PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of genannJournal InfoAuthorsPermissionsJournals.ASM.orggenomeA ArticleGenome Announcements
 
Genome Announc. 2017 April; 5(14): e01720-16.
Published online 2017 April 6. doi:  10.1128/genomeA.01720-16
PMCID: PMC5383906

Genome Sequence of a Spontaneous Nonhemolytic Mutant of Mannheimia haemolytica 16041065 GH

ABSTRACT

We report here the draft genome sequence of a spontaneous nonhemolytic mutant of Mannheimia haemolytica 16041065 GH. This mutant arose during routine passage and was devoid of hemolytic activity on standard blood agars. This genome sequence had a total size of 2.7 Mb with an N50 of 117 kb.

GENOME ANNOUNCEMENT

Several bacteria, including Mannheimia haemolytica and numerous viruses, contribute to what is commonly referred to as bovine respiratory disease complex. However, the bacterial pathogen that is most commonly associated with the acute, severe form of the disease, and that is readily isolated from lung lesions, is M. haemolytica (1). Mannheimia (formerly Pasteurella) haemolytica is a Gram-negative coccobacillus that is readily isolated from nasal swabs of domestic ruminants such as cattle, sheep, and goats, where it constitutes the normal flora of the upper respiratory tract (2). Numerous serotypes of this bacterium have been isolated and characterized (3, 4). This otherwise innocuous opportunistic pathogen aggressively replicates when the host’s defense mechanisms are compromised by any number of stressful factors (5). Under these conditions, M. haemolytica can be inhaled into the lower respiratory tract and cause severe, and often fatal, pneumonia (1). Studies have shown that several characteristics or activities of this bacterium contribute to its virulence. An exotoxin, leukotoxin, is considered the most important virulence factor associated with M. haemolytica (6). Leukotoxin is responsible for M. haemolytica hemolysis on blood agar and causes leukocyte necrosis and apoptosis in vivo, thus allowing the bacterium to escape phagocytosis and killing by neutrophils and macrophages (7, 8).

One of the cultural characteristics of M. haemolytica on brain heart infusion (BHI) blood agar plates is its ability to cause clearing around the colonies, a feature also known as β-hemolysis (8). During routine transfers in our laboratory of an otherwise β-hemolytic strain, a spontaneous γ-hemolytic (nonhemolytic) mutant was observed. The identity of this γ-hemolytic mutant was confirmed as M. haemolytica serotype 1 by matrix-assisted laser desorption ionization–time of flight mass spectrometry and serology (9, 10).

Genomic DNA was extracted from a well-isolated colony grown in BHI broth using a Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). The purity of the genomic DNA data was tested by measuring absorbance at 260 and 280 nm (ratio of 260/280). The integrity of the data was further confirmed by resolving uncut and EcoRI digested forms on 1% agarose gel.

All sequence reads for M. haemolytica 16041065 GH were quality-filtered with standard Illumina filtering settings, resulting in 250 Mb of quality sequence data. Quality-filtered reads were assembled to produce the genome using the short-read de Brujin graph assembly (11) program Velvet (12). Run-time settings for Velvet were a k-mer value of 77 and a minimum contig coverage value of 7×. All gene models were created using the prokaryotic gene-calling software package Prodigal (13) on the Velvet genome assembly. The Velvet genome assembly had a total size of 2,675,629 bp, 68 scaffolds, a scaffold N50 of 117 kb, and 2,588 predicted proteins. All called gene models were annotated using a combination of homology comparison, domain prediction, and annotation. Programs used for analysis of all gene models were NCBI BLAST C++ homology search (14) and HMMER version 3.0 hmmscan (15) against the Pfam version 26.0 database (16). All genes were annotated for functional significance using the UniProt database (17).

Accession number(s).

This whole-genome shotgun project has been deposited in GenBank under the accession number MLYM00000000. The version described in this paper is the first version, MLYM01000000.

ACKNOWLEDGMENT

This work was supported by USDA NIFA phase I SBIR grant 2014-33610-21937.

Footnotes

Citation Ayalew S, Confer AW, Hansen RD, Couger MB. 2017. Genome sequence of a spontaneous nonhemolytic mutant of Mannheimia haemolytica 16041065 GH. Genome Announc 5:e01720-16. https://doi.org/10.1128/genomeA.01720-16.

