A Rapid and Specific Method for the Detection of Indole in Complex Biological Samples

Indole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 produces 3.3 ± 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota.     

Minerals Affect the Specific Diversity of Forest Soil Bacterial Communities

Minerals constitute an ecological niche poorly investigated in the soil, in spite of their important role in biogeochemical cycles and plant nutrition. To evaluate the impact of minerals on the structure of the soil bacterial communities, we compared the bacterial diversity on mineral surfaces and in the surrounding soil. Three pure and calibrated minerals (apatite, plagioclase and a mix of phlogopite-quartz) were buried into the organo-mineral layer of a forest soil. After a 4-year incubation in soil conditions, mineral weathering and microbial colonization were evaluated. Apatite and plagioclase were the only two significantly weathered minerals. The analysis of the 16S rRNA gene sequences generated by the cloning-sequencing procedure revealed that bacterial diversity was higher in the surrounding soil and on the unweathered phlogopite-quartz samples compared with the other minerals. Moreover, a multivariate analysis based on the relative abundance of the main taxonomic groups in each compartments of origin demonstrated that the bacterial communities from the bulk soil differed from that colonizing the minerals. A significant correlation was obtained between the dissolution rate of the minerals and the relative abundance of Beta-proteobacteria detected. Notably, many sequences coming from bacteria colonizing the mineral surfaces, whatever the mineral, harbored high similarity with efficient mineral weathering bacteria belonging to Burkholderia and Collimonas genera, previously isolated on the same experimental site. Taken together, the present results provide new highlights concerning the bacterial communities colonizing minerals surfaces in the soil and suggests that the minerals create true ecological niches: the mineralosphere.     

Dynamics of Fungal Communities in Bulk and Maize Rhizosphere Soil in the Tropics

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.     

产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

目的:采用亚硝基胍(NTG)诱变结合96孔板高通量筛选方法筛选产耐高温谷氨酰胺转胺酶(MTG)的茂原链霉菌(Streptomyces mobaraensis)。方法:通过优化96孔板高通量测定MTG活性的方法、确定筛选温度和时间,建立了产耐高温MTG菌株的快速筛选方法;通过优化NTG诱变条件建立了筛选突变库;通过96孔板高通量初筛、摇瓶复筛获得了产耐高温MTG的突变株12-82,并通过摇瓶发酵对12-82所产MTG进行热稳定性分析。结果:采用2mg/ml NTG、p H8.0、60min的诱变条件获得突变株,将突变株的发酵上清液于70℃水浴7.5min,再在37℃空气浴、反应10min的条件下测定MTG活性,从5 200株突变株中筛选出5株产耐高温MTG的突变株,其中突变株12-82在50℃水浴60min以及70℃水浴1.5min的酶活残留率均比出发株高出近20%,且80℃保温2min仍有11.9%的酶活残留率。结论:利用NTG诱变结合96孔板高通量筛选的方法筛选到5株所产MTG热稳定性相对较高的突变株,其中突变株12-82在50℃、70℃和80℃的酶活残留率均有10%~20%的提高。这为高温食品加工领域所需耐高温MTG生产菌株的高效筛选提供了可行性方案。     

Specific Lysogenicity in Streptomyces azureus

Slope (or plate) cultures of thiostrepton-producing Streptomyces azureus (ATCC 14921) often showed spontaneously developing plaques. Plaques increased in number during serial subcultures. The production of aerial mycelia and sporulating aerial hyphae was interrupted by the overlapping plaques, whereas the growth of substrate mycelia continued in the plaques. These abnormal (eroded) cultures were easily restored to their normal conditions once they were passed through liquid cultures under shaking conditions. A few phage particles were found in the plaques, together with some headless tails and numerous tail tips which formed a hexagonal crystal or a large crystal mass when viewed in an electron microscope. No lytic phenomenon and no phage production were found in the liquid cultures, although all mycelia and spores harbored phage-producing abilities. It was also found that the propagation of phages was successful in solid culture, but not in liquid culture. The whole phage was named SAt2, which belongs to group B of Bradley's morphological classification. From these results, it is considered that S. azureus is lysogenic with temperate phage SAt2, of which virulent mutants are able to infect the aerial mycelia and sporulating hyphae of their lysogenic host.     

In Situ Dynamics and Spatial Heterogeneity of Soil Bacterial Communities Under Different Crop Residue Management

The effect of the location of wheat residues (soil surface vs. incorporated in soil) on their decomposition and on soil bacterial communities was investigated by the means of a field experiment. Bacterial-automated ribosomal intergenic spacer analysis of DNA extracts from residues, detritusphere (soil adjacent to residues), and bulk soil evidenced that residues constitute the zone of maximal changes in bacterial composition. However, the location of the residues influenced greatly their decomposition and the dynamics of the colonizing bacterial communities. Sequencing of 16S rRNA gene in DNA extracts from the residues at the early, middle, and late stages of degradation confirmed the difference of composition of the bacterial community according to the location. Bacteria belonging to the γ-subgroup of proteobacteria were stimulated when residues were incorporated whereas the α-subgroup was stimulated when residues were left at the soil surface. Moreover, Actinobacteria were more represented when residues were left at the soil surface. According to the ecological attributes of the populations identified, our results suggested that climatic fluctuations at the soil surface select populations harboring enhanced catabolic and/or survival capacities whereas residues characteristics likely constitute the main determinant of the composition of the bacterial community colonizing incorporated residues.     

DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, ³²P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 × 10⁴ cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-μg DNA sample.     

