Three colony types of Lactobacillus were isolated from the stomach of LL colony stock Peromyscus leucopus deermice, a reservoir for several human zoonoses. Genome sequences revealed two isolates to be new strains of Lactobacillus animalis and Lactobacillus reuteri The third was distinct from known species and was provisionally designated Lactobacillus sp. strain LL6

Barbour, Alan G

Bassam, Khalil

Milovic, Ana

English

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UC IrvineUC Irvine Previously Published WorksTitleGenome Sequences of Three Lactobacillus Species Strains of the Stomach of the White-Footed Deermouse (Peromyscus leucopus)Permalinkhttps://escholarship.org/uc/item/8cm5f88kJournalMicrobiology Resource Announcements, 8(40)ISSN2576-098XAuthorsBassam, KhalilMilovic, AnaBarbour, Alan GPublication Date2019-10-03DOI10.1128/mra.00963-19Copyright InformationThis work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaGenome Sequences of Three Lactobacillus Species Strains ofthe Stomach of the White-Footed Deermouse (Peromyscusleucopus)Khalil Bassam,a Ana Milovic,a Alan G. Barboura,baDepartment of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, USAbDepartment of Medicine, University of California Irvine, Irvine, California, USAABSTRACT Three colony types of Lactobacillus were isolated from the stomach ofLL colony stock Peromyscus leucopus deermice, a reservoir for several human zoono-ses. Genome sequences revealed two isolates to be new strains of Lactobacillus ani-malis and Lactobacillus reuteri. The third was distinct from known species and wasprovisionally designated Lactobacillus sp. strain LL6.Peromyscus leucopus, a widely distributed, abundant mammal in North America, is animportant reservoir for agents of tick-borne zoonoses, including Lyme disease,babesiosis, anaplasmosis, and a viral encephalitis (1). The genus Peromyscus clusterswith hamsters and voles rather than with murids such as Mus musculus (2). P. leucopusis the target for field-delivered oral vaccines (3), but little is known about the gastro-intestinal (GI) microbiota of this or other Peromyscus species.During study of the GI tract of animals of P. leucopus LL stock (Peromyscus GeneticStock Center, University of South Carolina), we identified three types of Lactobacillus inthe stomachs, ceca, and feces of both females and males. The organisms were isolatedfrom stomach contents on Rogosa SL agar (Sigma-Aldrich) plates in a candle jar at 37°Cand were distinguished by colony morphologies. Single colonies of each were culti-vated in Difco MRS broth (Becton, Dickinson). High-molecular-weight genomic DNAwas extracted from harvested cells with a Qiagen Genomic-tip 100/G kit.Their genomes were sequenced by two methods, (i) for long reads on an OxfordNanopore Technology (ONT) MinION Mk1B instrument with a ligation sequencing kit,R9.4.1 flow cell, MinKNOW v. 19.6.8 for primary data acquisition, and Guppy v. 3.2.4 forbase calling with default settings and (ii) for short reads on an Illumina MiSeq instru-ment with paired-end v2 microchemistry and 150 cycles. The library was constructedusing the NEXTflex rapid DNA-seq kit (Bioo Scientific), the quality of sequencing readswas analyzed using FastQC (Babraham Bioinformatics), and reads were trimmed ofPhred scores of 15 and corrected for poor-quality bases using Trimmomatic (4).A hybrid assembly of each genome was carried out with Unicycler v. 0.4.7 (5) withdefault settings and 16 threads on the high-performance computing cluster of theUniversity of California Irvine. Complete, circularly permuted chromosomes were ob-tained for two of the isolates (LL1 and LL7), and two contigs of 1.75 Mb and 0.32 Mb,respectively, were obtained for the third (LL6) (Table 1). Annotation was provided bythe NCBI Prokaryotic Genome Annotation Pipeline (6). By the criterion of 16S ribosomalsequence identity of 99%, isolates LL1 and LL7 were identified as Lactobacillusanimalis and Lactobacillus reuteri, respectively. Other sequences of these organismsconfirmed those classifications.L. reuteri LL7 had another replicon (Table 1) that had coding sequences for a Rep_3(pfam01051) plasmid replication initiation protein and multiple transposases. This isconsistent with a type of megaplasmid observed in other lactobacilli (7).Citation Bassam K, Milovic A, Barbour AG.2019. Genome sequences of threeLactobacillus species strains of the stomach ofthe white-footed deermouse (Peromyscusleucopus). Microbiol Resour Announc 8:e00963-19. https://doi.org/10.1128/MRA.00963-19.Editor Julie C. Dunning Hotopp, University ofMaryland School of MedicineCopyright © 2019 Bassam et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Alan G. Barbour,abarbour@uci.edu.Received 8 August 2019Accepted 10 September 2019Published 3 October 2019GENOME SEQUENCEScrossmVolume 8 Issue 40 e00963-19 mra.asm.org 1TABLE1Resourcesfromthisstudy,sequencingstatistics,andcharacteristicsofchromosomesandplasmidsofthreeLactobacillusspp.SpeciesStrainBioProjectaccessionno.BioSampleaccessionno.SRAaccessionno.