3) The spores have smooth surfaces and the

3). The spores have smooth surfaces and the MK1775 chains are spiral in shape. A blast search with partial 16S rRNA gene sequences (∼1252 bp) of BE74 showed similarity (93–99%) to members of the genus Nocardiopsis in the Nocardiopsaceae family. In a phylogenetic tree based on the neighbor-joining

algorithm, BE74 is clustered with all Nocardiopsis typing species (Tamura et al., 2008). The closest strain to BE74 is N. alba DSM 43377 (99% identity). The two formed a clade that was strongly supported by a high bootstrap value (100%). The N. alba strain BE74 was susceptible to rifamycin (∼2 μg mL−1) on AIA. It was isolated from bee guts in all four seasons. However, we can only ascertain that 23% of the sampled bees (N=40) at this location in the winter carried the N. alba strain. The isolate produced medium levels of antagonism (clearing zones ∼3–7 mm) against the B. marisflavi strain. It showed no activities against other organisms in Table 1 except B. cereus. Nocardiopsis species have been isolated from marine sediments (Engelhardt et al., 2010). Antibiotic biosynthetic genes were searched in a draft of the genome of Nocardiopsis dassonvillei DSM 43111 (Wu et al., 2009). One gene cluster proposed for an involvement in

phenazine biosynthesis has been identified in this organism (Mentel et al., 2009). Phenazines are a family of nitrogen-containing tricyclic pigments produced by rhizosphere bacteria including Pseudomonas and Streptomyces BAY 57-1293 (Pierson & Pierson, 2010). Interestingly, it has been shown that some secreted phenazines of Pseudomonas aeruginosa can promote the anaerobic survival of the producer itself via extracellular electron transfer (Wang et al., 2010). Therefore, we were interested in whether N. alba from the honeybee gut has the phenazine biosynthetic genes and whether they are expressed. The phenazine biosynthetic pathway

is branched from the shikimate pathway in bacteria (Mentel et al., 2009). Five genes, phzB, phzD, phzE, phzF and phzG, are enough required for biosynthesis of the core structure, and they are highly conserved in all known phenazine biosynthetic gene clusters. phzF has been used as a genetic marker for analyzing the diversity and evolution of phenazine biosynthetic pathways in many Gram-negative bacteria, most of which are pseudomonads (Mavrodi et al., 2010). The PCR primers for phzF were tested with BE74 genomic DNA but the reactions did not yield products under the suggested conditions. Instead, PCR primers based on the alignments of phzD genes encoding an isochorismatase from Streptomcyes cinnamonensis DSM 1042, Streptomyces anulatus LU9663 and N. dassonvillei DSM 43111 yielded an ∼340-bp fragment with the BE74 DNA. Putative protein sequences encoded by this DNA fragment showed the highest homology to a part of PhzD from N. dassonvillei DSM 43111 and other homologs (similarity ∼70–90%) involved in isochorismate metabolism.

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