Field Application of a Rapid Spectrophotometric Method for Determination of Persulfate in Soil

Remediation of hydrocarbon contaminated soils can be performed both in situ and ex situ using chemical oxidants such as sodium persulfate. Standard methods for quantifying persulfate require either centrifugation or prolonged settling times. An optimized soil extraction procedure was developed for persulfate involving simple water extraction using a modified disposable syringe. This allows considerable saving of time and removes the need for centrifugation. The extraction time was reduced to only 5 min compared to 15 min for the standard approach. A comparison of the two approaches demonstrated that each provides comparable results. Comparisons were made using high (93 g kg⁻¹ soil) and low (9.3 g kg⁻¹ soil) additions of sodium persulfate to a petroleum hydrocarbon-contaminated soil, as well as sand spiked with diesel. Recoveries of 95±1% and 96±10% were observed with the higher application rate in the contaminated soil and spiked sand, respectively. Corresponding recoveries of 86±5% and 117±19% were measured for the lower application rate. Results were obtained in only 25 min and the method is well suited to batch analyses. In addition, it is suitable for application in a small field laboratory or even a mobile, vehicle-based system, as it requires minimal equipment and reagents.     

放牧对赖草草地土壤呼吸日、季动态的影响

土壤呼吸是土壤碳向大气排放的关键过程,受土地利用变化的强烈影响。利用LI-840a静态箱法,对放牧利用下赖草(Leymus secalinus)草地土壤呼吸速率日、季动态进行为期2年(2012–2013)的观测,并分析其与大气、土壤温度和土壤含水量的相关性,旨在为合理利用赖草草地提供依据。结果表明,赖草草地土壤呼吸日、季动态均呈单峰型变化,一天中的最高值出现在午间13点,凌晨4点最低,在生长季初的5–6月和生长季末的9月较低,在生长旺盛期7–8月较高。放牧降低了土壤呼吸速率,但并不改变土壤呼吸速率的变化趋势。土壤呼吸速率日变化与大气温度呈显著相关(P0.05),季节变化主要受0–10 cm土壤温度的调控。围封和放牧草地土壤呼吸速率可以分别用下列方程拟合:Rs=1.040 8e0.086Ts(R2=0.91,P0.01);Rs=1.016e0.075 2Ts(R2=0.95,P0.01)。经综合分析得出如下结论:温度是影响赖草草地土壤呼吸速率的主要因素,放牧通过改变土壤表层温度而降低土壤呼吸速率。     

Rapid Method of Determining Factors Limiting Bacterial Growth in Soil

A technique to determine which nutrients limit bacterial growth in soil was developed. The method was based on measuring the thymidine incorporation rate of bacteria after the addition of C, N, and P in different combinations to soil samples. First, the thymidine incorporation method was tested in two different soils: an agricultural soil and a forest humus soil. Carbon (as glucose) was found to be the limiting substance for bacterial growth in both of these soils. The effect of adding different amounts of nutrients was studied, and tests were performed to determine whether the additions affected the soil pH and subsequent bacterial activity. The incubation time required to detect bacterial growth after adding substrate to the soil was also evaluated. Second, the method was used in experiments in which three different size fractions of straw (1 to 2, 0.25 to 1, and <0.25 mm) were mixed into the agricultural soil in order to induce N limitation for bacterial growth. When the straw fraction was small enough (<0.25 mm), N became the limiting nutrient for bacterial growth after about 3 weeks. After the addition of the larger straw fractions (1 to 2 and 0.25 to 1 mm), the soil bacteria were C limited throughout the incubation period (10 weeks), although an increase in the thymidine incorporation rate after the addition of C and N together compared with adding them separately was seen in the sample containing the size fraction from 0.25 to 1 mm. Third, soils from high-pH, limestone-rich areas were examined. P limitation was observed in one of these soils, while tendencies toward P limitation were seen in some of the other soils.     

Soil Communities Promote Temporal Stability and Species Asynchrony in Experimental Grassland Communities

Background

Over the past two decades many studies have demonstrated that plant species diversity promotes primary productivity and stability in grassland ecosystems. Additionally, soil community characteristics have also been shown to influence the productivity and composition of plant communities, yet little is known about whether soil communities also play a role in stabilizing the productivity of an ecosystem.

Methodology/Principal Findings

Here we use microcosms to assess the effects of the presence of soil communities on plant community dynamics and stability over a one-year time span. Microcosms were filled with sterilized soil and inoculated with either unaltered field soil or field soil sterilized to eliminate the naturally occurring soil biota. Eliminating the naturally occurring soil biota not only resulted in lower plant productivity, and reduced plant species diversity, and evenness, but also destabilized the net aboveground productivity of the plant communities over time, which was largely driven by changes in abundance of the dominant grass Lolium perenne. In contrast, the grass and legumes contributed more to net aboveground productivity of the plant communities in microcosms where soil biota had been inoculated. Additionally, the forbs exhibited compensatory dynamics with grasses and legumes, thus lowering temporal variation in productivity in microcosms that received the unaltered soil inocula. Overall, asynchrony among plant species was higher in microcosms where an unaltered soil community had been inoculated, which lead to higher temporal stability in community productivity.

Conclusions/Significance

Our results suggest that soil communities increase plant species asynchrony and stabilize plant community productivity by equalizing the performance among competing plant species through potential antagonistic and facilitative effects on individual plant species.     

Rapid Plate Test for Evaluating Phage Induction Capacity

An agar plate test is presented as a screening test for phage induction capacity of various chemicals.