(Illumina,ONT)No.ofONTreads(106)/N50(kb)Meancoverage()Size(bp)GCcontent(%)GenBankaccessionno.ONTIlluminaChromosomePlasmidChromosomePlasmidChromosome(s)PlasmidLactobacillusanimalisLL1PRJNA533837SAMN11468794SRX5831611,SRX58316122.31/6.112,0981822,280,5774,01641.037.1CP039849MK858222LactobacillusreuteriLL7PRJNA554696SAMN12280509SRX6494060,SRX64940611.52/0.874232182,205,740178,97738.938.1CP041676CP041677Lactobacillussp.LL6PRJNA554698SAMN12280527SRX6492358,SRX64923572.73/6.212,6012782,067,2366,96933.536.4VLLR01000001(contig1),VLLR01000002(contig1)VLLR01000003Bassam et al.Volume 8 Issue 40 e00963-19 mra.asm.org 2Isolate LL6 could be not be identified with a known Lactobacillus species in thegenome database. Over its full-length 16S rRNA gene sequence, the closest organismwas Lactobacillus amylolyticus at 95.6% nucleotide identity. The identities of LL6’scoding sequences for the core genome genes recA, rpoB, gyrA, and rplB were no morethan 83%, 87%, 81%, and 94%, respectively, with any other Lactobacillus sp. Thesedistances suggest that this represents a previously unknown taxon, provisionallydesignated Lactobacillus sp. strain LL6.Data availability. The strains described here are available from the correspondingauthor. Table 1 provides the publicly available resources from this study with hyperlinksto repositories.ACKNOWLEDGMENTSThis work was supported by National Institutes of Health grant AI-136523. A portionof the work was also supported by NIH S10 instrumentation grants OD010794 andRR024679 and made possible, in part, through access to the Genomic High ThroughputFacility Shared Resource of the Cancer Center Support Grant (CA-62203) at the Univer-sity of California Irvine.REFERENCES1. Barbour AG. 2017. Infection resistance and tolerance in Peromyscus spp.,natural reservoirs of microbes that are virulent for humans. Semin CellDev Biol 61:115–122. https://doi.org/10.1016/j.semcdb.2016.07.002.2. Bedford NL, Hoekstra HE. 2015. The natural history of model organisms:Peromyscus mice as a model for studying natural variation. Elife 4:e06813.https://doi.org/10.7554/eLife.06813.3. Gomes-Solecki MJ, Brisson DR, Dattwyler RJ. 2006. Oral vaccine that breaksthe transmission cycle of the Lyme disease spirochete can be delivered viabait. Vaccine 24:4440–4449. https://doi.org/10.1016/j.vaccine.2005.08.089.4. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer forIllumina sequence data. Bioinformatics 30:2114 –2120. https://doi.org/10.1093/bioinformatics/btu170.5. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterialgenome assemblies from short and long sequencing reads. PLoS ComputBiol 13:e1005595. https://doi.org/10.1371/journal.pcbi.1005595.6. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O’Neill K, Li W,Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, SongJS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genomeannotation and curation. Nucleic Acids Res 46:D851–D860. https://doi.org/10.1093/nar/gkx1068.7. Claesson MJ, Li Y, Leahy S, Canchaya C, van Pijkeren JP, Cerdeño-TárragaAM, Parkhill J, Flynn S, O’Sullivan GC, Collins JK, Higgins D, Shanahan F,Fitzgerald GF, van Sinderen D, O’Toole PW. 2006. Multireplicon genomearchitecture of Lactobacillus salivarius. Proc Natl Acad Sci U S A 103:6718 – 6723. https://doi.org/10.1073/pnas.0511060103.Microbiology Resource AnnouncementVolume 8 Issue 40 e00963-19 mra.asm.org 3

Genome Sequences of Three Lactobacillus Species Strains of the Stomach of the White-Footed Deermouse (Peromyscus leucopus)

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Genome Sequences of Three Lactobacillus Species Strains of the Stomach of the White-Footed Deermouse (Peromyscus leucopus).

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