The ecosystem of the human gastrointestinal (GI) tract traverses a number of environmental, chemical, and physical conditions because it runs from the oral cavity to the anus. These differences in conditions along with food or other ingested substrates affect the composition and density of the microbiota as well as their functional roles by selecting those that are the most suitable for that environment. Previous studies have mostly focused on Bacteria, with the number of studies conducted on Archaea, Eukarya, and Viruses being limited despite their important roles in this ecosystem. Furthermore, due to the challenges associated with collecting samples directly from the inside of humans, many studies are still exploratory, with a primary focus on the composition of microbiomes. Thus, mechanistic studies to investigate functions are conducted using animal models. However, differences in physiology and microbiomes need to be clarified in order to aid in the translation of animal model findings into the context of humans. This review will highlight Bacteria, Archaea, Fungi, and Viruses, discuss differences along the GI tract of healthy humans, and perform comparisons with three common animal models: rats, mice, and pigs.
Root-associated bacterial communities are necessary for healthy plant growth. Nitrate is a signal molecule as well as a major nitrogen source for plant growth. In this study, nitrate-dependent alterations in root-associated bacterial communities and the relationship between nitrate signaling and root-associated bacteria in Arabidopsis were examined. The bacterial community was analyzed by a ribosomal RNA intergenic spacer analysis (RISA) and 16S rRNA amplicon sequencing. The Arabidopsis root-associated bacterial community shifted depending on the nitrate amount and timing of nitrate application. The relative abundance of operational taxonomic units of 25.8% was significantly changed by the amount of nitrate supplied. Moreover, at the family level, the relative abundance of several major root-associated bacteria including Burkholderiaceae, Paenibacillaceae, Bradyrhizobiaceae, and Rhizobiaceae markedly fluctuated with the application of nitrate. These results suggest that the application of nitrate strongly affects root-associated bacterial ecosystems in Arabidopsis. Bulk soil bacterial communities were also affected by the application of nitrate; however, these changes were markedly different from those in root-associated bacteria. These results also suggest that nitrate-dependent alterations in root-associated bacterial communities are mainly affected by plant-derived factors in Arabidopsis. T-DNA insertion plant lines of the genes for two transcription factors involved in nitrate signaling in Arabidopsis roots, NLP7 and TCP20, showed similar nitrate-dependent shifts in root-associated bacterial communities from the wild-type, whereas minor differences were observed in root-associated bacteria. Thus, these results indicate that NLP7 and TCP20 are not major regulators of nitrate-dependent bacterial communities in Arabidopsis roots.
Cyanobacteria are widely distributed in marine, aquatic, and terrestrial ecosystems, and play an important role in the global nitrogen cycle. In the present study, we examined the genome sequence of the thermophilic non-heterocystous N2-fixing cyanobacterium, Thermoleptolyngbya sp. O-77 (formerly known as Leptolyngbya sp. O-77) and characterized its nitrogenase activity. The genome of this cyanobacterial strain O-77 consists of a single chromosome containing a nitrogen fixation gene cluster. A phylogenetic analysis indicated that the NifH amino acid sequence from strain O-77 was clustered with those from a group of mesophilic species: the highest identity was found in Leptolyngbya sp. KIOST-1 (97.9% sequence identity). The nitrogenase activity of O-77 cells was dependent on illumination, whereas a high intensity of light of 40 μmol m−2 s−1 suppressed the effects of illumination.
RNA-based microbiological analyses, e.g., transcriptome and reverse transcription-quantitative PCR, require a relatively large amount of high quality RNA. RNA-based analyses on microbial communities in deep-sea hydrothermal environments often encounter methodological difficulties with RNA extraction due to the presence of unique minerals in and the low biomass of samples. In the present study, we assessed RNA extraction methods for deep-sea vent chimneys that had complex mineral compositions. Mineral-RNA adsorption experiments were conducted using mock chimney minerals and Escherichia coli total RNA solution, and showed that detectable RNA significantly decreased possibly due to adsorption onto minerals. This decrease in RNA was prevented by the addition of sodium tripolyphosphate (STPP), deoxynucleotide triphosphates (dNTPs), salmon sperm DNA, and NaOH. The addition of STPP was also effective for RNA extraction from the mixture of E. coli cells and mock chimney minerals when TRIzol reagent and the RNeasy column were used, but not when the RNeasy PowerSoil total RNA kit was used. A combination of STPP, TRIzol reagent, the RNeasy column, and sonication resulted in the highest RNA yield from a natural chimney. This indirect extraction procedure is simple, rapid, inexpensive, and may be used for large-scale RNA extraction.
Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA.
