We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology

Angen, Øystein

Bossé, Janine T

BRaDP1T consortium

Fernandez Crespo, Roberto

Holden, Matthew TG

Langford, Paul R

Li, Yanwen

Maskell, Duncan J

Rycroft, Andrew N

Tucker, Alexander W

Weinert, Lucy A

Wren, Brendan W

Microbiology Resource Announcements

English

LSHTM Research Online

LSHTM Research OnlineBossé, Janine T; Li, Yanwen; Fernandez Crespo, Roberto; Angen, Øystein; Holden, Matthew TG;Weinert, Lucy A; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, AndrewN; +2 more... Langford, Paul R; BRaDP1T consortium; (2020) Draft Genome Sequences of theType Strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), Two NAD-Dependent Bacterial Species Found in the Respiratory Tract of Pigs. MICROBIOLOGY RESOURCEANNOUNCEMENTS, 9 (1). ISSN 2576-098X DOI: https://doi.org/10.1128/MRA.00716-19Downloaded from: http://researchonline.lshtm.ac.uk/id/eprint/4656034/DOI: https://doi.org/10.1128/MRA.00716-19Usage Guidelines:Please refer to usage guidelines at https://researchonline.lshtm.ac.uk/policies.html or alternativelycontact researchonline@lshtm.ac.uk.Available under license: http://creativecommons.org/licenses/by/2.5/https://researchonline.lshtm.ac.ukDraft Genome Sequences of the Type Strains of Actinobacillusindolicus (46K2C) and Actinobacillus porcinus (NM319), TwoNAD-Dependent Bacterial Species Found in the RespiratoryTract of PigsJanine T. Bossé,a Yanwen Li,a Roberto Fernandez Crespo,a Øystein Angen,b Matthew T. G. Holden,c* Lucy A. Weinert,dDuncan J. Maskell,d Alexander W. Tucker,d Brendan W. Wren,e Andrew N. Rycroft,f Paul R. Langford,a on behalf ofthe BRaDP1T consortiumaSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, London, United KingdombDepartment of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, DenmarkcThe Wellcome Trust Sanger Institute, Cambridge, United KingdomdDepartment of Veterinary Medicine, University of Cambridge, Cambridge, United KingdomeFaculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United KingdomfDepartment of Pathology and Pathogen Biology, The Royal Veterinary College, Hatﬁeld, United KingdomABSTRACT We report here the draft genome sequences of the type strains of Acti-nobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract ofpigs and are occasionally associated with lung pathology.Bacteria belonging to the family Pasteurellaceae that inhabit the porcine respiratorytract and require NAD (also called the V factor) for growth include Actinobacilluspleuropneumoniae, Glaesserella parasuis (formerly Haemophilus parasuis), Actinobacillusindolicus, Actinobacillus porcinus, Actinobacillus minor, and a separate species tentativelydesignated “Actinobacillus porcitonsillarum,” which, although phenotypically more sim-ilar to A. pleuropneumoniae, is phylogenetically closer to A. minor (1–5). The ﬁrst twospecies are important pathogens, whereas the latter four are considered commensalsof the upper respiratory tract (1, 3, 6), though isolates of A. indolicus and A. porcinus,associated with lung pathology, have been identiﬁed (1). For A. pleuropneumoniae,there are two biotypes based on the requirement for NAD, with most isolates belongingto the NAD-dependent biovar 1 (7, 8).The phylogeny of the Pasteurellaceae is complicated, and there remain numerousspecies with uncertain taxonomic status (4, 9, 10). Whole-genome sequences, whichcan help clarify evolutionary relationships and the development of differential diag-nostics, are available for all of the above-mentioned bacteria except A. indolicus and A.porcinus. We therefore chose to sequence the type strains, 46K2C and NM319, respec-tively, of these species. Both 46K2CT and NM319T were originally isolated from theupper respiratory tract of pigs in Denmark (11) and Canada (12), respectively. They wereinitially identiﬁed as Haemophilus taxons D and F, respectively, prior to their designa-tion as the type strains of the newly assigned species in 1996 (1, 2).Strains 46K2CT and NM319T were obtained from the Culture Collection University ofGothenburg (CCUG 39029T and CCUG 38924T, respectively), stored frozen at 80°C in25% glycerol, and minimally passaged (up to four times) on brain heart infusion agar(Difco) supplemented with 0.01% NAD prior to genomic DNA extraction using aFastDNA spin kit (MP Biomedicals), according to the manufacturer’s protocol forbacterial cells. Paired-end libraries were prepared from 500 ng of genomic DNA, asCitation Bossé JT, Li Y, Fernandez Crespo R,Angen Ø, Holden MTG, Weinert LA, Maskell DJ,Tucker AW, Wren BW, Rycroft AN, Langford PR,on behalf of the BRaDP1T Consortium. 2020.Draft genome sequences of the type strains ofActinobacillus indolicus (46K2C) andActinobacillus porcinus (NM319), two NAD-dependent bacterial species found in therespiratory tract of pigs. Microbiol ResourAnnounc 9:e00716-19. https://doi.org/10.1128/MRA.00716-19.Editor Steven R. Gill, University of RochesterSchool of Medicine and DentistryCopyright © 2020 Bossé et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to Janine T. Bossé,j.bosse@imperial.ac.uk, or Paul R. Langford,p.langford@imperial.ac.uk.* Present address: Matthew T. G. Holden,School of Medicine, University of St. Andrews,St. Andrews, United Kingdom.Received 18 June 2019Accepted 14 November 2019Published 2 January 2020GENOME SEQUENCEScrossmVolume 9 Issue 1 e00716-19 mra.asm.org 1previously described (13), using the modiﬁed Illumina protocol developed by Quail etal. (14) for sequencing at the Wellcome Sanger Institute (Hinxton, UK) on an IlluminaHiSeq 2000 analyzer for 75 cycles. Draft genome sequences were assembled de novousing a previously reported pipeline (15). Brieﬂy, the 2  76-bp paired reads were usedto create multiple assemblies for each species’ genome using Velvet v1.2 (16) andVelvetOptimiser v2.2.5 (http://bioinformatics.net.au/software.velvetoptimiser.shtml).For each genome, the assembly with the best N50 value was further improved byscaffolding contigs using SSPACE (17), with any gaps ﬁlled by GapFiller (18). Automatedannotation was performed for both genome assemblies using Prokka v1.5 (19). Defaultparameters were used for all software programs.The draft genome of A. indolicus 46K2CT was assembled from 2,031,716 total reads,with an average quality score of 37.9, into 22 contigs (N50, 183,245; L50, 4), with a totallength of 2,103,350 bases. This sequence encodes 1,956 predicted proteins and has aGC content of 40.1%, which is higher than the 35.5% originally determined (2) butlower than the value of 42.6% calculated by Kuhnert and Korczak based on the GCcontent of selected genes (20). The draft genome of A. porcinus NM319T was assembledfrom 1,733,818 reads, with an average quality score of 37.8, into 29 contigs (N50,182,117; L50, 5), with a total length of 2,280,774 bases. This sequence encodes 2,095predicted