The number of copies of rRNA (rrn) operons in a bacterial genome differs greatly among bacterial species. Here we examined the phenotypic effects of variations in the number of copies of rRNA genes in the genome of Bacillus subtilis by analysis of eight mutant strains constructed to carry from two to nine copies of the rrn operon. We found that a decrease in the number of copies from ten to one increased the doubling time, and decreased the sporulation frequency and motility. The maximum levels for transformation activity were similar among the strains, although the competence development was significantly delayed in the strain with a single rrn operon. Normal sporulation only occurred if more than four copies of the rrn operon were present, although ten copies were needed for vegetative growth after germination of the spores. This behaviour was seen even though the intracellular level of ribosomes was similar among strains with four to ten copies of the rrn operon. Furthermore, ten copies of the rrn operon were needed for the highest swarming activity. We also constructed 21 strains that carried all possible combinations of two copies of the rrn operons, and found that these showed a range of growth rates and sporulation frequencies that all fell between those recorded for strains with one or three copies of the rrn operon. The results suggested that the copy number of the rrn operon has a major influence on cellular processes such as growth rate and sporulation frequency.

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/µL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

No abstract

Root-associated microbial communities are very important in the adaptation of halophytes to coastal environments. However, little has been reported on microbial community structures related to halophytes, or on comparisons of their compositions among halophytic plant species. Here, we studied the diversity and community structure of both rhizosphere and root endosphere bacteria in two halophytic plants: Glaux maritima and Salicornia europaea. We sampled the rhizosphere, the root endosphere, and bulk control soil samples, and performed bacterial 16S rRNA sequencing using the Illumina MiSeq platform to characterize the bacterial community diversities in the rhizosphere and root endosphere of both halophytes. Among the G. maritima samples, the richness and diversity of bacteria in the rhizosphere were higher than those in the root endosphere but were lower than those of the bulk soil. In contrast for S. europaea, the bulk soil, the rhizosphere, and the root endosphere all had similar bacterial richness and diversity. The number of unique operational taxonomic units within the root endosphere, the rhizosphere, and the bulk soil were 181, 366, and 924 in G. maritima and 126, 416, and 596 in S. europaea, respectively, implying habitat-specific patterns for each halophyte. In total, 35 phyla and 566 genera were identified. The dominant phyla across all samples were Proteobacteria and Bacteroidetes. Actinobacteria was extremely abundant in the root endosphere from G. maritima. Beneficial bacterial genera were enriched in the root endosphere and rhizosphere in both halophytes. Rhizobium, Actinoplanes, and Marinomonas were highly abundant in G. maritima, whereas Sulfurimonas and Coleofasciculus were highly abundant in S. europaea. A principal coordinate analysis demonstrated significant differences in the microbiota composition associated with the plant species and type of sample. These results strongly indicate that there are clear differences in bacterial community structure and diversity between G. maritima and S. europaea. This is the first report to characterize the root microbiome of G. maritima, and to compare the diversity and community structure of rhizosphere and root endosphere bacteria between G. maritima and S. europaea.

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activatorlike effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinaldeficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.