The availability of benthic O2 plays a crucial role in benthic microbial communities and regulates many important biogeochemical processes. Burrowing activities of macrobenthos in the sediment significantly affect O2 distribution and its spatial and temporal dynamics in burrows, followed by alterations of sediment microbiology. Consequently, numerous research groups have investigated O2 dynamics in macrofaunal burrows. The introduction of powerful tools, such as microsensors and planar optodes, to sediment analysis has greatly enhanced our ability to measure O2 dynamics in burrows at high spatial and temporal resolution with minimal disturbance of the physical structure of the sediment. In this review, we summarize recent studies of O2-concentration measurements in burrows with O2 microsensors and O2 planar optodes. This manuscript mainly focuses on the fundamentals of O2 microsensors and O2 planar optodes, and their application in the direct measurement of the spatial and temporal dynamics of O2 concentrations in burrows, which have not previously been reviewed, and will be a useful supplement to recent literature reviews on O2 dynamics in macrofaunal burrows.
An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost culturability by the addition of 3 mM H2O2 to cultures grown in Luria-Bertani (LB) medium; however, some H2O2-treated bacteria regained their culturability in M9 minimal medium, if sodium pyruvate was present at at least 0.3 mM. In addition, most pyruvate analogues, such as bromopyruvate or phenylpyruvate, did not show restoration activity similar to that of pyruvate, except in the case of α-ketobutyrate. Further analysis of the mechanism underlying the resuscitation by pyruvate revealed that although many of the bacteria showed respiratory activity on CTC (5-cyano-2,3-di-(p-tolyl) tetrazolium chloride) reduction with or without pyruvate, the biosynthesis of DNA and protein synthesis were quite different in the presence or absence of pyruvate, i.e., pyruvate endowed the cells with the ability to incorporate much more radio-label into precursors during the resuscitation process. These results suggest that pyruvate is one of the key molecules working in the resuscitation process by taking bacteria from the non-culturable state to the growing and colony-forming state by triggering the synthesis of macromolecules such as DNA and protein.
We investigated the effect of rainfall on the levels and sources of microbial contamination in the Han River, Korea. Thirty-four samples were collected at two sampling sites located upstream and downstream in the river from July 2010 to February 2011. Various fecal indicator microorganisms, including total coliform, fecal coliform, Escherichia coli, Enterococcus spp., somatic and male-specific (F+) coliphage, and four major enteric viruses were analyzed. Rainfall was positively correlated with the levels of fecal coliform and norovirus at both sampling sites. Additionally, rainfall was positively correlated with the levels of total coliform, E. coli, Enterococcus spp., and F+ coliphage at the upstream site. To identify the source of fecal contamination, microbial source tracking (MST) was conducted using both male-specific (F+) RNA coliphage and the Enterococcus faecium esp gene as previously described. Our results clearly indicated that the majority of fecal contamination at the downstream Han River site was from a human source. At the upstream sampling site, contamination from human fecal matter was very limited; however, fecal contamination from non-point animal sources increased following rainfall. In conclusion, our data suggest that rainfall significantly affects the level and source of fecal contamination in the Han River, Korea.
Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)-1. In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)-1 for P. nicotianae and from 13 to 233 pg (g soil)-1 for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.
We investigated communities of arbuscular mycorrhizal fungi (AMF) in the fine roots of Pyrus pyrifolia var. culta, and Plantago asiatica to consider the relationship between orchard trees and herbaceous plants in AMF symbioses. The AMF communities were analyzed on the basis of the partial fungal DNA sequences of the nuclear small subunit ribosomal RNA gene (SSU rDNA), which were amplified using the AMF-specific primers AML1 and AML2. Phylogenetic analysis showed that the obtained AMF sequences were divided into 23 phylotypes. Among them, 12 phylotypes included AMF from both host plants, and most of the obtained sequences (689/811) were affiliated to them. Canonical correspondence analysis showed that the host plant species did not have a significant effect on the distribution of AMF phylotypes, whereas the effects of sampling site, soil total C, soil total N and soil-available P were significant. It was also found that the mean observed overlaps of AMF phylotypes between the paired host plants in the same soil cores (27.1% of phylotypes shared) were significantly higher than the mean 1,000 simulated overlaps (14.2%). Furthermore, the same AMF sequences (100% sequence identity) were detected from both host plants in 8/12 soil cores having both roots. Accordingly, we concluded that Py. pyrifolia and Pl. asiatica examined shared some AMF communities, which suggested that understory herbaceous plants may function as AMF inoculum sources for orchard trees.
