Bacterial community and genome analysis of cytoplasmic incompatibility-inducing Wolbachia in American serpentine leafminer, Liriomyza trifolii.

Liriomyza trifolii, an agricultural pest, is occasionally infected by Wolbachia. A Wolbachia strain present in Liriomyza trifolii is associated with cytoplasmic incompatibility (CI) effects, leading to the death of embryos resulting from incompatible crosses between antibiotic-treated or naturally Wolbachia-free strain females and Wolbachia-infected males. In this study, high-throughput sequencing of hypervariable rRNA genes was employed to characterize the bacterial community in Wolbachia-infected L. trifolii without antibiotic treatment. The analysis revealed that Wolbachia dominates the bacterial community in L. trifolii, with minor presence of Acinetobacter, Pseudomonas, and Limnobacter. To elucidate the genetic basis of the CI phenotype, metagenomic sequencing was also conducted to assemble the genome of the Wolbachia strain. The draft-genome of the Wolbachia strain wLtri was 1.35 Mbp with 34% GC content and contained 1,487 predicted genes. Notably, within the wLtri genome, there are three distinct types of cytoplasmic incompatibility factor (cif) genes: Type I, Type III, and Type V cifA;B. These genes are likely responsible for inducing the strong cytoplasmic incompatibility observed in L. trifolii.

Synthetic engineering and biological containment of bacteriophages.

The serious threats posed by drug-resistant bacterial infections and recent developments in synthetic biology have fueled a growing interest in genetically engineered phages with therapeutic potential. To date, many investigations on engineered phages have been limited to proof of concept or fundamental studies using phages with relatively small genomes or commercially available "phage display kits". Moreover, safeguards supporting efficient translation for practical use have not been implemented. Here, we developed a cell-free phage engineering and rebooting platform. We successfully assembled natural, designer, and chemically synthesized genomes and rebooted functional phages infecting gram-negative bacteria and acid-fast mycobacteria. Furthermore, we demonstrated the creation of biologically contained phages for the treatment of bacterial infections. These synthetic biocontained phages exhibited similar properties to those of a parent phage against lethal sepsis in vivo. This efficient, flexible, and rational approach will serve to accelerate phage biology studies and can be used for many practical applications, including phage therapy.

Isolation and Characterization of a Novel Phage SaGU1 that Infects Staphylococcus aureus Clinical Isolates from Patients with Atopic Dermatitis.

The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

Discovery and Complete Genome Sequence of a Bacteriophage from an Obligate Intracellular Symbiont of a Cellulolytic Protist in the Termite Gut.

Termites depend nutritionally on their gut microbes, and protistan, bacterial, and archaeal gut communities have been extensively studied. However, limited information is available on viruses in the termite gut. We herein report the complete genome sequence (99,517 bp) of a phage obtained during a genome analysis of "Candidatus Azobacteroides pseudotrichonymphae" phylotype ProJPt-1, which is an obligate intracellular symbiont of the cellulolytic protist Pseudotrichonympha sp. in the gut of the termite Prorhinotermes japonicus. The genome of the phage, designated ProJPt-Bp1, was circular or circularly permuted, and was not integrated into the two circular chromosomes or five circular plasmids composing the host ProJPt-1 genome. The phage was putatively affiliated with the order Caudovirales based on sequence similarities with several phage-related genes; however, most of the 52 protein-coding sequences had no significant homology to sequences in the databases. The phage genome contained a tRNA-Gln (CAG) gene, which showed the highest sequence similarity to the tRNA-Gln (CAA) gene of the host "Ca. A. pseudotrichonymphae" phylotype ProJPt-1. Since the host genome lacked a tRNA-Gln (CAG) gene, the phage tRNA gene may compensate for differences in codon usage bias between the phage and host genomes. The phage genome also contained a non-coding region with high nucleotide sequence similarity to a region in one of the host plasmids. No other phage-related sequences were found in the host ProJPt-1 genome. To the best of our knowledge, this is the first report of a phage from an obligate, mutualistic endosymbiont permanently associated with eukaryotic cells.

Dysgonomonas termitidis sp. nov., isolated from the gut of the subterranean termite Reticulitermes speratus.

A facultatively anaerobic, Gram-stain-negative, non-motile and rod-shaped bacterium, strain N-10(T), was isolated from the gut of the termite Reticulitermes speratus. Strain N-10(T) was closely related to Dysgonomonas gadei JCM 16698(T) according to 16S rRNA gene sequence similarity analysis (98 %) and DNA-DNA relatedness value (≤61.3 %). The optimum growth temperature of strain N-10(T) was 30 °C, which was distinct from that (37 °C) of known species of the genus Dysgonomonas. Growth of strain N-10(T) was inhibited on medium containing 5 or 20 % bile, unlike other species of the genus Dysgonomonas. In addition, acid production in the API 20A system and enzymic reactions in the Rapid ID 32A system of strain N-10(T) differed from those of other species of the genus Dysgonomonas. Based on these characteristics, strain N-10(T) represents a novel species of the genus Dysgonomonas, for which the name Dysgonomonas termitidis sp. nov. is proposed. The type strain is N-10(T) ( = JCM 30204(T) = CCUG 66188(T)).