proteins and has a GC content of 41.1%, which is similar to values (41.4%and 41.6%) previously reported for this strain (2, 20).The availability of the draft genome sequences of the type strains of A. indolicus andA. porcinus will facilitate differentiation of these species from other NAD-dependentbacteria found in the porcine respiratory tract and contribute to the understanding ofPasteurellaceae phylogeny and host interactive biology.Data availability. The draft genome assemblies for A. indolicus 46K2CT and A.porcinus NM319T have been deposited in the European Nucleotide Archive (ENA) underBioProject number PRJEB31492. Raw sequence reads have been deposited in theENA under accession numbers ERS134282 and ERS134597, respectively. The ENAaccession numbers for the assembled contigs are GCA_901764975 (CABFKH010000001through CABFKH010000022) and GCA_901764995 (CAAGST010000001 throughCAAGST010000029), respectively.ACKNOWLEDGMENTSThe Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortiumcomprises the following members: Duncan J. Maskell, Alexander W. (Dan) Tucker, SarahE. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, Roy R. Chaudhuri(University of Cambridge; present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Shefﬁeld, Shefﬁeld, United Kingdom); AndrewN. Rycroft, Gareth A. Maglennon, Jessica Beddow (Royal Veterinary College); Brendan W.Wren, Jon Cuccui, Vanessa S. Terra (London School of Hygiene and Tropical Medicine);and Paul R. Langford, Janine T. Bossé, and Yanwen Li (Imperial College London).This work was supported by a Longer and Larger (LoLa) grant from the Biotechnol-ogy and Biological Sciences Research Council (BBSRC, grant numbers BB/G020744/1,BB/G019177/1, BB/G019274/1, BB/G018553/1, and, in part, BB/S000103/1 and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis(formerly Pﬁzer Animal Health) awarded to the BRaDP1T Consortium. M.T.G.H. wassupported by the Wellcome Trust (grant number 098051).The funders had no role in the study design, data collection and analysis, decisionto publish, or preparation of the manuscript.REFERENCES1. Kielstein P, Wuthe H, Angen Ø, Mutters R, Ahrens P. 2001. Phenotypicand genetic characterization of NAD-dependent Pasteurellaceae fromthe respiratory tract of pigs and their possible pathogenetic impor-tance. Vet Microbiol 81:243–255. https://doi.org/10.1016/s0378-1135(01)00351-0.2. Møller K, Fussing V, Grimont PA, Paster BJ, Dewhirst FE, Kilian M. 1996.Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Acti-nobacillus indolicus sp. nov., three new V factor-dependent species fromthe respiratory tract of pigs. Int J Syst Bacteriol 46:951–956. https://doi.org/10.1099/00207713-46-4-951.Bossé et al.Volume 9 Issue 1 e00716-19 mra.asm.org 23. Gottschalk M, Broes A, Mittal KR, Kobisch M, Kuhnert P, Lebrun A, Frey J.2003. Non-pathogenic Actinobacillus isolates antigenically and biochem-ically similar to Actinobacillus pleuropneumoniae: a novel species? VetMicrobiol 92:87–101. https://doi.org/10.1016/s0378-1135(02)00341-3.4. Korczak B, Christensen H, Emler S, Frey J, Kuhnert P. 2004. Phylogeny ofthe family Pasteurellaceae based on rpoB sequences. Int J Syst EvolMicrobiol 54:1393–1399. https://doi.org/10.1099/ijs.0.03043-0.5. Dickerman A, Bandara AB, Inzana TJ. Phylogenomic analysis of Haemo-philus parasuis and proposed reclassiﬁcation to Glaesserella parasuis,gen. nov., comb. nov. Int J Syst Evol Microbiol, in press. https://doi.org/10.1099/ijsem.0.003730.6. Chiers K, Haesebrouck F, Mateusen B, Van Overbeke I, Ducatelle R. 2001.Pathogenicity of Actinobacillus minor, Actinobacillus indolicus and Acti-nobacillus porcinus strains for gnotobiotic piglets. J Vet Med B Infect DisVet Public Health 48:127–131. https://doi.org/10.1111/j.1439-0450.2001.00433.x.7. Sassu EL, Bossé JT, Tobias TJ, Gottschalk M, Langford PR, Hennig-PaukaI. 2018. Update on Actinobacillus pleuropneumoniae—knowledge, gapsand challenges. Transbound Emerg Dis 65:72–90. https://doi.org/10.1111/tbed.12739.8. Maldonado J, Valls L, Martínez E, Riera P. 2009. Isolation rates, serovars,and toxin genotypes of nicotinamide adenine dinucleotide-independentActinobacillus pleuropneumoniae among pigs suffering from pleuro-pneumonia in Spain. J Vet Diagn Invest 21:854–857. https://doi.org/10.1177/104063870902100615.9. Christensen H, Kuhnert P, Busse H-J, Frederiksen WC, Bisgaard M. 2007.Proposed minimal standards for the description of genera, species andsubspecies of the Pasteurellaceae. Int J Syst Evol Microbiol 57:166–178.https://doi.org/10.1099/ijs.0.64838-0.10. Naushad S, Adeolu M, Goel N, Khadka B, Al-Dahwi A, Gupta RS. 2015.Phylogenomic and molecular demarcation of the core members of thepolyphyletic Pasteurellaceae genera Actinobacillus, Haemophilus, andPasteurella. Int J Genomics 2015:198560. https://doi.org/10.1155/2015/198560.11. Møller K, Kilian M. 1990. V factor-dependent members of the familyPasteurellaceae in the porcine upper respiratory tract. J Clin Microbiol28:2711–2716.12. Gilbride KA, Rosendal S. 1983. Evaluation of a selective medium forisolation of Haemophilus pleuropneumoniae. Can J Comp Med 47:445–450.13. Howell KJ, Weinert LA, Luan S-L, Peters SE, Chaudhuri RR, Harris D,Angen Ø, Aragon V, Parkhill J, Langford PR, Rycroft AN, Wren BW, TuckerAW, Maskell DJ, BRaDP1T Consortium. 2013. Gene content and diversityof the loci encoding biosynthesis of capsular polysaccharides of the15 serovar reference strains of Haemophilus parasuis. J Bacteriol 195:4264–4273. https://doi.org/10.1128/JB.00471-13.14. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, SwerdlowH, Turner D. 2008. A large genome center’s improvements to the Illu-mina sequencing system. Nat Methods 5:1005–1010. https://doi.org/10.1038/nmeth.1270.15. Page AJ, De Silva N, Hunt M, Quail MA, Parkhill J, Harris SR, Otto TD,Keane JA. 2016. Robust high-throughput prokaryote de novo assemblyand improvement pipeline for Illumina data. Microb Genom 2:e000083.https://doi.org/10.1099/mgen.0.000083.16. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short readassembly using de Bruijn graphs. Genome Res 18:821–829. https://doi.org/10.1101/gr.074492.107.17. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffoldingpre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https://doi.org/10.1093/bioinformatics/btq683.18. Boetzer M, Pirovano W. 2012. Toward almost closed genomes withGapFiller. Genome Biol 13:R56. https://doi.org/10.1186/gb-2012-13-6-r56.19. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.20. Kuhnert P, Korczak BM. 2006. Prediction of whole-genome DNA–DNA similarity, determination of GC content and phylogeneticanalysis within the family Pasteurellaceae by multilocus sequence anal-ysis (MLSA). Microbiology 152:2537–2548. https://doi.org/10.1099/mic.0.28991-0.Microbiology Resource AnnouncementVolume 9 Issue 1 e00716-19 mra.asm.org 3

