While Lactobacillus crispatus contributes to the stability of normal vaginal microbiota, its role in urinary health remains unclear. As part of an on-going attempt to characterize the female urinary microbiota, we report the genome sequence of an L. crispatus strain isolated from a woman displaying no lower urinary tract symptoms

Hilt, Evann Elizabeth

Kalesinskas, Laurynas

Malki, Kema

Price, Travis Kyle

Putonti, Catherine

Shaheen, Majed

Wolfe, Alan J

English

Loyola eCommons

Loyola University Chicago Loyola eCommons Bioinformatics Faculty Publications Faculty Publications 2016 Draft Genome for a Urinary Isolate of Lactobacillus crispatus Travis Kyle Price Loyola University Chicago Majed Shaheen Loyola University Chicago Laurynas Kalesinskas Loyola University Chicago Kema Malki Loyola University Chicago Evann Elizabeth Hilt Loyola University Chicago See next page for additional authors Follow this and additional works at: https://ecommons.luc.edu/bioinformatics_facpub  Part of the Bacteriology Commons, and the Genomics Commons Recommended Citation Price, Travis Kyle; Shaheen, Majed; Kalesinskas, Laurynas; Malki, Kema; Hilt, Evann Elizabeth; Putonti, Catherine; and Wolfe, Alan J.. Draft Genome for a Urinary Isolate of Lactobacillus crispatus. Genome Announcements, 4, 6: e01278-16, 2016. Retrieved from Loyola eCommons, Bioinformatics Faculty Publications, http://dx.doi.org/10.1128/genomeA.01248-16 This Article is brought to you for free and open access by the Faculty Publications at Loyola eCommons. It has been accepted for inclusion in Bioinformatics Faculty Publications by an authorized administrator of Loyola eCommons. For more information, please contact ecommons@luc.edu. This work is licensed under a Creative Commons Attribution 4.0 License. © 2016 Price et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Authors Travis Kyle Price, Majed Shaheen, Laurynas Kalesinskas, Kema Malki, Evann Elizabeth Hilt, Catherine Putonti, and Alan J. Wolfe This article is available at Loyola eCommons: https://ecommons.luc.edu/bioinformatics_facpub/20 Draft Genome Sequence of a Urinary Isolate of Lactobacillus crispatusTravis K. Price,a Majed Shaheen,b Laurynas Kalesinskas,b,c Kema Malki,c Evann E. Hilt,a Catherine Putonti,b,c,d Alan J. WolfeaHealth Sciences Division, Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USAa; BioinformaticsProgram, Loyola University Chicago, Chicago, Illinois, USAb; Department of Biology, Loyola University Chicago, Chicago, Illinois, USAc; Department of Computer Science,Loyola University Chicago, Chicago, Illinois, USAdWhile Lactobacillus crispatus contributes to the stability of normal vaginal microbiota, its role in urinary health remains un-clear. As part of an on-going attempt to characterize the female urinary microbiota, we report the genome sequence of anL. crispatus strain isolated from a woman displaying no lower urinary tract symptoms.Received 17 September 2016 Accepted 7 October 2016 Published 1 December 2016Citation Price TK, Shaheen M, Kalesinskas L, Malki K, Hilt EE, Putonti C, Wolfe AJ. 2016. Draft genome sequence of a urinary isolate of Lactobacillus crispatus. Genome Announc4(6):e01278-16. doi:10.1128/genomeA.01278-16.Copyright © 2016 Price et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Address correspondence to Alan J. Wolfe, awolfe@luc.edu.As part of an attempt to characterize the newly discovered fe-male urinary microbiota (1–8), we report the genome se-quence and annotation of a strain of Lactobacillus crispatus iso-lated from the bladder of an adult female. This is the first genomereport of a urinary isolate of L. crispatus, a species associated withbladder health (8).Using the expanded spectrum version (9) of the enhancedquantitative urine