuela. Several types of soils were used for bacterial isolation and nodules were sampled from environments characterized by abiotic stressors, such as high temperatures, high concentrations of NaCl, and acidic or alkaline pH. Venezuelan Vigna-rhizobia were mainly fast-growing. Sequencing of several housekeeping genes showed that in contrast to other continents, Venezuelan Vigna species were nodulated by rhizobia genus including Burkholderia, containing bacteria from several new phylogenetic lineages within the genus Bradyrhizobium. Some Rhizobium and Bradyrhizobium isolates were tolerant of high salinity and Al toxicity. The stress tolerance of strains was dependent on the type of rhizobia, soil origin, and cultivation history. An isolate classified as R. phaseoli showed the highest plant biomass, nitrogen fixation, and excellent abiotic stress response, suggesting a novel promising inoculant for Vigna cultivation in Venezuela.
Accretionary prisms are thick masses of sedimentary material scraped from the oceanic crust and piled up at convergent plate boundaries found across large regions of the world. Large amounts of anoxic groundwater and natural gas, mainly methane (CH4), are contained in deep aquifers associated with these accretionary prisms. To identify the subsurface environments and potential for CH4 production by the microbial communities in deep aquifers, we performed chemical and microbiological assays on groundwater and natural gas derived from deep aquifers associated with an accretionary prism and its overlying sedimentary layers. Physicochemical analyses of groundwater and natural gas suggested wide variations in the features of the six deep aquifers tested. On the other hand, a stable carbon isotope analysis of dissolved inorganic carbon in the groundwater and CH4 in the natural gas showed that the deep aquifers contained CH4 of biogenic or mixed biogenic and thermogenic origins. Live/dead staining of microbial cells contained in the groundwater revealed that the cell density of live microbial cells was in the order of 104 to 106 cells mL–1, and cell viability ranged between 7.5 and 38.9%. A DNA analysis and anoxic culture of microorganisms in the groundwater suggested a high potential for CH4 production by a syntrophic consortium of hydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. These results suggest that the biodegradation of organic matter in ancient sediments contributes to CH4 production in the deep aquifers associated with this accretionary prism as well as its overlying sedimentary layers.
Diverse members of Bradyrhizobium diazoefficiens, B. japonicum, and B. ottawaense were isolated from the roots of field-grown sorghum plants in Fukushima, and classified into “Rhizobia” with nodulated soybeans, “Free-living diazotrophs”, and “Non-diazotrophs” by nitrogen fixation and nodulation assays. Genome analyses revealed that B. ottawaense members possessed genes for N2O reduction, but lacked those for the Type VI secretion system (T6SS). T6SS is a new bacterial weapon against microbial competitors. Since T6SS-possessing B. diazoefficiens and B. japonicum have mainly been isolated from soybean nodules in Japan, T6SS-lacking B. ottawaense members may be a cryptic lineage of soybean bradyrhizobia in Japan.
Chitin amendment is an agricultural management strategy for controlling soil-borne plant disease. We previously reported an exponential decrease in chitin added to incubated upland soil. We herein investigated the transition of the bacterial community structure in chitin-degrading soil samples over time and the characteristics of chitinolytic bacteria in order to elucidate changes in the chitinolytic bacterial community structure during chitin degradation. The addition of chitin to soil immediately increased the population of bacteria in the genus Streptomyces, which is the main decomposer of chitin in soil environments. Lysobacter, Pseudoxanthomonas, Cellulosimicrobium, Streptosporangium, and Nonomuraea populations increased over time with decreases in that of Streptomyces. We isolated 104 strains of chitinolytic bacteria, among which six strains were classified as Lysobacter, from chitin-treated soils. These results suggested the involvement of Lysobacter as well as Streptomyces as chitin decomposers in the degradation of chitin added to soil. Lysobacter isolates required yeast extract or casamino acid for significant growth on minimal agar medium supplemented with glucose. Further nutritional analyses demonstrated that the six chitinolytic Lysobacter isolates required methionine (Met) to grow, but not cysteine or homocysteine, indicating Met auxotrophy. Met auxotrophy was also observed in two of the five type strains of Lysobacter spp. tested, and these Met auxotrophs used d-Met as well as l-Met. The addition of Met to incubated upland soil increased the population of Lysobacter. Met may be a factor increasing the population of Lysobacter in chitin-treated upland soil.