Draft Genome Sequences of the Type Strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), Two NAD-Dependent Bacterial Species Found in the Respiratory Tract of Pigs.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC, grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, BB/G018553/1, and, in part, BB/S000103/1 and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the BRaDP1T Consortium. M.T.G.H. was supported by the Wellcome Trust (grant number 098051).We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.Peer reviewe

Holden, Matthew T G

BRaDP1T Consortium

St Andrews Research Repository

Draft Genome Sequences of the Type Strains of Actinobacillusindolicus (46K2C) and Actinobacillus porcinus (NM319), TwoNAD-Dependent Bacterial Species Found in the RespiratoryTract of PigsJanine T. Bossé,a Yanwen Li,a Roberto Fernandez Crespo,a Øystein Angen,b Matthew T. G. Holden,c* Lucy A. Weinert,dDuncan J. Maskell,d Alexander W. Tucker,d Brendan W. Wren,e Andrew N. Rycroft,f Paul R. Langford,a on behalf ofthe BRaDP1T consortiumaSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, London, United KingdombDepartment of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, DenmarkcThe Wellcome Trust Sanger Institute, Cambridge, United KingdomdDepartment of Veterinary Medicine, University of Cambridge, Cambridge, United KingdomeFaculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United KingdomfDepartment of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United KingdomABSTRACT We report here the draft genome sequences of the type strains of Acti-nobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract ofpigs and are occasionally associated with lung pathology.Bacteria belonging to the family Pasteurellaceae that inhabit the porcine respiratorytract and require NAD (also called the V factor) for growth include Actinobacilluspleuropneumoniae, Glaesserella parasuis (formerly Haemophilus parasuis), Actinobacillusindolicus, Actinobacillus porcinus, Actinobacillus minor, and a separate species tentativelydesignated “Actinobacillus porcitonsillarum,” which, although phenotypically more sim-ilar to A. pleuropneumoniae, is phylogenetically closer to A. minor (1–5). The first twospecies are important pathogens, whereas the latter four are considered commensalsof the upper respiratory tract (1, 3, 6), though isolates of A. indolicus and A. porcinus,associated with lung pathology, have been identified (1). For A. pleuropneumoniae,there are two biotypes based on the requirement for NAD, with most isolates belongingto the NAD-dependent biovar 1 (7, 8).The phylogeny of the Pasteurellaceae is complicated, and there remain numerousspecies with uncertain taxonomic status (4, 9, 10). Whole-genome sequences, whichcan help clarify evolutionary relationships and the development of differential diag-nostics, are available for all of the above-mentioned bacteria except A. indolicus and A.porcinus. We therefore chose to sequence the type strains, 46K2C and NM319, respec-tively, of these species. Both 46K2CT and NM319T were originally isolated from theupper respiratory tract of pigs in Denmark (11) and Canada (12), respectively. They wereinitially identified as Haemophilus taxons D and F, respectively, prior to their designa-tion as the type strains of the newly assigned species in 1996 (1, 2).Strains 46K2CT and NM319T were obtained from the Culture Collection University ofGothenburg (CCUG 39029T and CCUG 38924T, respectively), stored frozen at 80°C in25% glycerol, and minimally passaged (up to four times) on brain heart infusion agar(Difco) supplemented with 0.01% NAD prior to genomic DNA extraction using aFastDNA spin kit (MP Biomedicals), according to the manufacturer’s protocol forbacterial cells. Paired-end libraries were prepared from 500 ng of genomic DNA, asCitation Bossé JT, Li Y, Fernandez Crespo R,Angen Ø, Holden MTG, Weinert LA, Maskell DJ,Tucker AW, Wren BW, Rycroft AN, Langford PR,on behalf of the BRaDP1T Consortium. 2020.Draft genome sequences of the type strains ofActinobacillus indolicus (46K2C) andActinobacillus porcinus (NM319), two NAD-dependent bacterial species found in therespiratory tract of pigs. Microbiol ResourAnnounc 9:e00716-19. https://doi.org/10.1128/MRA.00716-19.Editor Steven R. Gill, University of RochesterSchool of Medicine and DentistryCopyright © 2020 Bossé et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to Janine T. Bossé,j.bosse@imperial.ac.uk, or Paul R. Langford,p.langford@imperial.ac.uk.* Present address: Matthew T. G. Holden,School of Medicine, University of St. Andrews,St. Andrews, United Kingdom.Received 18 June 2019Accepted 14 November 2019Published 2 January 2020GENOME SEQUENCEScrossmVolume 9 Issue 1 e00716-19 mra.asm.org 1 on January 10, 2020 at ST ANDREWS UNIVhttp://mra.asm.org/Downloaded from previously described (13), using the modified Illumina protocol developed by Quail etal. (14) for sequencing at the Wellcome Sanger Institute (Hinxton, UK) on an IlluminaHiSeq 2000 analyzer for 75 cycles. Draft genome sequences were assembled de novousing a previously reported pipeline (15). Briefly, the 2  76-bp paired reads were usedto create multiple assemblies for each species’ genome using Velvet v1.2 (16) andVelvetOptimiser v2.2.5 (http://bioinformatics.net.au/software.velvetoptimiser.shtml).For each genome, the assembly with the best N50 value was further improved byscaffolding contigs using SSPACE (17), with any gaps filled by GapFiller (18). Automatedannotation was performed for both genome assemblies using Prokka v1.5 (19). Defaultparameters were used for all software programs.The draft genome of A. indolicus 46K2CT was assembled from 2,031,716 total reads,with an average quality score of 37.9, into 22 contigs (N50, 183,245; L50, 4), with a totallength of 2,103,350 bases. This sequence encodes 1,956 predicted proteins and has aGC content of 40.1%, which is higher than the 35.5% originally determined (2) butlower than the value of 42.6% calculated by Kuhnert and Korczak based on the GCcontent of selected genes (20). The draft genome of A. porcinus NM319T was assembledfrom 1,733,818 reads, with an average quality score of 37.8, into 29 contigs (N50,182,117; L50, 5), with a total length of 2,280,774 bases. This sequence encodes 2,095predicted proteins and has a GC content of 41.1%, which is similar to values (41.4%and 41.6%) previously reported for this strain (2, 20).The availability of the draft genome sequences of the type strains of A. indolicus andA. porcinus will facilitate differentiation of these species from other NAD-dependentbacteria found in the porcine respiratory tract and contribute to the understanding ofPasteurellaceae phylogeny and host interactive biology.Data availability. The draft genome assemblies for A. indolicus 46K2CT and A.porcinus NM319T have been deposited in the European Nucleotide Archive (ENA) underBioProject number PRJEB31492. Raw sequence reads have been deposited in theENA under accession numbers ERS134282 and ERS134597, respectively. The ENAaccession numbers for the assembled contigs are GCA_901764975 (CABFKH010000001through CABFKH010000022) and GCA_901764995 (CAAGST010000001 throughCAAGST010000029), respectively.ACKNOWLEDGMENTSThe Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortiumcomprises the following members: Duncan J. Maskell, Alexander W. (Dan) Tucker, SarahE. