\ua9 2024 American Society for Microbiology. All rights reserved.In this paper, the draft genomes of Micromonospora RTGN7 and RTP1Z1, derived from Alnus glutinosa root nodules, are reported. The assembly of RTGN7 is 6.6 Mbp, composed of 59 contigs, with an N50 of 321,872. RTP1Z1’s assembly is 6.3 Mbp, composed of 151 contigs, with an N50 of 76,442 bp

Fox EM

Montero-Calasanz MDC

Thompson RM

English

Newcastle University E-Prints

 | Bacteriology | AnnouncementDraft genome sequences of two Micromonospora strains isolated from the root nodules of Alnus glutinosaRyan Michael Thompson,1 Edward M. Fox,2 Maria del Carmen Montero-Calasanz1,3AUTHOR AFFILIATIONS See affiliation list on p. 2.ABSTRACT In this paper, the draft genomes of Micromonospora RTGN7 and RTP1Z1, derived from Alnus glutinosa root nodules, are reported. The assembly of RTGN7 is 6.6 Mbp, composed of 59 contigs, with an N50 of 321,872. RTP1Z1’s assembly is 6.3 Mbp, composed of 151 contigs, with an N50 of 76,442 bp.KEYWORDS Micromonospora, endophytes, Alnus glutinosa, Alnus, genomesA lnus glutinosa nodules are hosts of Micromonospora, with isolation of two strains reported (1, 2). Isolation was conducted from the nodules of a single A. glutinosa within Saltwell Park, United Kingdom (54.944723–1.605852). Nodules were washed with tap water, and 4–15 lobes were surface sterilized in 1 mL of 25% strength household bleach (~1.125% sodium hypochlorite) for 5 minutes. After removing the bleach, the lobes were washed five times in 1 mL of sterile distilled water and homogenized in 0.5 mL of ¼ strength Ringer’s solution. To the homogenate, 14.5 mL of ¼ strength Ringer’s solution was added and plated upon tap water yeast extract agar (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1625.pdf) and Zhang starch soil extract agar (both containing 50 mg/mL nystatin) and incubated at 28°C (3).RTGN7 and RTP1Z1 were recovered from tap water yeast extract and Zhang starch soil extract, respectively, after 30 and 40 days of respective incubation. Single colonies were streaked upon their corresponding media and incubated at 28°C for 24 and 14 days individually, with three subculturing iterations performed from single colonies.From a 10-day old culture of RTGN7, grown upon GYM agar (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium65.pdf), DNA was extracted using a GenEluteBacterial Genomic DNA Kit (Sigma-Aldrich, USA) with an additional ethanol precipitation step. Sequencing was conducted by Novogene Co. Ltd., with a DNA library prepared using a Novogene NGS DNA library prep set (catalog no. Pt004), in which the DNA was randomly sheared into short fragments, end repaired, A tailed then ligated with the Illumina adaptor. These sequences were amplified using PCR, size selected for 350 bp, purified, and then sequenced using 150-bp Illumina paired-end sequencing upon an Illumina NovaSeq, with raw reads filtered using FastP (version 0.23.1) (4).From an 11-day-old nutrient broth culture of RTP1Z1, DNA extraction and sequenc­ing were conducted by MicrobesNG (http://www.microbesng.com). The material was incubated in 120 µL of TE buffer containing lysozyme (0.1 mg/mL) and RNase A (0.1 mg/mL) at 37°C for 25 minutes. After which, proteinase K and SDS (final concentra­tion 0.1 mg/mL and 0.5%, respectively) were added and incubated for 5 minutes at 65°C. Genomic DNA was purified using equal volume solid-phase reversible immobilisation beads and elution buffer, with a DNA library prepared using a Nextera XT library prep kit (Illumina, USA). Two modifications were made according to the manufacturer’s protocol, the DNA amount was increased twofold, and the PCR elongation was extended to 45 seconds. The library was sequenced using a 250-bp paired-end protocol upon an March 2024  Volume 13  Issue 3 10.1128/mra.01131-23 1Editor David A. Baltrus, The University of Arizona, Tucson, Arizona, USAAddress correspondence to Ryan Michael Thompson, R.thompson12@newcastle.ac.uk, or Maria del Carmen Montero-Calasanz, mariac.montero.calasanz@juntadeandalucia.es.The authors declare no conflict of interest.See the funding table on p. 2.Received 21 November 2023Accepted 17 January 2024Published 1 February 2024Copyright © 2024 Thompson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Downloaded from https://journals.asm.org/journal/mra on 02 April 2024 by 92.9.186.133.Illumina NovaSeq 6000. The reads were trimmed using Trimmomatic (version 0.30) with a sliding window quality cutoff of Q15 (5).The following analysis was conducted using the program’s standard settings unless otherwise noted. Both isolates reads were uploaded to Galaxy Europe (https://usega­laxy.eu/) (6), assessed using FASTQC (Galaxy Version 0.73 + galaxy0) (http://www.bioin­formatics.babraham.ac.uk/projects/fastqc/), and assembled using Shovill (Galaxy Version 1.1.0 + galaxy1) (https://github.com/tseemann/shovill), and the assembly quality was assessed using Quast (Galaxy Version 5.0.2 + galaxy5) (7–9), with contigs <200 bp removed. The taxonomic relationship of RTGN7 