Diversity of Transcripts of Nitrite Reductase Genes ( nirK and nirS ) in Rhizospheres of Grain Legumes

Abstract

Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba, Lupinus albus, and Pisum sativum. The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pisum rhizospheres, while patterns 6, 8, and 12 were absent from the Vicia library. Pattern 1, which was the most dominant in the Vicia and Lupinus libraries, constituted about 25% of all clones. The Lupinus library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described nirK sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.

Effect of Sulfadiazine-Contaminated Pig Manure on the Abundances of Genes and Transcripts Involved in Nitrogen Transformation in the Root-Rhizosphere Complexes of Maize and Clover

“…It has been shown in several studies that microbes harboring the nirK gene form the major part of nitrite reducers in different rhizospheres (14,21) and show increased activity compared to bacteria harboring the nirS gene (45). In our study, we confirmed higher nirK gene copy numbers than nirS gene copy numbers in both treatments at all sampling time points and observed that nirK-harboring microbes were more affected by SDZ than nirS-denitrifying bacteria.…”

Diversity of nirK Denitrifying Genes and Transcripts in an Agricultural Soil

“…Beyond assessing the diversity of the denitrifying population, very few studies have assessed the diversity of denitrifying gene transcripts to determine which members of the denitrifying community actively contribute to denitrification (23,32) or have compared the diversity of the community with that of the active component (5,33). Bacteria that use denitrification as an alternative respiratory process belong to different taxonomic groups and under identical conditions may exhibit different growth rates and levels of denitrification activity.…”

Extraction of mRNA from Soil

“…However, rRNA surveys provide no or only indirect information on the functional status of a microbial community. Therefore, monitoring the environmental expression of key genes of particular metabolic pathways, such as the genes for nitrogen fixation (nifH), nitrite reduction (nirS, nirK), ammonia oxidation (amoA), or methane oxidation (pmoA), received increasing attention (3,5,8,16,17,25). Most recently, new sequencing platforms, such as 454 pyrosequencing, allowed the metatranscriptome analysis of complex microbial communities (9, 10, 23, 31).…”