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, Roy R. Chaudhuri(University of Cambridge; present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Sheffield, Sheffield, United Kingdom); AndrewN. Rycroft, Gareth A. Maglennon, Jessica Beddow (Royal Veterinary College); Brendan W.Wren, Jon Cuccui, Vanessa S. Terra (London School of Hygiene and Tropical Medicine);and Paul R. Langford, Janine T. Bossé, and Yanwen Li (Imperial College London).This work was supported by a Longer and Larger (LoLa) grant from the Biotechnol-ogy and Biological Sciences Research Council (BBSRC, grant numbers BB/G020744/1,BB/G019177/1, BB/G019274/1, BB/G018553/1, and, in part, BB/S000103/1 and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis(formerly Pfizer Animal Health) awarded to the BRaDP1T Consortium. M.T.G.H. wassupported by the Wellcome Trust (grant number 098051).The funders had no role in the study design, data collection and analysis, decisionto publish, or preparation of the manuscript.REFERENCES1. Kielstein P, Wuthe H, Angen Ø, Mutters R, Ahrens P. 2001. Phenotypicand genetic characterization of NAD-dependent Pasteurellaceae fromthe respiratory tract of pigs and their possible pathogenetic impor-tance. Vet Microbiol 81:243–255. https://doi.org/10.1016/s0378-1135(01)00351-0.2. Møller K, Fussing V, Grimont PA, Paster BJ, Dewhirst FE, Kilian M. 1996.Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Acti-nobacillus indolicus sp. nov., three new V factor-dependent species fromthe respiratory tract of pigs. Int J Syst Bacteriol 46:951–956. https://doi.org/10.1099/00207713-46-4-951.Bossé et al.Volume 9 Issue 1 e00716-19 mra.asm.org 2 on January 10, 2020 at ST ANDREWS UNIVhttp://mra.asm.org/Downloaded from 3. Gottschalk M, Broes A, Mittal KR, Kobisch M, Kuhnert P, Lebrun A, Frey J.2003. Non-pathogenic Actinobacillus isolates antigenically and biochem-ically similar to Actinobacillus pleuropneumoniae: a novel species? VetMicrobiol 92:87–101. https://doi.org/10.1016/s0378-1135(02)00341-3.4. Korczak B, Christensen H, Emler S, Frey J, Kuhnert P. 2004. Phylogeny ofthe family Pasteurellaceae based on rpoB sequences. Int J Syst EvolMicrobiol 54:1393–1399. https://doi.org/10.1099/ijs.0.03043-0.5. Dickerman A, Bandara AB, Inzana TJ. Phylogenomic analysis of Haemo-philus parasuis and proposed reclassification to Glaesserella parasuis,gen. nov., comb. nov. Int J Syst Evol Microbiol, in press. https://doi.org/10.1099/ijsem.0.003730.6. Chiers K, Haesebrouck F, Mateusen B, Van Overbeke I, Ducatelle R. 2001.Pathogenicity of Actinobacillus minor, Actinobacillus indolicus and Acti-nobacillus porcinus strains for gnotobiotic piglets. J Vet Med B Infect DisVet Public Health 48:127–131. https://doi.org/10.1111/j.1439-0450.2001.00433.x.7. Sassu EL, Bossé JT, Tobias TJ, Gottschalk M, Langford PR, Hennig-PaukaI. 2018. Update on Actinobacillus pleuropneumoniae—knowledge, gapsand challenges. Transbound Emerg Dis 65:72–90. https://doi.org/10.1111/tbed.12739.8. Maldonado J, Valls L, Martínez E, Riera P. 2009. Isolation rates, serovars,and toxin genotypes of nicotinamide adenine dinucleotide-independentActinobacillus pleuropneumoniae among pigs suffering from pleuro-pneumonia in Spain. J Vet Diagn Invest 21:854–857. https://doi.org/10.1177/104063870902100615.9. Christensen H, Kuhnert P, Busse H-J, Frederiksen WC, Bisgaard M. 2007.Proposed minimal standards for the description of genera, species andsubspecies of the Pasteurellaceae. Int J Syst Evol Microbiol 57:166–178.https://doi.org/10.1099/ijs.0.64838-0.10. Naushad S, Adeolu M, Goel N, Khadka B, Al-Dahwi A, Gupta RS. 2015.Phylogenomic and molecular demarcation of the core members of thepolyphyletic Pasteurellaceae genera Actinobacillus, Haemophilus, andPasteurella. Int J Genomics 2015:198560. https://doi.org/10.1155/2015/198560.11. Møller K, Kilian M. 1990. V factor-dependent members of the familyPasteurellaceae in the porcine upper respiratory tract. J Clin Microbiol28:2711–2716.12. Gilbride KA, Rosendal S. 1983. Evaluation of a selective medium forisolation of Haemophilus pleuropneumoniae. Can J Comp Med 47:445–450.13. Howell KJ, Weinert LA, Luan S-L, Peters SE, Chaudhuri RR, Harris D,Angen Ø, Aragon V, Parkhill J, Langford PR, Rycroft AN, Wren BW, TuckerAW, Maskell DJ, BRaDP1T Consortium. 2013. Gene content and diversityof the loci encoding biosynthesis of capsular polysaccharides of the15 serovar reference strains of Haemophilus parasuis. J Bacteriol 195:4264–4273. https://doi.org/10.1128/JB.00471-13.14. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, SwerdlowH, Turner D. 2008. A large genome center’s improvements to the Illu-mina sequencing system. Nat Methods 5:1005–1010. https://doi.org/10.1038/nmeth.1270.15. Page AJ, De Silva N, Hunt M, Quail MA, Parkhill J, Harris SR, Otto TD,Keane JA. 2016. Robust high-throughput prokaryote de novo assemblyand improvement pipeline for Illumina data. Microb Genom 2:e000083.https://doi.org/10.1099/mgen.0.000083.16. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short readassembly using de Bruijn graphs. Genome Res 18:821–829. https://doi.org/10.1101/gr.074492.107.17. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffoldingpre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https://doi.org/10.1093/bioinformatics/btq683.18. Boetzer M, Pirovano W. 2012. Toward almost closed genomes withGapFiller. Genome Biol 13:R56. https://doi.org/10.1186/gb-2012-13-6-r56.19. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.20. Kuhnert P, Korczak BM. 2006. Prediction of whole-genome DNA–DNA similarity, determination of GC content and phylogeneticanalysis within the family Pasteurellaceae by multilocus sequence anal-ysis (MLSA). Microbiology 152:2537–2548. https://doi.org/10.1099/mic.0.28991-0.Microbiology Resource AnnouncementVolume 9 Issue 1 e00716-19 mra.asm.org 3 on January 10, 2020 at ST ANDREWS UNIVhttp://mra.asm.org/Downloaded from 

Draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), two NAD-dependent bacterial species found in the respiratory tract of pigs

Bossé, J T

Li, Y

Fernandez Crespo, R

Angen, Ø

Holden, M T G

Weinert, L A

Maskell, D J

Tucker, A W

Wren, B W

Rycroft, A N

Langford, P R

RVC Research Online

Draft Genome Sequences of the Type Strains of Actinobacillusindolicus (46K2C) and Actinobacillus porcinus (NM319), TwoNAD-Dependent Bacterial Species Found in the RespiratoryTract of PigsJanine T. Bossé,a Yanwen Li,a Roberto Fernandez Crespo,a Øystein Angen,b Matthew T. G. Holden,c* Lucy A. Weinert,dDuncan J. Maskell,d Alexander W. Tucker,d Brendan W. Wren,e Andrew N. Rycroft,f Paul R. Langford,a on behalf ofthe BRaDP1T consortiumaSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, London, United KingdombDepartment of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, DenmarkcThe Wellcome Trust Sanger Institute, Cambridge, United KingdomdDepartment of Veterinary Medicine, University of Cambridge, Cambridge, United KingdomeFaculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United KingdomfDepartment of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United KingdomABSTRACT We report here the draft genome sequences of the type strains of Acti-nobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract ofpigs and are occasionally associated with lung pathology.Bacteria belonging to the family Pasteurellaceae that inhabit the porcine respiratorytract and require NAD (also called the V factor) for growth include Actinobacilluspleuropneumoniae, Glaesserella parasuis (formerly Haemophilus parasuis), Actinobacillusindolicus, Actinobacillus porcinus, Actinobacillus minor, and a separate species tentativelydesignated “Actinobacillus porcitonsillarum,” which, although phenotypically more sim-ilar to A. pleuropneumoniae, is phylogenetically closer to A. minor (1–5). The first twospecies are important pathogens, whereas the latter four are considered commensalsof the upper respiratory tract (1, 3, 6), though isolates of A. indolicus and A. porcinus,associated with lung pathology, have been identified (1). For A. pleuropneumoniae,there are two biotypes based on the requirement for NAD, with most isolates belongingto the NAD-dependent biovar 1 (7, 8).The phylogeny of the Pasteurellaceae is complicated, and there remain numerousspecies with uncertain taxonomic status (4, 9, 10). Whole-genome sequences, whichcan help clarify evolutionary relationships and the development of differential diag-nostics, are available for all of the above-mentioned bacteria except A. indolicus and A.porcinus. We therefore chose to sequence the type strains, 46K2C and NM319, respec-tively, of these species. Both 46K2CT and NM319T were originally isolated from theupper respiratory tract of pigs in Denmark (11) and Canada (12), respectively. They wereinitially identified as Haemophilus taxons D and F, respectively, prior to their designa-tion as the type strains of the newly assigned species in 1996 (1, 2).Strains 46K2CT and NM319T were obtained from the Culture Collection University ofGothenburg (CCUG 39029T and CCUG 38924T, respectively), stored frozen at 80°C in25% glycerol, and minimally passaged (up to four times) on brain heart infusion agar(Difco) supplemented with 0.01% NAD prior to genomic DNA extraction using aFastDNA spin kit (MP Biomedicals), according to the manufacturer’s protocol forbacterial cells. Paired-end libraries were prepared from 500 ng of genomic DNA, asCitation Bossé JT, Li Y, Fernandez Crespo R,Angen Ø, Holden MTG, Weinert LA, Maskell DJ,Tucker AW, Wren BW, Rycroft AN, Langford PR,on behalf of the BRaDP1T Consortium. 2020.Draft genome sequences of the type strains ofActinobacillus indolicus (46K2C) andActinobacillus porcinus (NM319), two NAD-dependent bacterial species found in therespiratory tract of pigs. Microbiol ResourAnnounc 9:e00716-19. https://doi.org/10.1128/MRA.00716-19.Editor Steven R. Gill, University of RochesterSchool of Medicine and DentistryCopyright © 2020 Bossé et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to Janine T. Bossé,j.bosse@imperial.ac.uk, or Paul R. Langford,p.langford@imperial.ac.uk.* Present address: Matthew T. G. Holden,School of Medicine, University of St. Andrews,St. Andrews, United Kingdom.Received 18 June 2019Accepted 14 November 2019Published 2 January 2020GENOME SEQUENCEScrossmVolume 9 Issue 1 e00716-19 mra.asm.org 1 on January 17, 2020 at Royal Veterinary Collegehttp://mra.asm.org/Downloaded from previously described (13), using the modified Illumina protocol developed by Quail etal. (14) for sequencing at the Wellcome Sanger Institute (Hinxton, UK) on an IlluminaHiSeq 2000 analyzer for 75 cycles. Draft genome sequences were assembled de novousing a previously reported pipeline (15). Briefly, the 2  76-bp paired reads were usedto create multiple assemblies for each species’ genome using Velvet v1.2 (16) andVelvetOptimiser v2.2.5 (http://bioinformatics.net.au/software.velvetoptimiser.shtml).For each genome, the assembly with the best N50 value was further improved byscaffolding contigs using SSPACE (17), with any gaps filled by GapFiller (18). Automatedannotation was performed for both genome assemblies using Prokka v1.5 (19). Defaultparameters were used for all software programs.The draft genome of A. indolicus 46K2CT was assembled from 2,031,716 total reads,with an average quality score of 37.9, into 22 contigs (N50, 183,245; L50, 4), with a totallength of 2,103,350 bases. This sequence encodes 1,956 predicted proteins and has aGC content of 40.1%, which is higher than the 35.5% originally determined (2) butlower than the value of 42.6% calculated by Kuhnert and Korczak based on the GCcontent of selected genes (20). The draft genome of A. porcinus NM319T was assembledfrom 1,733,818 reads, with an average quality score of 37.8, into 29 contigs (N50,182,117; L50, 5), with a total length of 2,280,774 bases. This sequence encodes 2,095predicted proteins and has a GC content of 41.1%, which is similar to values (41.4%and 41.6%) previously reported for this strain (2, 20).The availability of the draft genome sequences of the type strains of A. indolicus andA. porcinus will facilitate differentiation of these species from other NAD-dependentbacteria found in the porcine respiratory tract and contribute to the understanding ofPasteurellaceae phylogeny and host interactive biology.Data availability. The draft genome assemblies for A. indolicus 46K2CT and A.porcinus NM319T have been deposited in the European Nucleotide Archive (ENA) underBioProject number PRJEB31492. Raw sequence reads have been deposited in theENA under accession numbers ERS134282 and ERS134597, respectively. The ENAaccession numbers for the assembled contigs are GCA_901764975 (CABFKH010000001through CABFKH010000022) and GCA_901764995 (CAAGST010000001 throughCAAGST010000029), respectively.ACKNOWLEDGMENTSThe Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortiumcomprises the following members: Duncan J. Maskell, Alexander W. (Dan) Tucker, SarahE. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, Roy R. Chaudhuri(University of Cambridge; present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Sheffield, Sheffield, United Kingdom); AndrewN. Rycroft, Gareth A. Maglennon, Jessica Beddow (Royal Veterinary College); Brendan W.Wren, Jon Cuccui, Vanessa S. Terra (London School of Hygiene and Tropical Medicine);and Paul R. Langford, Janine T. Bossé, and Yanwen Li (Imperial College London).This work was supported by a Longer and Larger (LoLa) grant from the Biotechnol-ogy and Biological Sciences Research Council (BBSRC, grant numbers BB/G020744/1,BB/G019177/1, BB/G019274/1, BB/G018553/1, and, in part, BB/S000103/1 and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis(formerly Pfizer Animal Health) awarded to the BRaDP1T Consortium. M.T.G.H. wassupported by the Wellcome Trust (grant number 098051).The funders had no role in the study design, data collection and analysis, decisionto publish, or preparation of the manuscript.REFERENCES1. Kielstein P, Wuthe H, Angen Ø, Mutters R, Ahrens P. 2001. Phenotypicand genetic characterization of NAD-dependent Pasteurellaceae fromthe respiratory tract of pigs and their possible pathogenetic impor-tance. Vet Microbiol 81:243–255. https://doi.org/10.1016/s0378-1135(01)00351-0.2. Møller K, Fussing V, Grimont PA, Paster BJ, Dewhirst FE, Kilian M. 1996.Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Acti-nobacillus indolicus sp. nov., three new V factor-dependent species fromthe respiratory tract of pigs. Int J Syst Bacteriol 46:951–956. https://doi.org/10.1099/00207713-46-4-951.Bossé et al.Volume 9 Issue 1 e00716-19 mra.asm.org 2 on January 17, 2020 at Royal Veterinary Collegehttp://mra.asm.org/Downloaded from 3. Gottschalk M, Broes A, Mittal KR, Kobisch M, Kuhnert P, Lebrun A, Frey J.2003. Non-pathogenic Actinobacillus isolates antigenically and biochem-ically similar to Actinobacillus pleuropneumoniae: a novel species? VetMicrobiol 92:87–101. https://doi.org/10.1016/s0378-1135(02)00341-3.4. Korczak B, Christensen H, Emler S, Frey J, Kuhnert P. 2004. Phylogeny ofthe family Pasteurellaceae based on rpoB sequences. Int J Syst EvolMicrobiol 54:1393–1399. https://doi.org/10.1099/ijs.0.03043-0.5. Dickerman A, Bandara AB, Inzana TJ. Phylogenomic analysis of Haemo-philus parasuis and proposed reclassification to Glaesserella parasuis,gen. nov., comb. nov. Int J Syst Evol Microbiol, in press. https://doi.org/10.1099/ijsem.0.003730.6. Chiers K, Haesebrouck F, Mateusen B, Van Overbeke I, Ducatelle R. 2001.Pathogenicity of Actinobacillus minor, Actinobacillus indolicus and Acti-nobacillus porcinus strains for gnotobiotic piglets. J Vet Med B Infect DisVet Public Health 48:127–131. https://doi.org/10.1111/j.1439-0450.2001.00433.x.7. Sassu EL, Bossé JT, Tobias TJ, Gottschalk M, Langford PR, Hennig-PaukaI. 2018. Update on Actinobacillus pleuropneumoniae—knowledge, gapsand challenges. Transbound Emerg Dis 65:72–90. https://doi.org/10.1111/tbed.12739.8. Maldonado J, Valls L, Martínez E, Riera P. 2009. Isolation rates, serovars,and toxin genotypes of nicotinamide adenine dinucleotide-independentActinobacillus pleuropneumoniae among pigs suffering from pleuro-pneumonia in Spain. J Vet Diagn Invest 21:854–857. https://doi.org/10.1177/104063870902100615.9. Christensen H, Kuhnert P, Busse H-J, Frederiksen WC, Bisgaard M. 2007.Proposed minimal standards for the description of genera, species andsubspecies of the Pasteurellaceae. Int J Syst Evol Microbiol 57:166–178.https://doi.org/10.1099/ijs.0.64838-0.10. Naushad S, Adeolu M, Goel N, Khadka B, Al-Dahwi A, Gupta RS. 2015.Phylogenomic and molecular demarcation of the core members of thepolyphyletic Pasteurellaceae genera Actinobacillus, Haemophilus, andPasteurella. Int J Genomics 2015:198560. https://doi.org/10.1155/2015/198560.11. Møller K, Kilian M. 1990. V factor-dependent members of the familyPasteurellaceae in the porcine upper respiratory tract. J Clin Microbiol28:2711–2716.12. Gilbride KA, Rosendal S. 1983. Evaluation of a selective medium forisolation of Haemophilus pleuropneumoniae. Can J Comp Med 47:445–450.13. Howell KJ, Weinert LA, Luan S-L, Peters SE, Chaudhuri RR, Harris D,Angen Ø, Aragon V, Parkhill J, Langford PR, Rycroft AN, Wren BW, TuckerAW, Maskell DJ, BRaDP1T Consortium. 2013. Gene content and diversityof the loci encoding biosynthesis of capsular polysaccharides of the15 serovar reference strains of Haemophilus parasuis. J Bacteriol 195:4264–4273. https://doi.org/10.1128/JB.00471-13.14. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, SwerdlowH, Turner D. 2008. A large genome center’s improvements to the Illu-mina sequencing system. Nat Methods 5:1005–1010. https://doi.org/10.1038/nmeth.1270.15. Page AJ, De Silva N, Hunt M, Quail MA, Parkhill J, Harris SR, Otto TD,Keane JA. 2016. Robust high-throughput prokaryote de novo assemblyand improvement pipeline for Illumina data. Microb Genom 2:e000083.https://doi.org/10.1099/mgen.0.000083.16. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short readassembly using de Bruijn graphs. Genome Res 18:821–829. https://doi.org/10.1101/gr.074492.107.17. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffoldingpre-assembled contigs using SSPACE. Bioinformatics 27:578–579. https://doi.org/10.1093/bioinformatics/btq683.18. Boetzer M, Pirovano W. 2012. Toward almost closed genomes withGapFiller. Genome Biol 13:R56. https://doi.org/10.1186/gb-2012-13-6-r56.19. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.20. Kuhnert P, Korczak BM. 2006. Prediction of whole-genome DNA–DNA similarity, determination of GC content and phylogeneticanalysis within the family Pasteurellaceae by multilocus sequence anal-ysis (MLSA). Microbiology 152:2537–2548. https://doi.org/10.1099/mic.0.28991-0.Microbiology Resource AnnouncementVolume 9 Issue 1 e00716-19 mra.asm.org 3 on January 17, 2020 at Royal Veterinary Collegehttp://mra.asm.org/Downloaded from 