We addressed the challenges of analyzing next-generation 16S rRNA gene deep sequencing data from the uncharacterized microbial majority. This was performed using a novel de novo semi-alignment approach. The semi-alignments were based on Orthologous Tri-Nucleotides (OTNs), which are identical trinucleotides located in the same sequence region. OTNs in high error homopolymeric tracts were excluded to avoid overestimation of genetic distances. Phylogenetic information was derived assuming an exponential decay in shared OTNs between pairs of bacteria. OTN relatedness was also explored through principal component analysis (PCA). In evaluating the OTN approach we reanalyzed a dataset consisting of triplicate GS FLX titanium pyrosequencing runs for each of two experimental soil samples, in addition to analyses of the Greengenes core dataset. The conclusion from these comparisons was that the OTN approach was superior to traditional alignments both with respect to speed and accuracy. We therefore believe that our OTN-based semi-alignment approach will be a valuable contribution to future exploration of deep sequencing data.
Elevated atmospheric CO2 can influence the structure and function of rhizoplane and rhizosphere microorganisms by altering root growth and the quality and quantity of compounds released into the rhizoplane and rhizosphere via root exudation. In these studies we investigated the transcriptional responses of Bradyrhizobium japonicum cells growing in the rhizoplane of soybean plants exposed to elevated atmospheric CO2. The results of microarray analyses indicated that elevated atmospheric CO2 concentration indirectly influenced the expression of a large number of genes in Bradyrhizobium attached to soybean roots. In addition, relative to plants and bacteria grown under ambient CO2 growth conditions, genes involved in C1 metabolism, denitrification and FixK2-associated genes, including those involved in nitrogen fixation, microaerobic respiration, respiratory nitrite reductase, and heme biosynthesis, were significantly up-regulated under conditions of elevated CO2 in the rhizosphere. The expression profile of genes involved in lipochitooligosaccharide Nod factor biosynthesis and negative transcriptional regulators of nodulation genes, nolA and nodD2, were also influenced by plant growth under conditions of elevated CO2. Taken together, the results of these studies indicate that the growth of soybeans under conditions of elevated atmospheric CO2 influences gene expressions in B. japonicum in the soybean rhizoplane, resulting in changes to carbon/nitrogen metabolism, respiration, and nodulation efficiency.
A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7T, was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7T was isolated from an enrichment culture at a high dilution rate. Strain SW7T was an obligately anaerobic heterotroph that grew with fermentation and had non-motile thin rods 3.5–16.5 μm in length and 0.2 μm in width constituting multicellular filaments. Growth was observed between 37–65°C (optimum 60°C), pH 5.5–7.3 (optimum pH 6.0), and 0.5–3.5% (w/v) NaCl concentration (optimum 1.0%). Based on the physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7T (=JCM15506T=KCTC5908T).
Nitrification is an important step in nitrogen removal in biological wastewater treatment processes. Recently, Nitrospira have been recognized as the numerically dominant nitrite-oxidizing bacterial genus primarily responsible for the second step of aerobic nitrification; however, Nitrospira usually resist cultivation under laboratory conditions and only one species enriched from activated sludge has been described. In this study, a novel enrichment method for Nitrospira was successfully developed using continuous feeding bioreactors. By controlling nitrite concentrations strictly in the bioreactor at low levels below 10 mg-N L-1, coexisting members of sublineages I and II of the genus Nitrospira were enriched selectively. The maximum ratios of sublineages I and II to total microbial cells achieved 88.3% and 53.8%, respectively. This enrichment method is potentially applicable to other uncultured Nitrospira.
The class Thermoplasmata harbors huge uncultured archaeal lineages at the order level, so-called Groups E2 and E3. A novel archaeon Kjm51a affiliated with Group E2 was enriched from anaerobic sludge in the present study. Clone library analysis of the archaeal 16S rRNA and mcrA genes confirmed a unique archaeal population in the enrichment culture. The 16S rRNA gene-based phylogeny revealed that the enriched archaeon Kjm51a formed a distinct cluster within Group E2 in the class Thermoplasmata together with Methanomassiliicoccus luminyensis B10T and environmental clone sequences derived from anaerobic digesters, bovine rumen, and landfill leachate. Archaeon Kjm51a showed 87.7% 16S rRNA gene sequence identity to the closest cultured species, M. luminyensis B10T, indicating that archaeon Kjm51a might be phylogenetically novel at least at the genus level. In fluorescence in situ hybridization analysis, archaeon Kjm51a was observed as coccoid cells completely corresponding to the archaeal cells detected, although bacterial rod cells still coexisted. The growth of archaeon Kjm51a was dependent on the presence of methanol and yeast extract, and hydrogen and methane were produced in the enrichment culture. The addition of 2-bromo ethanesulfonate to the enrichment culture completely inhibited methane production and increased hydrogen concentration, which suggested that archaeon Kjm51a is a methanol-reducing hydrogenotrophic methanogen. Taken together, we propose the provisional taxonomic assignment, named Candidatus Methanogranum caenicola, for the enriched archaeon Kjm51a belonging to Group E2. We also propose to place the methanogenic lineage of the class Thermoplasmata in a novel order, Methanomassiliicoccales ord. nov.