REFERENCES

1. Gagea MI, Bateman KG, van Dreumel T, McEwen BJ, Carman S, Archambault M, Shanahan RA, Caswell JL 2006. Diseases and pathogens associated with mortality in Ontario beef feedlots. J Vet Diagn Invest 18:18–28. doi:.10.1177/104063870601800104 [PubMed] [Cross Ref]
2. Rice JA, Carrasco-Medina L, Hodgins DC, Shewen PE 2007. Mannheimia haemolytica and bovine respiratory disease. Anim Health Res Rev 8:117–128. doi:.10.1017/S1466252307001375 [PubMed] [Cross Ref]
3. Frank GH. 1985. Serotype-specific resistance to nasal colonization by Pasteurella haemolytica in cattle. Am J Vet Res 46:2245–2248. [PubMed]
4. Frank GH. 1982. Serotypes of Pasteurella haemolytica in sheep in the midwestern United States. Am J Vet Res 43:2035–2037. [PubMed]
5. Caswell JL. 2014. Failure of respiratory defenses in the pathogenesis of bacterial pneumonia of cattle. Vet Pathol 51:393–409. doi:.10.1177/0300985813502821 [PubMed] [Cross Ref]
6. Czuprynski CJ, Leite F, Sylte M, Kuckleburg C, Schultz R, Inzana T, Behling-Kelly E, Corbeil L 2004. Complexities of the pathogenesis of Mannheimia haemolytica and Haemophilus somnus infections: challenges and potential opportunities for prevention? Anim Health Res Rev 5:277–282. doi:.10.1079/AHR200483 [PubMed] [Cross Ref]
7. Singh K, Ritchey JW, Confer AW 2011. Mannheimia haemolytica: bacterial-host interactions in bovine pneumonia. Vet Pathol 48:338–348. doi:.10.1177/0300985810377182 [PubMed] [Cross Ref]
8. Murphy GL, Whitworth LC, Clinkenbeard KD, Clinkenbeard PA 1995. Hemolytic activity of the Pasteurella haemolytica leukotoxin. Infect Immun 63:3209–3212. [PMC free article] [PubMed]
9. Frank GH, Wessman GE 1978. Rapid plate agglutination procedure for serotyping Pasteurella haemolytica. J Clin Microbiol 7:142–145. [PMC free article] [PubMed]
10. Risch M, Radjenovic D, Han JN, Wydler M, Nydegger U, Risch L 2010. Comparison of MALDI TOF with conventional identification of clinically relevant bacteria. Swiss Med Wkly 140:w13095. doi:.10.4414/smw.2010.13095 [PubMed] [Cross Ref]
11. Compeau PE, Pevzner PA, Tesler G 2011. How to apply de Bruijn graphs to genome assembly. Nat Biotechnol 29:987–991. doi:.10.1038/nbt.2023 [PubMed] [Cross Ref]
12. Zerbino DR. 2010. Using the Velvet de novo assembler for short-read sequencing technologies. Curr Protoc Bioinformatics Chapter 11:Unit 11.5. doi:.10.1002/0471250953.bi1105s31 [PMC free article] [PubMed] [Cross Ref]
13. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. doi:.10.1186/1471-2105-11-119 [PMC free article] [PubMed] [Cross Ref]
14. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. doi:.10.1186/1471-2105-10-421 [PMC free article] [PubMed] [Cross Ref]
15. Finn RD, Clements J, Arndt W, Miller BL, Wheeler TJ, Schreiber F, Bateman A, Eddy SR 2015. HMMER web server: 2015 update. Nucleic Acids Res 43:W30–W38. doi:.10.1093/nar/gkv397 [PMC free article] [PubMed] [Cross Ref]
16. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M 2014. Pfam: the protein families database. Nucleic Acids Res 42:D222–D230. doi:.10.1093/nar/gkt1223 [PMC free article] [PubMed] [Cross Ref]
17. UniProt Consortium 2015. UniProt: a hub for protein information. Nucleic Acids Res 43:D204–D212. doi:.10.1093/nar/gku989 [PMC free article] [PubMed] [Cross Ref]

Articles from Genome Announcements are provided here courtesy of American Society for Microbiology (ASM)