Draft Genome Sequences of the Type Strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), Two NAD-Dependent Bacterial Species Found in the Respiratory Tract of Pigs

Apollo (Cambridge)

Draft Genome Sequences of the Type Strains of Actinobacillusindolicus (46K2C) and Actinobacillus porcinus (NM319), TwoNAD-Dependent Bacterial Species Found in the RespiratoryTract of PigsJanine T. Bossé,a Yanwen Li,a Roberto Fernandez Crespo,a Øystein Angen,b Matthew T. G. Holden,c* Lucy A. Weinert,dDuncan J. Maskell,d Alexander W. Tucker,d Brendan W. Wren,e Andrew N. Rycroft,f Paul R. Langford,a on behalf ofthe BRaDP1T consortiumaSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, London, United KingdombDepartment of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, DenmarkcThe Wellcome Trust Sanger Institute, Cambridge, United KingdomdDepartment of Veterinary Medicine, University of Cambridge, Cambridge, United KingdomeFaculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United KingdomfDepartment of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, United KingdomABSTRACT We report here the draft genome sequences of the type strains of Acti-nobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract ofpigs and are occasionally associated with lung pathology.Bacteria belonging to the family Pasteurellaceae that inhabit the porcine respiratorytract and require NAD (also called the V factor) for growth include Actinobacilluspleuropneumoniae, Glaesserella parasuis (formerly Haemophilus parasuis), Actinobacillusindolicus, Actinobacillus porcinus, Actinobacillus minor, and a separate species tentativelydesignated “Actinobacillus porcitonsillarum,” which, although phenotypically more sim-ilar to A. pleuropneumoniae, is phylogenetically closer to A. minor (1–5). The first twospecies are important pathogens, whereas the latter four are considered commensalsof the upper respiratory tract (1, 3, 6), though isolates of A. indolicus and A. porcinus,associated with lung pathology, have been identified (1). For A. pleuropneumoniae,there are two biotypes based on the requirement for NAD, with most isolates belongingto the NAD-dependent biovar 1 (7, 8).The phylogeny of the Pasteurellaceae is complicated, and there remain numerousspecies with uncertain taxonomic status (4, 9, 10). Whole-genome sequences, whichcan help clarify evolutionary relationships and the development of differential diag-nostics, are available for all of the above-mentioned bacteria except A. indolicus and A.porcinus. We therefore chose to sequence the type strains, 46K2C and NM319, respec-tively, of these species. Both 46K2CT and NM319T were originally isolated from theupper respiratory tract of pigs in Denmark (11) and Canada (12), respectively. They wereinitially identified as Haemophilus taxons D and F, respectively, prior to their designa-tion as the type strains of the newly assigned species in 1996 (1, 2).Strains 46K2CT and NM319T were obtained from the Culture Collection University ofGothenburg (CCUG 39029T and CCUG 38924T, respectively), stored frozen at 80°C in25% glycerol, and minimally passaged (up to four times) on brain heart infusion agar(Difco) supplemented with 0.01% NAD prior to genomic DNA extraction using aFastDNA spin kit (MP Biomedicals), according to the manufacturer’s protocol forbacterial cells. Paired-end libraries were prepared from 500 ng of genomic DNA, asCitation Bossé JT, Li Y, Fernandez Crespo R,Angen Ø, Holden MTG, Weinert LA, Maskell DJ,Tucker AW, Wren BW, Rycroft AN, Langford PR,on behalf of the BRaDP1T Consortium. 2020.Draft genome sequences of the type strains ofActinobacillus indolicus (46K2C) andActinobacillus porcinus (NM319), two NAD-dependent bacterial species found in therespiratory tract of pigs. Microbiol ResourAnnounc 9:e00716-19. https://doi.org/10.1128/MRA.00716-19.Editor Steven R. Gill, University of RochesterSchool of Medicine and DentistryCopyright © 2020 Bossé et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to Janine T. Bossé,j.bosse@imperial.ac.uk, or Paul R. Langford,p.langford@imperial.ac.uk.* Present address: Matthew T. G. Holden,School of Medicine, University of St. Andrews,St. Andrews, United Kingdom.Received 18 June 2019Accepted 14 November 2019Published 2 January 2020GENOME SEQUENCEScrossmVolume 9 Issue 1 e00716-19 mra.asm.org 1previously described (13), using the modified Illumina protocol developed by Quail etal. (14) for sequencing at the Wellcome Sanger Institute (Hinxton, UK) on an IlluminaHiSeq 2000 analyzer for 75 cycles. Draft genome sequences were assembled de novousing a previously reported pipeline (15). Briefly, the 2  76-bp paired reads were usedto create multiple assemblies for each species’ genome using Velvet v1.2 (16) andVelvetOptimiser v2.2.5 (http://bioinformatics.net.au/software.velvetoptimiser.shtml).For each genome, the assembly with the best N50 value was further improved byscaffolding contigs using SSPACE (17), with any gaps filled by GapFiller (18). Automatedannotation was performed for both genome assemblies using Prokka v1.5 (19). Defaultparameters were used for all software programs.The draft genome of A. indolicus 46K2CT was assembled from 2,031,716 total reads,with an average quality score of 37.9, into 22 contigs (N50, 183,245; L50, 4), with a totallength of 2,103,350 bases. This sequence encodes 1,956 predicted