culture protocol (2), L. crispatus strain C037was isolated from a healthy female not displaying any urinarysymptoms. The strain was subcultured to purity, analyzed bymatrix-assisted laser desorption/ionization-time-of-flight massspectrometry, and pure cultures were stored at 80°C in 2 mLCryoSaver Brucella broth with 10% glycerol, no beads, cryovials(Hardy Diagnostics). For genome extraction, the preserved pureculture isolate was grown on 5% sheep blood agar (BDBBL™ pre-pared plated media) under 5% CO2 at 35°C for 24 h.To extract genomic DNA, cells were resuspended in 0.5 mLDNA extraction buffer (20 mM tris-Cl, 2 mM EDTA, 1.2% TritonX-100, pH 8) followed by addition of 50L lysozyme (20 mg/mL),30 L mutanolysin, and 5 L RNase (10 mg/mL). After a 1-hincubation at 37°C, 80 L 10% SDS and 20 L proteinase K wereadded followed by a 2-h incubation at 55°C. Then, 210 L of6M NaCl and 700 L phenol-chloroform were added. After a30-min incubation with rotation, the solution was centrifugedat 13,500 rpm for 10 min, and the aqueous phase extracted. Anequivalent volume of isopropanol was added; after a 10-min in-cubation, the solution was centrifuged at 13,500 rpm for 10 min.The supernatant was decanted and the DNA pellet precipitatedusing 600 L 70% ethanol. Following ethanol evaporation, theDNA pellet was resuspended in tris-EDTA and stored at20°C.Genomic DNA was diluted in water to a concentration of0.2 ng/l. Library preparation was performed using the NexteraXT DNA library preparation kit (Illumina) according to manufac-turer’s instructions with 1 ng of input DNA. The isolate was se-quenced twice, on two separate runs, using the Illumina MiSeqplatform and the MiSeq reagent kit v2 (300-cycles). Sequence as-sembly was performed using Velvet (10) (k  99) followed bySSPACE (11) for scaffolding. L. crispatus C037 was assembled into96 scaffolds with a genome coverage of 113. The scaffolds in-clude 2.147 Mbp of sequence with a GC content of 36.6%. Geneannotations were performed using GLIMMER (12) and tRNA-Scan (13) identifying 2,096 protein coding genes, 65 RNA (tRNAand rRNA) genes, and four clustered regularly interspaced shortpalindromic repeats (CRISPR) (14). The 16S rRNA gene sequenceof the urinary isolate C037 was identical to that of the species’ typestrain L. crispatus ST1 (NR_074986), an avian enteric strain. Onescaffold (10,704 bp in length) produced a hit to the 16,663 bpL. crispatus plasmid pLc17 (KR052811); while a significant pro-portion of the plasmid sequence was detected, a complete, circu-larized assembly was not possible.Accession number(s). The draft whole-genome project forL. crispatus C037 has been deposited at DDBJ/EMBL/GenBankunder accession number MAKH00000000.ACKNOWLEDGMENTSWe acknowledge Gina Kuffel and Michael Zilliox for sequencing this ge-nome.FUNDING INFORMATIONThis work, including the efforts of Alan J. Wolfe, was funded byHHS | National Institutes of Health (NIH) (RO1DK104718). Thiswork, including the efforts of Catherine Putonti, was funded byNational Science Foundation (NSF) (1149387). This work, includingthe efforts of Catherine Putonti and Alan J. Wolfe, was funded byLoyola University Chicago (LUC) (Multidisciplinary Research Award).REFERENCES1. Fouts DE, Pieper R, Szpakowski S, Pohl H, Knoblach S, Suh MJ, HuangST, Ljungberg I, Sprague BM, Lucas SK, Torralba M, Nelson KE, GroahSL. 