Airborne bacteria are important biological components of the aerosols and have a close relationship with human health as they can have adverse effects through infection and toxicity; higher concentrations can result in various microbial diseases. Moreover, they have a great influence on air quality in Beijing. In this study, a systematic survey on culturable airborne bacteria was carried out for 1 year at a slaughtering plant in Beijing. Bacterial samples were collected with FA-1 sampler for 3 min, three times each day, for three consecutive days of each month from three sampling sites using BIOLOG identification technology. Results showed that Gram-positive bacteria contributed 80%–85% and were much more prevalent than Gram-negative bacteria. Amongst 47 genera of bacteria, including 31 Gram-positive bacteria and 16 Gram-negative bacteria, Micrococcus, Staphylococcus, Bacillus, Corynebacterium, and Pseudomonas were dominant, and Micrococcus, which contributed 20%–30%, was the most dominant genus. The concentration of airborne bacteria was significantly higher in shed used to stay chicken waiting for slaughtering (SSC) and entrances to personnel and transport vehicles with products (EPV) than in green belt (GB). During the year, bacterial concentrations in summer and autumn were much higher than in winter and spring in SSC and EPV, and there were no significant variations in bacterial concentrations in GB. In different periods, a lower concentration of airborne bacteria was found at 13:00.
Antimony (Sb) is a naturally occurring toxic element commonly associated with arsenic (As) in the environment and both elements have similar chemistry and toxicity. Increasing numbers of studies have focused on microbial As transformations, while microbial Sb interactions are still not well understood. To gain insight into microbial roles in the geochemical cycling of Sb and As, soils from Sb mine tailing were examined for the presence of Sb- and As-oxidizing bacteria. After aerobic enrichment culturing with AsIII (10 mM) or SbIII (100 μM), pure cultures of Pseudomonas- and Stenotrophomonas-related isolates with SbIII oxidation activities and a Sinorhizobium-related isolate capable of AsIII oxidation were obtained. The AsIII-oxidizing Sinorhizobium isolate possessed the aerobic arsenite oxidase gene (aioA), the expression of which was induced in the presence of AsIII or SbIII. However, no SbIII oxidation activity was detected from the Sinorhizobium-related isolate, suggesting the involvement of different mechanisms for Sb and As oxidation. These results demonstrate that indigenous microorganisms associated with Sb mine soils are capable of Sb and As oxidation, and potentially contribute to the speciation and mobility of Sb and As in situ.
Microbiological monitoring is important to assure microbiological safety, especially in long-duration space habitation. We have been continuously monitoring the abundance and diversity of bacteria in the International Space Station (ISS)-“Kibo” module to accumulate knowledge on microbes in the ISS. In this study, we used a new sampling device, a microbe-collecting adhesive sheet developed in our laboratory. This adhesive sheet has high operability, needs no water for sampling, and is easy to transport and store. We first validated the adhesive sheet as a sampling device to be used in a space habitat with regard to the stability of the bacterial number on the sheet during prolonged storage of up to 12 months. Bacterial abundance on the surfaces in Kibo was then determined and was lower than on the surfaces in our laboratory (105 cells [cm2]-1), except for the return air grill, and the bacteria detected in Kibo were human skin microflora. From these aspects of microbial abundance and their phylogenetic affiliation, we concluded that Kibo has been microbiologically well maintained; however, microbial abundance may increase with the prolonged stay of astronauts. To ensure crew safety and understand bacterial dynamics in space habitation environments, continuous bacterial monitoring in Kibo is required.
A PCR-DGGE primer pair, Tyl2F-Tyl4R, specific to plant parasitic and fungivorous nematodes was designed based on the 18S rRNA gene. The results of community analysis using the primers showed that they are specific to the order Tylenchida. This primer pair detected species belonging to Tylenchida with high sensitivity and high resolution. The number of detected species of plant parasitic and fungivorous nematodes and their band intensity were much improved compared with PCR-DGGE analysis using the SSU18A-SSU9R primer, which is commonly used for nematode community analysis. It was confirmed that using a group-specific primer was effective for nematode community analysis with PCR-DGGE.
To shed light on the breadth of the host range of Mesorhizobium loti strain NZP2037, we determined the sequence of the NZP2037 symbiosis island and compared it with those of strain MAFF303099 and R7A islands. The determined 533 kb sequence of NZP2037 symbiosis island, on which 504 genes were predicted, implied its integration into a phenylalanine-tRNA gene and subsequent genome rearrangement. Comparative analysis revealed that the core regions of the three symbiosis islands consisted of 165 genes. We also identified several NZP2037-specific genes with putative functions in nodulation-related events, suggesting that these genes contribute to broaden the host range of NZP2037.