and RTP1Z1 to their nearest neighbours was determined using TYGS (v389) (10, 11) (Table 1).ACKNOWLEDGMENTSWe gratefully thank the Gateshead council and Jayne Calvert for their assistance with sample collection within Saltwell Park.This research was funded by the Natural Environment Research Council’s ONE Planet Doctoral Training Partnership (grant number NE/S007512/1). M.C.M.C. is grateful for the funding received from the Ramón y Cajal Research Grant (RYC2019-028468-I) from the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO).AUTHOR AFFILIATIONS1School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom2Department of Applied Sciences, Northumbria University, Newcastle upon Tyne, United Kingdom3IFAPA Las Torres-Andalusian Institute of Agricultural and Fisheries Research and Training, Junta de Andalucía, Cra. Sevilla-Cazalla, Seville, SpainAUTHOR ORCIDsRyan Michael Thompson  http://orcid.org/0000-0002-7349-7658Maria del Carmen Montero-Calasanz  http://orcid.org/0000-0002-2373-3683FUNDINGFunder Grant(s) Author(s)UKRI | Natural Environment Research Council (NERC)NE/S007512/1 Ryan Michael ThompsonSpanish Ministry of Economic, Industry and CompetitivenessRYC2019-028468-I Maria del Carmen Montero-CalasanzAUTHOR CONTRIBUTIONSRyan Michael Thompson, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Visualization, Writing – original draft, Writing – review and editing | Edward M. Fox, Methodology, Project administration, Supervision, Writing – review and editing | Maria del Car­TABLE 1 Summary of read and assembly statistics of the isolates RTGN7 and RTP1Z1Isolate Raw read numberFiltered read Assembly length (bp)Contig numberN50 value GC% Estimated sequencing depthNearest neighbor (percentage identity)RTGN7 9,080,972 9,041,320 6,649,740 84 321,872 72.18 205× Micromonospora rubida NEAU-HG-1 (37.8%)RTP1Z1 1,332,626 1,253,492 6,386,282 151 76,442 72.28 66× Micromonospora kangleipaken­sis DSM 45612 (48.2%)Announcement Microbiology Resource AnnouncementsMarch 2024  Volume 13  Issue 3 10.1128/mra.01131-23 2Downloaded from https://journals.asm.org/journal/mra on 02 April 2024 by 92.9.186.133.men Montero-Calasanz, Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review and editingDATA AVAILABILITYThe draft genomes of Micromonospora sp. RTGN7 and RTP1Z1 were deposited in INSDC under the accession numbers JAPZLF000000000 and JAPFQV000000000, respectively. The SRA data of Micromonospora sp. RTGN7 and RTP1Z1 were deposited in INSDC under the accession numbers SRR22064616 and SRR22354992, respectively.REFERENCES1. Carro L, Pujic P, Trujillo ME, Normand P. 2013. Micromonospora is a normal occupant of actinorhizal nodules. J Biosci 38:685–693. https://doi.org/10.1007/s12038-013-9359-y2. Ghodhbane-Gtari F, Essoussi I, Chattaoui M, Chouaia B, Jaouani A, Daffonchio D, Boudabous A, Gtari M. 2010. Isolation and characteriza­tion of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia. Symbiosis 50:51–57. https://doi.org/10.1007/s13199-009-0029-73. Zhang J, Zhang L. 2011. Improvement of an isolation medium for actinomycetes. MAS 5:124–127. https://doi.org/10.5539/mas.v5n2p1244. Chen S, Zhou Y, Chen Y, Gu J. 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/bioinformatics/bty5605. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer of illumina sequencing data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu1706. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Grüning BA, Guerler A, Hillman-Jackson J, Hiltemann S, Jalili V, Rasche H, Soranzo N, Goecks J, Taylor J, Nekrutenko A, Blankenberg D. 2018. The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46:W537–W544. https://doi.org/10.1093/nar/gky3797. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. Quast: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. https://doi.org/10.1093/bioinformatics/btt0868. Mikheenko A, Prjibelski A, Saveliev V, Antipov D, Gurevich A. 2018. Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34:i142–i150. https://doi.org/10.1093/bioinformatics/bty2669. Mikheenko A, Valin G, Prjibelski A, Saveliev V, Gurevich A. 2016. Icarus: visualizer for de novo assembly evaluation. Bioinformatics 32:3321–3323. https://doi.org/10.1093/bioinformatics/btw37910. Meier-Kolthoff JP, Göker M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182. https://doi.org/10.1038/s41467-019-10210-311. Meier-Kolthoff JP, Carbasse JS, Peinado-Olarte RL, Göker M. 2022. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res 50:D801–D807. https://doi.org/10.1093/nar/gkab902Announcement Microbiology Resource AnnouncementsMarch 2024  Volume 13  Issue 3 10.1128/mra.01131-23 3Downloaded from https://journals.asm.org/journal/mra on 02 April 2024 by 92.9.186.133.

Draft genome sequences of two <em>Micromonospora </em>strains isolated from the root nodules of <em>Alnus glutinosa</em>

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Draft genome sequences of two <em>Micromonospora </em>strains isolated from the root nodules of <em>Alnus glutinosa</em>

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