Two bacterial strains, designated KV-962 T and KV-963, were isolated from soil collected from a field in Japan. Cells of both strains were Gram-staining-positive, non-spore-forming, short rodshaped and motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains were related to Conexibacter woesei DSM 14684 T , with a similarity value of 98.6 %. These strains possessed MK-7 (H 4 ) as the sole menaquinone and contained C 18 : 1 v9c, C 17 : 1 v6c and iso-C 16 : 0 as the major fatty acids. On the basis of genotypic and phenotypic characteristics, strain KV-962 T and KV-963 were indicated as a novel species of the genus Conexibacter, for which the name Conexibacter arvalis sp. nov. is proposed. The type strain is KV-962 T (5DSM 23288 T 5NBRC 106558 T ). Kageyama et al. (2005Kageyama et al. ( , 2007Kageyama et al. ( , 2008) isolated many strains using agar media supplemented with oxidant scavengers and proposed several new genera. Patulibacter minatonensis KV-614 T , belonging to the order Solirubrobacterales (Reddy & Garcia-Pichel, 2009), is a novel species of genus Patulibacter, strains of which were also isolated using medium supplemented with superoxide dismutase (Takahashi et al., 2006). The order Solirubrobacterales is positioned within a deeply branching lineage of the class Actinobacteria and contains only three genera and five species. In the present study, we attempted to isolate new strains related to these five species; however, as all species in this order grow slowly, isolation can prove difficult using standard methods and media. With this in mind, the present study extended to the use of poor medium, on which most bacteria grow slowly and where oxidative stress is weak (Takahashi et al., 2003), allowing the isolation of two novel strains, designated KV-962 T and KV-963. Phylogenetic and chemotaxonomic characteristics indicated that these strains belong to the genus Conexibacter (Monciardini et al., 2003). In this study, we describe the taxonomic position of strains KV-962 T and KV-963, which represent the second novel species of genus Conexibacter.Strains KV-962 T and KV-963 were isolated from a soil sample collected from a field in Saitama prefecture, Japan, by incubation at 27 u C for 21 days on 1/5 strength nutrient agar (1/5 NA; Difco) with 0.002 % benlate (DuPont). The cultural characteristics of the two strains were examined after cultivation at 27 u C for 14 days on 1/5 NA, nutrient agar, ISP medium 5 (DAIGO; Nihon Pharmaceutical), brain heart infusion agar (BHI; Difco), 1/5 strength BHI agar, R2A agar (Difco), 1/5 strength R2A agar, tryptic soy agar (TSA; Difco), Todd-Hewitt agar (THA; Difco), 1/5 strength THA and GPM agar (1 % glucose, 0.5 % peptone, 0.5 % meat extract, 0.3 % NaCl, 1.2 % agar; pH 7). The motility of cells, incubated at 27 u C for 21 days on 1/5 NA, was determined by using light microscopy and morphological characteristics of the strains were determined using a transmission electron microscope (JEM-1200EXII; JEOL) after staining with 1 % uranyl a...

Three novel strains were isolated from a soil sample collected in Japan using GPM agar plates supplemented with superoxide dismutase and/or catalase. The strains were Gram-positive, catalase-positive, irregular rod-shaped bacteria with meso-diaminopimelic acid as a peptidoglycan diagnostic diamino acid, and the acyl type of the peptidoglycan was acetyl. The major menaquinone was MK-8(H4). Mycolic acids were not detected. The G+C content of the DNA was 72–73 mol%. On the basis of morphological and chemotaxonomic properties and a phylogenetic analysis using 16S rRNA gene sequences, these strains were classified as a novel genus and species, Oryzihumus leptocrescens gen. nov., sp. nov., in the family Intrasporangiaceae of the order Actinomycetales. The type strain is KV-628T (=NRRL B-24347T=JCM 12835T=NBRC 100762T).

The number of prokaryotes on earth is estimated to be in the order of 10 30 cells, 1 and it is well known that most microorganisms in the environment are as-yet-uncultured. 2 Many approaches have been tried to isolate unknown bacterial strains. Davis et al. 3 reported that medium choice and incubation time are significant for isolation of rare soil bacteria. Patulibacter minatonensis, belonging to the order Solirubrobacterales, was isolated using medium supplemented with superoxide dismutase and proposed as a novel genus in 2006. 4 At that time, the order Solirubrobacterales 5 consisted of only three families, three genera and three species; Patulibacter minatonensis, Conexibacter woesei 6 and Solirubrobacter pauli, 7 and presently there are still 11 species including the one species that is isolated in this study. We believe that as-yet-uncultured bacteria may exist within this taxon, and it is possible that additional strains could be used as an untapped resource. As the genera Patulibacter, Conexibacter and Solirubrobacter grow slowly, we tried to investigate their distribution and isolate them by cultivation over a long period of time in order to isolate strains belonging to the order Solirubrobacterales.The strategy of detection and isolation of strains in the order Solirubrobacterales is shown in Figure 1. We designed specific primers for detecting Solirubrobacterales strains using the sequences of nine most closely related strains. The strains were as follows: Patulibacter minatonensis KV-614 T , Conexibacter woesei DSM 14684 T , 6 Solirubrobacter pauli B33D1 T , 7 Rubrobacter radiotolerans DSM 5868 T , 8 R. taiwanensis LS-286, 9 R. xylanophilus PRD-1 T , 10 Symbiobacterium toebii SC-1 T , 11 Thermoleophilum album ATCC 35266 TM and T. minutam ATCC 35268 T . 12,13 These gene sequences were aligned to obtain specific sequences and then visually compared to identify regions showing a high degree of conservation within the target order. The primer length was adjusted to give an appropriate T m range to minimize T m differences within primer pairs. Specific primer sets for strains related to the order Solirubrobacterales were designed as follows: 423PF: 5′-TCAGTTGGGACGAAGCTTC-3′ and 1012PR: 5′-AGG GAAGACGTGTTTCCAC-3′. PCR was performed initially with universal primers, and then nested PCR was performed for detection of target DNA in soil samples. PCR with universal primers (11F:5′-AGTTTGATCATGGCTCAG-3′, 1100R:5′-GGGTTGCGCTCGTTG-3′ or 1115R:5′-AGGGTTGCGCTCGTTG-3′) was performed as follows: initial denaturation at 95°C for 1 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 1.5 min and an additional extension step at 72°C for 2 min. Reaction mixtures (50 μl) containing 0.4 μl of Taq polymerase (TaKaRa, Shiga, Japan), 5.0 μl of 10 × Taq buffer, 2.0 μl of dNTP mixtures (2.5 μM), 29.6 μl of dH 2 O, 4.0 μl of each primer (5 μM) and 5.0 μl of DNA were prepared. As a second step, PCR with specific primers (423PF-1012PR) was performed as follows: initial ...