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Draft Genome Sequence of Ureolytic Environmental Isolate Staphylococcus sp. NA309 Jonathan R. Gaiero, Tom Hsiang, Rob W. Nicol, Marc Habash School of Environmental Sciences, University of Guelph, Guelph, Ontario, Canada

We report the 2.7 Mb draft genome sequence of Staphylococcus sp. NA309 isolated from poultry litter. The isolate was a dominant member of the cultivable aerobic bacteria identified to have ureolytic activity, responsible for ammonia generation in poultry litter residue. Received 9 August 2016 Accepted 12 August 2016 Published 6 October 2016 Citation Gaiero JR, Hsiang T, Nicol RW, Habash M. 2016. Draft genome sequence of ureolytic environmental isolate Staphylococcus sp. NA309. Genome Announc 4(5):e0106616. doi:10.1128/genomeA.01066-16. Copyright © 2016 Gaiero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Marc Habash, [email protected].

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taphylococcus sp. NA309 was isolated in 2013 from a survey of cultivable aerobic bacteria present in poultry litter in southern Ontario, Canada (1). Isolate NA309 was identified as ureolytic based on a color change on Christensen’s urea agar indicator medium. PCR amplification and sequencing was performed on the alpha subunit (ureC) of the urease CDS and was identified as closely matching the PLUP group of urease sequences (2). This group, previously identified only by culture-independent sequencing, was found to be a dominant group in several poultry litters tested in the United States (2). Staphylococcus spp. are common in poultry litter (3), and known to be ureolytic (Staphylococcus equorum subsp. linens, S. succinus subsp. succinus) (4, 5). Based on identification by 16S rRNA gene sequencing, Staphylococcus sp. NA309 was determined to be the dominant member of the cultivable ureolytic bacteria, comprising 25% of the community. Genomic DNA from NA309 was isolated from a pure culture grown in nutrient broth at 30°C. DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Germantown, MD) according to the manufacturer’s recommended protocol for Grampositive bacteria. Two micrograms of DNA were sent for sequencing using an Illumina HiSeq 2000 platform and 100-bp paired-end reads at the Genome Quebec facility in Montreal, Quebec, Canada. A total of 22,200,944 reads were generated, with a genome sequence coverage of 1,531⫻. Genome assembly was performed using ABYSS (version 1.5.2), generating 54 contigs. The draft genome comprised 24 contigs that were greater than 200 bp in length, was 2,730,867 bp in size, with an average G⫹C% content of 33.1%. The NCBI prokaryotic genome pipeline (GeneMarkS⫹ version 2.1) was used to predict a total of 2,491 protein-encoding genes, 16 rRNA genes, 38 tRNA genes, and 106 pseudogenes. The scaffold assembly was also uploaded to the Rapid Annotation using Subsystem Technology (RAST) (6) (http://rast.nmpdr .org/rast.cgi). Closely related sequenced organisms were found to be Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305 (genome: 342451.11) with a score of 518, and Staphylococcus equorum subsp. equorum Mu2 with a score of 512 (genome: 1159488.5). Identification of the urease (EC 3.5.1.5) encoding operon (ureABCDEFG) supports the experimental evidence of

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ureolytic activity. The operon included genes encoding the alpha subunit (ureC), beta (ureB), and gamma subunits (ureA), as well as accessory proteins (ureDEFG). Additionally, genes encoding an alternate pathway for urea degradation and ammonia generation, involving allophanate hydrolase (EC 3.5.1.54) and urea carboxylase (EC 6.3.4.6) were found. Antimicrobial resistance genes were identified and similar to those found in other Staphylococcus spp. such as S. aureus (7). Particular genes of interest were unique to this isolate compared to S. aureus RF122 likely resulting from stressors in the poultry litter environment; genes for arsenic and copper resistance (ARC3 and protein D, respectively), ammonia assimilation (ammonium transporter), phosphorus metabolism (exopolyphosphatase), and stress response genes (detoxification, osmotic, oxidative). Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. LGPC00000000. The version described in this paper is the first version, LGPC01000000. FUNDING INFORMATION This work, including the efforts of Marc Habash, was funded by Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC). This work, including the efforts of Rob W. Nicol, was funded by Gouvernement du Canada | Agriculture and Agri-Food Canada (AAFC). This work was supported in part by the CanAdvance program, funded by Agriculture and Agri-Food Canada (AAFC).

REFERENCES 1. Gaiero JR. 2014. Microbiology and nitrogen mineralization in composted poultry litter amended with biodiesel wash water. M.S. thesis. University of Guelph, Guelph, Ontario, Canada. 2. Rothrock MJ, Cook KL, Lovanh N, Warren JG, Sistani K. 2008. Development of a quantitative real-time polymerase chain reaction assay to target a novel group of ammonia-producing bacteria found in poultry litter. Poult Sci 87:1058 –1067. http://dx.doi.org/10.3382/ps.2007-00350. 3. Fries R, Akcan M, Bandick N, Kobe A. 2005. Microflora of two different types of poultry litter. Br Poult Sci 46:668 – 672. http://dx.doi.org/10.1080/ 00071660500395483. 4. Lambert LH, Cox T, Mitchell K, Rosselló-Mora RA, Del Cueto C, Dodge

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DE Orkand P, Cano RJ. 1998. Staphylococcus succinus sp. nov., isolated from Dominican amber. Int J Syst Bacteriol 48:511–518. http://dx.doi.org/ 10.1099/00207713-48-2-511. 5. Place RB, Hiestand D, Gallmann HR, Teuber M. 2003. Staphylococcus equorum subsp. linens, subsp. nov., a starter culture component for surface ripened semi-hard cheeses. Syst Appl Microbiol 26:30 –37. http:// dx.doi.org/10.1078/072320203322337281. 6. Aziz RK, Bartels D, Best AA, Dejongh M, Disz T, Edwards RA,

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Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, Mcneil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:. http://dx.doi.org/10.1186/ 1471-2164-9-75. 7. Lowy FD. 2003. Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest 111:1265–1273. http://dx.doi.org/10.1172/JCI18535.

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September/October 2016 Volume 4 Issue 5 e01066-16

Draft Genome Sequence of Ureolytic Environmental Isolate Staphylococcus sp. NA309.

We report the 2.7 Mb draft genome sequence of Staphylococcus sp. NA309 isolated from poultry litter. The isolate was a dominant member of the cultivab...
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