Sho Morimoto

Community Structure of Ammonia-Oxidizing Bacteria under Long-Term Application of Mineral Fertilizer and Organic Manure in a Sandy Loam Soil

ABSTRACT The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the α subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils.

Complete genome sequence of Bradyrhizobium sp. S23321: insights into symbiosis evolution in soil oligotrophs.

Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

A new approach to retrieve full lengths of functional genes from soil by PCR-DGGE and metagenome walking

Metagenomes are a vast genetic resource, and various approaches have been developed to explore them. Here, we present a new approach to retrieve full lengths of functional genes from soil DNA using PCR-denaturing gradient gel electrophoresis (DGGE) followed by metagenome walking. Partial fragments of benzoate 1,2-dioxygenase alpha subunit gene (benA) were detected from a 3-chlorobenzoate (3CB)-dosed soil by PCR-DGGE, and one DGGE band induced by 3CB was used as a target fragment for metagenome walking. The walking retrieved the flanking regions of the target fragment from the soil DNA, resulting in recovery of the full length of benA and also downstream gene (benB). The same strategy retrieved another gene, tfdC, and a complete tfdC and two downstream genes were obtained from the same soil. PCR-DGGE allows screening for target genes based on their potential for degrading contaminants in the environment. This feature provides an advantage over other existing metagenomic approaches.

An improved method to extract RNA from soil with efficient removal of humic acids.

Aims:  To remove humic substances from RNA extracted from soil for the study of bacterial gene expression in soil.

Detection of bphAa Gene Expression of Rhodococcus sp. Strain RHA1 in Soil Using a New Method of RNA Preparation from Soil

To understand the response of soil bacteria to the surrounding environment, it is necessary to examine the gene expression profiles of the bacteria in the soil. For this purpose, we developed a new method of extracting RNA from soil reproducibly. Using this new method, we extracted RNA from a field soil, which was sterilized and inoculated with Rhodococcus sp. strain RHA1, a biphenyl degrader isolated from γ-hexachlorocyclohexane-contaminated soil. Data from agarose gel electrophoresis indicated that the extracted RNA was purified properly. This new method can be applied easily in the preparation of large amounts of RNA. Real-time reverse transcription-polymerase chain reaction (RT-PCR) experiments performed by the TaqMan method suggested that the bphAa gene in this strain, which is involved in the degradation of biphenyl, was induced in the biphenyl amended soil.

Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment.

New protocols for the extraction of nucleic acids from soil - a response to Peršoh and Rambold's letter.

Sir, At first, we would like to make one point clear. In Peršoh and Rambold’s (2009) letter to editor, it was thought that our improved method (Wang et al. 2009) increased the amount of extracted RNA ten-fold compared to the original method. Actually, in our paper, we stated that the signal detected by real-time RT-PCR increased ten-fold. This means that enzyme inhibitors (humic acids) were removed efficiently. Considering the binding between humic acids and nucleic acids, we agree that the method developed by Peršoh et al. (2008) is an excellent method of soil nucleic acid extraction, especially when comparing the data based on DNA extracted from different soil types or horizons. However, the situation is quite different with regard to soil RNA extraction for the study of gene expression. First, the common design of a soil RNA study is to compare the gene expression before and after a certain treatment in the same soil. In such case, the binding between humic acids and nucleic acids could be ignored since the loss of nucleic acids, if there is, is the same in all soil samples. Secondly, the half-life of bacterial mRNA is rather short, from less than 30 s to more than 20 min (Ehretsmann et al. 1992). For this reason, it is critical to finish RNA extraction rapidly (Ambion, Austin, TX, USA, http://www.ambion.com/techlib/basics/rnaisol/index.html). In the method developed by Peršoh et al. (2008), even for an experienced researcher, it will take at least 5 min between the first treatment and cell disruption, which is a rather long time compared to the half-life of bacterial mRNA. During that time, soil and the bacterial cells in that soil are subjected to two vigorous shaking in a beads beating instrument. As we mentioned before (Wang et al. 2008), we are afraid that gene expression in bacterial cells might be affected or altered during treatment prior to cell disruption. Thus, in our method, we gave priority to intactness of cells and quickness of the procedure prior to cell disruption, even though it might not be advantageous for entire removal of humic substances from soil samples prior to cell disruption.