Frankia is a representative genus of nitrogen-fixing (N2-fixing) actinobacteria; however, the molecular mechanisms underlying various phenomena such as the differentiation of a N2 fixation-specific structure (vesicle) and the regulation of N2 fixation (nif) genes, have yet to be elucidated in detail. In the present study, we screened hyphal fragments of Frankia casuarinae that were mutagenized by 1-methyl-3-nitro-1-nitrosoguanidine or gamma rays, and isolated 49 candidate N2 fixation mutants. Twelve of these mutants were selected for further study, and their abilities to grow in NH3-deficient (N-) liquid media and their rates of acetylene reduction activities were evaluated. Eleven mutant strains were confirmed to lack the ability to fix N2. Five mutant strains formed significantly reduced numbers of vesicles, while some failed to form large mature vesicles. These vesicle mutants also exhibited an aberrant hyphal morphology, suggesting a relationship between vesicle differentiation and hyphal branching. Ten mutants showed significant reductions in the expression of nifE, nifH, and nifV genes under N- conditions. The genome sequencing of eight mutants identified 20 to 400 mutations. Although mutant strains N3H4 and N6F4 shared a large number of mutations (108), most were unique to each strain. Mutant strain N7C9 had 3 mutations in the nifD and nifH genes that may result in the inability to fix N2. The other mutant strains did not have any mutations in any known N2 fixation-related genes, indicating that they are novel N2 fixation mutants.
Geosmin and 2-methylisoborneol (MIB) outbreaks in tropical water bodies, such as Southeast Asia, by actinomycetes have not yet been elucidated in detail. Six Streptomyces isolates from lowland environments in Malaysia were selected and evaluated for their odor production under different temperatures. The gene responsible for the production of geosmin, geoA, was detected in all isolates, while only two isolates harbored tpc, which is responsible for 2-MIB production. This result suggested that geosmin and 2-MIB synthesis pathway genes already existed in the environment in the Tropics of Southeast Asia. Furthermore, our isolates produced musty odor compounds at 30°C, and differences were observed in musty odor production between various temperatures. This result indicated the potential for odor episodes in water bodies of the tropical countries of Southeast Asia throughout the year due to the mean annual ambient temperature of 27°C in the lowlands.
The results of marine bacterial community succession from a short-term study of seawater incubations at 4°C to North Sea crude oil are presented herein. Oil was used alone (O) or in combination with a dispersant (OD). Marine bacterial communities resulting from these incubations were characterized by a fingerprinting analysis and pyrosequencing of the 16S rRNA gene with the aim of 1) revealing differences in bacterial communities between the control, O treatment, and OD treatment and 2) identifying the operational taxonomic units (OTUs) of early responders in order to define the bacterial gene markers of oil pollution for in situ monitoring.
After an incubation for 1 d, the distribution of the individual ribotypes of bacterial communities in control and oil-treated (O and OD) tanks differed. Differences related to the structures of bacterial communities were observed at later stages of the incubation. Among the early responders identified (Pseudoalteromonas, Sulfitobacter, Vibrio, Pseudomonas, Glaciecola, Neptunomonas, Methylophaga, and Pseudofulvibacter), genera that utilize a disintegrated biomass or hydrocarbons as well as biosurfactant producers were detected. None of these genera included obligate hydrocarbonoclastic bacteria (OHCB). After an incubation for 1 d, the abundances of Glaciecola and Pseudofulvibacter were approximately 30-fold higher in the OD and O tanks than in the control tank. OTUs assigned to the Glaciecola genus were represented more in the OD tank, while those of Pseudofulvibacter were represented more in the O tank. We also found that 2 to 3% of the structural community shift originated from the bacterial community in the oil itself, with Polaribacter being a dominant bacterium.
Carbonyl sulfide (COS) is one of the major sources of stratospheric sulfate aerosols, which affect the global radiation balance and ozone depletion. COS-degrading microorganisms are ubiquitous in soil and important for the global flux of COS. We examined the sulfur isotopic fractionation during the enzymatic degradation of COS by carbonyl sulfide hydrolase (COSase) from Thiobacillus thioparus THI115. The isotopic fractionation constant (34ɛ value) was −2.2±0.2‰. Under experimental conditions performed at parts per million by volume level of COS, the 34ɛ value for intact cells of T. thioparus THI115 was −3.6±0.7‰, suggesting that, based on Rees’ model, the 34ɛ value mainly depended on COS transport into the cytoplasm. The 34ɛ value for intact cells of T. thioparus THI115 was similar to those for Mycobacterium spp. and Williamsia sp., which are known to involve the conserved region of nucleotide sequences encoding the clade D of β-class carbonic anhydrase (β-CA) including COSase. On the other hand, the 34ɛ value was distinct from those for bacteria in the genus Cupriavidus. These results provide an insight into biological COS degradation, which is indispensable for estimating the COS global budget based on the isotope because of the significant contribution of COS degradation by microorganisms harboring β-CA family enzymes.