2012. Integrated next-generation sequencing of 16S rDNA and meta-proteomics differentiate the healthy urine microbiome from asymptom-atic bacteriuria in neuropathic bladder associated with spinal cord injury.J Transl Med 10:174. http://dx.doi.org/10.1186/1479-5876-10-174.2. Hilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, MuellerER, Brubaker L, Gai X, Wolfe AJ, Schreckenberger PC. 2014. Urine isnot sterile: use of enhanced urine culture techniques to detect residentbacterial flora in the adult female bladder. J Clin Microbiol 52:871– 876.http://dx.doi.org/10.1128/JCM.02876-13.3. Khasriya R, Sathiananthamoorthy S, Ismail S, Kelsey M, Wilson M,crossmarkGenome AnnouncementsNovember/December 2016 Volume 4 Issue 6 e01278-16 genomea.asm.org 1 on January 18, 2017 by guesthttp://genomea.asm.org/Downloaded from Rohn JL, Malone-Lee J. 2013. Spectrum of bacterial colonization associ-ated with urothelial cells from patients with chronic lower urinary tractsymptoms. J Clin Microbiol 51:2054 –2062. http://dx.doi.org/10.1128/JCM.03314-12.4. Lewis DA, Brown R, Williams J, White P, Jacobson SK, Marchesi JR,Drake MJ. 2013. The human urinary microbiome; bacterial DNA invoided urine of asymptomatic adults. Front Cell Infect Microbiol 3:41.http://dx.doi.org/10.3389/fcimb.2013.00041.5. Nienhouse V, Gao X, Dong Q, Nelson DE, Toh E, McKinley K,Schreckenberger P, Shibata N, Fok CS, Mueller ER, Brubaker L, WolfeAJ, Radek KA. 2014. Interplay between bladder microbiota and urinaryantimicrobial peptides: mechanisms for human urinary tract infectionrisk and symptom severity. PLoS One 9:e114185. http://dx.doi.org/10.1371/journal.pone.0114185.6. Siddiqui H, Nederbragt AJ, Lagesen K, Jeansson SL, Jakobsen KS. 2011.Assessing diversity of the female urine microbiota by high throughputsequencing of 16S rDNA amplicons. BMC Microbiol 11:244. http://dx.doi.org/10.1186/1471-2180-11-244.7. Wolfe AJ, Toh E, Shibata N, Rong R, Kenton K, Fitzgerald M, MuellerER, Schreckenberger P, Dong Q, Nelson DE, Brubaker L. 2012. Evi-dence of uncultivated bacteria in the adult female bladder. J Clin Micro-biol 50:1376 –1383. http://dx.doi.org/10.1128/JCM.05852-11.8. Pearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, FokC, Kliethermes S, Schreckenberger PC, Brubaker L, Gai X, Wolfe AJ.2014. The female urinary microbiome: a comparison of women with andwithout urgency urinary incontinence. mBio 5:e01283-14. http://dx.doi.org/10.1128/mBio.01283-14.9. Price TK, Dune T, Hilt EE, Thomas-White KJ, Kliethermes S, BrincatC, Brubaker L, Wolfe AJ, Mueller ER, Schreckenberger PC. 2016. Theclinical urine culture: enhanced techniques improve detection of clinicallyrelevant microorganisms. J Clin Microbiol 54:1216 –1222. http://dx.doi.org/10.1128/JCM.00044-16.10. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short readassembly using de Bruijn graphs. Genome Res 18:821– 829. http://dx.doi.org/10.1101/gr.074492.107.11. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaf-folding pre-assembled contigs using SSPACE. Bioinformatics 27:578 –579. http://dx.doi.org/10.1093/bioinformatics/btq683.12. Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifyingbacterial genes and endosymbiont DNA with glimmer. Bioinformatics23:673– 679. http://dx.doi.org/10.1093/bioinformatics/btm009.13. Schattner P, Brooks AN, Lowe TM. 2005. The tRNAscan-SE, snoscanand snoGPS web servers for the detection of tRNAs and snoRNAs. NucleicAcids Res 33:W686 –W689. http://dx.doi.org/10.1093/nar/gki366.14. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool toidentify clustered regularly interspaced short palindromic repeats. NucleicAcids Res 35:W52–W57. http://dx.doi.org/10.1093/nar/gkm360.Price et al.Genome Announcements2 genomea.asm.org November/December 2016 Volume 4 Issue 6 e01278-16 on January 18, 2017 by guesthttp://genomea.asm.org/Downloaded from 

Draft Genome for a Urinary Isolate of Lactobacillus crispatus

Name not available

https://ecommons.luc.edu/cgi/viewcontent.cgi?article=1025&amp;context=bioinformatics_facpub

Draft Genome for a Urinary Isolate of Lactobacillus crispatus

Abstract

Similar works

Full text

Available Versions

Loyola eCommons

Name not available