ABSTRACT
Nitrite reductase is a key enzyme for denitrification. There are two types of nitrite reductases: copper-containing NirK and cytochrome cd1-containing NirS. Most denitrifiers possess either nirK or nirS, although a few strains been reported to possess both genes. We herein report the presence of nirK and nirS in the soil-denitrifying bacterium Bradyrhizobium sp. strain TSA1T. Both nirK and nirS were identified and actively transcribed under denitrification conditions. Based on physiological, chemotaxonomic, and genomic properties, strain TSA1T (=JCM 18858T=KCTC 62391T) represents a novel species within the genus Bradyrhizobium, for which we propose the name Bradyrhizobium nitroreducens sp. nov.
Subject(s)
Bradyrhizobium/classification , Bradyrhizobium/enzymology , Denitrification/genetics , Nitrite Reductases/genetics , Soil Microbiology , Bradyrhizobium/genetics , Bradyrhizobium/physiology , DNA, Bacterial/genetics , DNA, Ribosomal Spacer/genetics , Gene Expression Regulation, Enzymologic , Genome, Bacterial/genetics , Molecular Sequence Annotation , Nitrates/metabolism , Oxygen , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
Thirty-nine denitrifying bacterial strains closely related to one another, represented by strains TSA40T and TSA66T, were isolated from rice paddy soils. Strains TSA40T and TSA66T were Gram-stain-negative, slightly curved rod-shaped, and motile by means of polar flagella. They were able to reduce nitrate, nitrite and nitrous oxide, but unable to fix atmospheric N2. While strain TSA66T was able to grow autotrophically by H2-dependent denitrification, strain TSA40T could not. Phylogenetic analysis suggested that they belong to the family Oxalobacteraceae, the order Burkholderiales in the class Betaproteobacteria. Major components in the fatty acids (C16â:â0, C17â:â0 cyclo, C18â:â1ω7c and summed feature 3) and quinone (Q-8) also supported the affiliation of strains TSA40T and TSA66T to the family Oxalobacteraceae. Based on 16S rRNA gene sequence comparisons, strains TSA40T and TSA66T showed the greatest degree of similarity to Herbaspirillum massiliense JC206T, Noviherbaspirillum malthae CC-AFH3T, Noviherbaspirillum humi U15T, Herbaspirillum seropedicae Z67T and Paucimonas lemoignei LMG 2207T, and lower similarities to the members of other genera. Average nucleotide identity values between the genomes of strain TSA40T, TSA66T and H. massiliense JC206T were 75-77â%, which was lower than the threshold value for species discrimination (95-96â%). Based on the 16S rRNA gene sequence analysis in combination with physiological, chemotaxonomic and genomic properties, strains TSA40T (=JCM 17722T=ATCC TSD-69T) and TSA66T (=JCM 17723T=DSM 25787T) are the type strains of two novel species within the genus Noviherbaspirillum, for which the names Noviherbaspirillum denitrificans sp. nov. and Noviherbaspirillum autotrophicum sp. nov. are proposed, respectively. We also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.
Subject(s)
Herbaspirillum/classification , Oryza , Phylogeny , Soil Microbiology , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Denitrification , Fatty Acids/chemistry , Herbaspirillum/genetics , Herbaspirillum/isolation & purification , RNA, Ribosomal, 16S/genetics , Republic of Korea , Sequence Analysis, DNAABSTRACT
Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N(2)O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.
Subject(s)
Azospirillum/genetics , Bradyrhizobium/genetics , Denitrification , Genes, Bacterial , Herbaspirillum/genetics , Metabolic Networks and Pathways/genetics , Azospirillum/isolation & purification , Azospirillum/metabolism , Bradyrhizobium/isolation & purification , Bradyrhizobium/metabolism , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Herbaspirillum/isolation & purification , Herbaspirillum/metabolism , Molecular Sequence Data , Nitrite Reductases/genetics , Oxidoreductases/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.