proteins and has aGC content of 40.1%, which is higher than the 35.5% originally determined (2) butlower than the value of 42.6% calculated by Kuhnert and Korczak based on the GCcontent of selected genes (20). The draft genome of A. porcinus NM319T was assembledfrom 1,733,818 reads, with an average quality score of 37.8, into 29 contigs (N50,182,117; L50, 5), with a total length of 2,280,774 bases. This sequence encodes 2,095predicted proteins and has a GC content of 41.1%, which is similar to values (41.4%and 41.6%) previously reported for this strain (2, 20).The availability of the draft genome sequences of the type strains of A. indolicus andA. porcinus will facilitate differentiation of these species from other NAD-dependentbacteria found in the porcine respiratory tract and contribute to the understanding ofPasteurellaceae phylogeny and host interactive biology.Data availability. The draft genome assemblies for A. indolicus 46K2CT and A.porcinus NM319T have been deposited in the European Nucleotide Archive (ENA) underBioProject number PRJEB31492. Raw sequence reads have been deposited in theENA under accession numbers ERS134282 and ERS134597, respectively. The ENAaccession numbers for the assembled contigs are GCA_901764975 (CABFKH010000001through CABFKH010000022) and GCA_901764995 (CAAGST010000001 throughCAAGST010000029), respectively.ACKNOWLEDGMENTSThe Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortiumcomprises the following members: Duncan J. Maskell, Alexander W. (Dan) Tucker, SarahE. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, Roy R. Chaudhuri(University of Cambridge; present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Sheffield, Sheffield, United Kingdom); AndrewN. Rycroft, Gareth A. Maglennon, Jessica Beddow (Royal Veterinary College); Brendan W.Wren, Jon Cuccui, Vanessa S. Terra (London School of Hygiene and Tropical Medicine);and Paul R. Langford, Janine T. Bossé, and Yanwen Li (Imperial College London).This work was supported by a Longer and Larger (LoLa) grant from the Biotechnol-ogy and Biological Sciences Research Council (BBSRC, grant numbers BB/G020744/1,BB/G019177/1, BB/G019274/1, BB/G018553/1, and, in part, BB/S000103/1 and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis(formerly Pfizer Animal Health) awarded to the BRaDP1T Consortium. M.T.G.H. wassupported by the Wellcome Trust (grant number 098051).The funders had no role in the study design, data collection and analysis, decisionto publish, or preparation of the manuscript.REFERENCES1. Kielstein P, Wuthe H, Angen Ø, Mutters R, Ahrens P. 2001. Phenotypicand genetic characterization of NAD-dependent Pasteurellaceae fromthe respiratory tract of pigs and their possible pathogenetic impor-tance. Vet Microbiol 81:243–255. https://doi.org/10.1016/s0378-1135(01)00351-0.2. Møller K, Fussing V, Grimont PA, Paster BJ, Dewhirst FE, Kilian M. 1996.Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Acti-nobacillus indolicus sp. nov., three new V factor-dependent species fromthe respiratory tract of pigs. Int J Syst Bacteriol 46:951–956. https://doi.org/10.1099/00207713-46-4-951.Bossé et al.Volume 9 Issue 1 e00716-19 mra.asm.org 23. Gottschalk M, Broes A, Mittal KR, Kobisch M, Kuhnert P, Lebrun A, Frey J.2003. Non-pathogenic Actinobacillus isolates antigenically and biochem-ically similar to Actinobacillus pleuropneumoniae: a novel species? VetMicrobiol 92:87–101. https://doi.org/10.1016/s0378-1135(02)00341-3.4. Korczak B, Christensen H, Emler S, Frey J, Kuhnert P. 2004. Phylogeny ofthe family Pasteurellaceae based on rpoB sequences. Int J Syst EvolMicrobiol 54:1393–1399. https://doi.org/10.1099/ijs.0.03043-0.5. Dickerman A, Bandara AB, Inzana TJ. Phylogenomic analysis of Haemo-philus parasuis and proposed reclassification to Glaesserella parasuis,gen. nov., comb. nov. Int J Syst Evol Microbiol, in press. https://doi.org/10.1099/ijsem.0.003730.6. Chiers K, Haesebrouck F, Mateusen B, Van Overbeke I, Ducatelle R. 2001.Pathogenicity of Actinobacillus minor, Actinobacillus indolicus and Acti-nobacillus porcinus strains for gnotobiotic piglets. J Vet Med B Infect DisVet Public Health 48:127–131. https://doi.org/10.1111/j.1439-0450.2001.00433.x.7. Sassu EL, Bossé JT, Tobias TJ, Gottschalk M, Langford PR, Hennig-PaukaI. 2018. Update on Actinobacillus pleuropneumoniae—knowledge, gapsand challenges. Transbound Emerg Dis 65:72–90. https://doi.org/10.1111/tbed.12739.8. Maldonado J, Valls L, Martínez E, Riera P. 2009. Isolation rates, serovars,and toxin genotypes of nicotinamide adenine dinucleotide-independentActinobacillus pleuropneumoniae among pigs suffering from pleuro-pneumonia in Spain. 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We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.</p

University of St. Andrews - Pure

Draft genome sequences of the type strains of <i>Actinobacillus indolicus</i> (46K2C) and <i>Actinobacillus porcinus</i> (NM319), two NAD-dependent bacterial species found in the respiratory tract of pigs

https://www.repository.cam.ac.uk/bitstream/1810/301733/2/article.pdf

Draft Genome Sequences of the Type Strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), Two NAD-Dependent Bacterial Species Found in the Respiratory Tract of Pigs.

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University of St. Andrews - Pure

University of St. Andrews - Pure