Biocontrol agents (BCA) effectively suppress soil-borne disease symptoms using natural antagonistic prokaryotes or eukaryotes. The main issue associated with the application of BCA is that disease reduction effects are unstable under different field conditions. In order to identify potentially effective BCA among several fields, we compared prokaryotic and eukaryotic communities in soil with and without tomato bacterial wilt from three different fields, each of which had the same field management and similar soil characteristics. Soil samples were collected from three fields and two depths because bacterial wilt pathogens were present in soil at a depth greater than 40 cm. We classified soil samples based on the presence or absence of the bacterial phcA gene, a key gene for bacterial wilt pathogenicity and tomato disease symptoms. Pyrosequencing of the prokaryotic 16S rRNA gene and eukaryotic internal transcribed spacer region sequences showed that the diversity and richness of the communities mostly did not correlate with disease symptoms. Prokaryotic and eukaryotic community structures were affected more by regional differences than the appearance of disease. Several prokaryotes and eukaryotes were more abundant in soil that lacked disease symptoms, and eight prokaryotes and one eukaryote of this group were commonly detected among the three fields. Some of these taxa were not previously found in disease-suppressive soil. Our results suggest that several prokaryotes and eukaryotes control plant disease symptoms.
The aim of the present study was to identify major bacteria associated with the swim bladder in the rainbow trout, Oncorhynchus mykiss. We extracted DNA from the swim bladder and gut contents in order to perform a temporal temperature gradient gel electrophoresis (TTGE) analysis of 16S rRNA amplicons for bacterial identification to further compare both profiles. Arthrobacter and Cellulosimicrobium were the major genera observed in the swim bladder in fish, but were not present in fish gut contents; Mycoplasma were instead observed in these samples. Further research to investigate the possible symbiotic roles of the swim bladder-associated microbiota in salmonids is needed.
Archaeal communities in mineral soils were compared between a boreal forest in Finland and cold-temperate forest in Japan using 16S rRNA gene-targeted high-throughput sequencing. In boreal soils, Thaumarchaeota Group 1.1c archaea predominated and Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea were also detected. In temperate soils, Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea were dominant members at the subsurface, whereas their dominancy was replaced by Thermoplasmata archaea at the subsoil. An analysis of the ammonia monooxygenase subunit A gene of Archaea also indicated the distribution of Thaumarchaeota Group 1.1a-associated and Group 1.1b archaea in these soils.
The plant symbiotic α-proteobacterium Sinorhizobium meliloti has two RpoH-type sigma factors, RpoH1 and RpoH2. The former induces the synthesis of heat shock proteins and optimizes interactions with the host. Using a Western blot analysis, we examined time course changes in the intracellular contents of these factors upon a temperature upshift. The RpoH1 level was relatively high and constant, suggesting that its regulatory role in the heat shock response is attained through the activation of the pre-existing RpoH1 protein. In contrast, the RpoH2 level was initially undetectable, and gradually increased. These differential patterns reflect the functional diversification of these factors.
When soil oxygen levels decrease, some bradyrhizobia use denitrification as an alternative form of respiration. Bradyrhizobium diazoefficiens (nos+) completely denitrifies nitrate (NO3−) to dinitrogen, whereas B. japonicum (nos−) is unable to reduce nitrous oxide to dinitrogen. We found that anaerobic growth with NO3− as the electron acceptor was significantly lower in B. japonicum than in B. diazoefficiens, and this was not explained by the absence of nos in B. japonicum. Our results indicate that the reason for the limited growth of B. japonicum is weak NO3− reduction due to impaired periplasmic nitrate reductase activity, which may rely on posttranscriptional events.
We isolated Enterococcus species that colonized in the African buffalo (Syncerus caffer) in order to investigate their genetic relatedness and antimicrobial susceptibility. A total of 219 isolates were obtained and a 16S rRNA gene sequence analysis showed they were classified into Enterococcus avium, E. casseliflavus, E. faecalis, E. faecium, E. hirae, or E. mundtii. Multilocus sequence typing of E. faecalis and E. faecium isolates indicated that some of the isolates showed an evolutionary distance that was far from the primary founders. The antimicrobial susceptibility of the enterococcal isolates suggested that the significant transmission of antimicrobial resistance via human intervention had not yet occurred.