Abstract 1262 Symbiosis of carpenter bees with uncharacterized lactic acid bacteria showing NAD auxotrophy

Summary NAD is a coenzyme central to metabolism in all living organisms. In this study, we identified three novel species of lactic acid bacteria that lacked genes to synthesize NAD in the gut microbes of three different Xylocopa species (carpenter bees) inhabiting Japan. Carpenter bees are classified as the solitary bees that usually make nests by tunneling into old timber and bamboo with a small social group. We analyzed the gut microbiomes of three Xylocopa species between 2016 and 2021 and found that all species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known bacterial species. 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. Although most of them were difficult to isolate a pure colony, we isolated three novel LAB species from three different Xylocopa species by supplementing bacterial culture supernatants. We performed whole-genome sequencing of the isolates and revealed that the strains lacked NAD biosynthesis; however, the genome size and number of coding sequences were not significantly reduced, which is a rare character among not only known LAB but also obligate symbionts in insects. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. Methods & Results We analyzed the gut microbiomes of three carpenter bee (Xylocopa) species with honey bees and bumble bees that visit the same flowers between 2016 and 2021 by V1-V2 based 16S rDNA amplicon sequencing. High-quality genomic DNA was extracted from cultured bacteria using standard protocols, and the genome was sequenced with the PacBio Sequel or RSII sequencing platform. All genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Colonies develop well on MRS agar plates supplemented with NAD under anaerobic conditions, and not developed under aerobic conditions. NAD auxotrophs were shown to lack the genes for NAD biosynthesis, specifically the genes for nicotinate phosphoribosyltransferase (pncB or nadC) and NAD+ synthetase (nadE). In some LAB species, the two genes comprise an operon, and both genes are well conserved in other Lactobacillaceae species. Conclusion Although obligate symbionts usually shared reduced genomes that lack genes in almost all functional categories, the genome size of the NAD auxotrophs showed average genome sizes of free-living LAB, and all categories of genes were normal or rather high in carbohydrate utilization. These results indicated that the isolated two strains are unable to reproduce independently unless NAD is provided under symbiosis conditions. Although the merit of acquisition of the NAD auxotroph LAB in Xylocopa is largely unknown, a phylogenetic distance of these core LAB that cannot reproduce themselves suggested that the symbiont acquisition is ancient and specific, and this may be maintained by maternal transmission in the unique lifestyle of carpenter bees. Reference: Kawasaki et al. Microbiology Spectrum 11(4):e00782-23 This study was supported in part by a grant-in-aid from the Japan Society for Promotion of Science (grant no.: 22K07058).