Distribution and isolation of strains belonging to the order Solirubrobacterales

Abstract

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 ...

Resuscitation of the microbial seed bank alters plant‐soil interactions

“…Specifically, OTUs belonging to the Solirubrobacterales were associated with the +Rpf treatment. Members of this group are aerobic, nonspore‐forming, and nonmotile bacteria that are often recovered in forest and agricultural soils (Kim et al, 2007; Reddy & Garcia‐Pichel, 2009; Seki et al, 2015). Little is known about the role of Solirubrobacterales in the context of plant‐microbe interactions, but some representatives assimilate simple organic compounds (e.g., glucose, maltose, sucrose, xylose, and arginine) that are commonly found in the rhizosphere (Reddy & Garcia‐Pichel, 2009).…”

Resuscitation of the microbial seed bank alters plant-soil interactions

“…Specifically, OTUs belonging to the Solirubrobacterales were associated with the +Rpf treatment. Members of this group are aerobic, non-spore-forming, and non-motile bacteria that are often recovered in forest and agricultural soils (Kim et al, 2007; Reddy & Garcia-Pichel, 2009; Seki, Matsumoto, Ōmura, & Takahashi, 2015). Little is known about the role of Solirubrobacterales in the context of plant-microbe interactions, but some representatives assimilate simple organic compounds (e.g., glucose, maltose, sucrose, xylose, and arginine) that are commonly found in the rhizosphere (Reddy & Garcia-Pichel, 2009).…”

Continuing fascination of exploration in natural substances from microorganisms†

“…The strains were detected in 31 samples (72%) and found to be widely distributed. 13) The novel species Conexibacter arvalis KV-962 T and KV-963 were isolated using fivefold diluted nutrient agar without radical scavenger after 21 days cultivation (Table 1).…”