Complete genome sequence of Promicromonospora sp. strain Populi, an actinobacterium isolated from Populus trichocarpa rhizosphere

Members of the bacterial phylum Actinomycetota are abundant and diverse colonizers of plant rhizosphere and various tissues, many of them promoting growth and contributing to stress adaptation or resistance to pathogens (1–4). Among them, Promicromonospora species have been characterized from soils and the roots and leaves of a variety of plants and can confer drought tolerance and resistance to fungal diseases (5–11).

We describe the complete genome sequence of Promicromonospora sp. strain Populi, isolated from the rhizosphere of a mature, wild Populus trichocarpa from the Tieton River watershed in Washington State, USA (latitude: 46^o42′11″ N, longitude: 120^o39′37″ W). A rhizosphere sample (fine roots and adhering soil) was used to obtain a microbial fraction by centrifugation on Histodenz (Sigma-Aldrich Inc., St. Louis, MO, USA) (12) and stained with 5 µM Syto59 (Fisher Scientific Inc., Waltham, MA, USA). A Cytopeia Influx cell sorter (BD, Franklin Lakes, NJ, USA) was used to deposit individual single cells (100 per plate) based on forward-side scatter and fluorescence intensity on various nutrient agar media, as we described previously (13). Following incubation at 28°C for 5 days, we performed taxonomic characterization of individual bacterial colonies by amplification of the small subunit rRNA gene with universal primers (27F-1492R), followed by Sanger sequencing of the PCR product (Eurofin Genomics, Louisville, KY, USA). The sequence was compared to those of other organisms by blastn search of the NCBI rRNA sequence archive (13). A white, rugous colony from a plate with TWYE medium (DSMZ 1625) was identified as a Promicromonospora strain, based on >99% pairwise sequence identity to described species (Promicromonospora alba, NR_148779; Promicromonospora kermanensis, NR_156057). Therefore, we designated the isolate as Promicromonospora sp. strain Populi.

We grew the bacterium in 5 mL of liquid R2A medium for 3 days at 28°C and purified genomic DNA using the Qiagen DNeasy Blood and Tissue kit following the manufacturer’s recommended protocol (Qiagen, Germantown, MD, USA). A non-sheared, unselected library was prepared using the Oxford Nanopore Ligation Sequencing Kit followed by long-read sequencing on a MinION R10.4.1 (Oxford Nanopore Technologies Inc., Cambridge, MA, USA), yielding 379,306 reads (2.9 Gb) with an N₅₀ of 7.6 kb. All data analyses were performed using software defaults. Base calling was performed using Dorado version 0.7.1 high-accuracy calling model. The reads were filtered to retain the top 1 Gbp of reads over 1 kb with Filtlong version 0.2.1, then used in the Trycycler version 0.5.4 workflow (14) together with Flye version 2.9.4 (15), Raven version 1.5.3 (16), and Miniasm/Minipolish version 0.3 (17). The final assembly was polished using Medaka version 1.5. The assembler output was a single circular contig, 5,224,924 bp in length, with 94-fold mean coverage and a G + C content of 70.9%. A protein phylogenetic tree using SpeciesTree version 2.2.0 (18) in KBase (19), using the default set of 49 core, universal bacterial genes places strain Populi closest to Promicromonospora thailandica, Promicromonospora kroppenstedtii, and Promicromonospora umidemergens as well as to other strains associated with Populus deltoides (AC04, CF082, and YR516) (13) (Fig. 1). Pairwise whole genome average nucleotide identity (ANI) among Promicromonospora species, including strain Populi, calculated using FastANI version 0.1.3 (20) ranged between 85% and 89% (Table 1). Gene prediction and functional annotation were performed using the NCBI PGAP version 6.7 (21).

This research was funded by the U.S. DOE Office of Biological and Environmental Research, Genomic Science Program as part of the Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.