Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome

BackgroundBrucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data.ResultsThe proteomic data was compiled from several independent comparative studies of Brucella abortus that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes.ConclusionsAn essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of Brucella abortus strain 2308.

[1]  G. Pacheco [The genus Brucella]. , 1953, Brasil-medico.

[2]  I. Moriyón,et al.  Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope , 1982, Journal of bacteriology.

[3]  Y. Sharma,et al.  Cloning of genes for proline and leucine biosynthesis from Brucella abortus by functional complementation in Escherichia coli. , 1993, Journal of general microbiology.

[4]  W. G. Miller,et al.  Characteristics of a Brucella Species from a Bottlenose Dolphin (Tursiops Truncatus) , 1994, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[5]  R. Reid,et al.  Brucella species infection in sea-mammals , 1994, Veterinary Record.

[6]  R. Essenberg Cloning and characterization of the glucokinase gene of Brucella abortus 19 and identification of three other genes , 1995, Journal of bacteriology.

[7]  E. Moreno,et al.  Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide , 1996, Journal of bacteriology.

[8]  R. Roop,et al.  The Brucella abortus host factor I (HF‐I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice , 1999, Molecular microbiology.

[9]  Natalia N. Ivanova,et al.  The genome sequence of the facultative intracellular pathogen Brucella melitensis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[10]  B Garin-Bastuji,et al.  Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus. , 2001, Microbes and infection.

[11]  J. Letesson,et al.  Effect of omp10 or omp19 Deletion on Brucella abortus Outer Membrane Properties and Virulence in Mice , 2002, Infection and Immunity.

[12]  J. Vandenhaute,et al.  Plasticity of a transcriptional regulation network among alpha‐proteobacteria is supported by the identification of CtrA targets in Brucella abortus , 2002, Molecular microbiology.

[13]  Ian T. Paulsen,et al.  The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Vidal,et al.  Generation of the Brucella melitensis ORFeome version 1.1. , 2004, Genome research.

[15]  J. Glasner,et al.  Comparative Whole-Genome Hybridization Reveals Genomic Islands in Brucella Species , 2004, Journal of bacteriology.

[16]  Jenn-Kang Hwang,et al.  Predicting subcellular localization of proteins for Gram‐negative bacteria by support vector machines based on n‐peptide compositions , 2004, Protein science : a publication of the Protein Society.

[17]  Rong Wang,et al.  Mass spectrometry of the M. smegmatis proteome: protein expression levels correlate with function, operons, and codon bias. , 2005, Genome research.

[18]  E. Bouza,et al.  Laboratory-acquired brucellosis: a Spanish national survey. , 2005, The Journal of hospital infection.

[19]  S. Malfatti,et al.  Whole-Genome Analyses of Speciation Events in Pathogenic Brucellae , 2005, Infection and Immunity.

[20]  G. Escobar,et al.  Unusual clinical presentation of brucellosis caused by Brucella canis. , 2005, Journal of medical microbiology.

[21]  M. Winkler,et al.  Role of HdeA in acid resistance and virulence in Brucella abortus 2308. , 2005, Veterinary microbiology.

[22]  Gajendra P. S. Raghava,et al.  PSLpred: prediction of subcellular localization of bacterial proteins , 2005, Bioinform..

[23]  Zhang Qing,et al.  Completion of the Genome Sequence of Brucella abortus and Comparison to the Highly Similar Genomes of Brucella melitensis and Brucella suis , 2005, Journal of bacteriology.

[24]  Martin Ester,et al.  Sequence analysis PSORTb v . 2 . 0 : Expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis , 2004 .

[25]  María A Rendón,et al.  Characterization of SP41, a surface protein of Brucella associated with adherence and invasion of host epithelial cells , 2006, Cellular microbiology.

[26]  Joseph P. Connolly,et al.  Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development , 2006, Proteomics.

[27]  O. Poch,et al.  Interrupted coding sequences in Mycobacterium smegmatis: authentic mutations or sequencing errors? , 2007, Genome Biology.

[28]  A. Bacher,et al.  Evolution of Vitamin B2 Biosynthesis: 6,7-Dimethyl-8-Ribityllumazine Synthases of Brucella , 2006 .

[29]  M. Wendl This is an Open Access article distribut... , 2007 .

[30]  P. Kearney,et al.  Extensive cell envelope modulation is associated with virulence in Brucella abortus. , 2007, Journal of proteome research.

[31]  Patrick G. A. Pedrioli,et al.  A high-quality catalog of the Drosophila melanogaster proteome , 2007, Nature Biotechnology.

[32]  C. Guzmán-Verri,et al.  BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence , 2007, Infection and Immunity.

[33]  Eric V Stabb,et al.  Comparative genomics-based investigation of resequencing targets in Vibrio fischeri: Focus on point miscalls and artefactual expansions , 2008, BMC Genomics.

[34]  A. Bacher,et al.  Structural and kinetic properties of lumazine synthase isoenzymes in the order Rhizobiales. , 2007, Journal of molecular biology.

[35]  Richard D. Smith,et al.  Whole proteome analysis of post-translational modifications: applications of mass-spectrometry for proteogenomic annotation. , 2007, Genome research.

[36]  Zhangjun Fei,et al.  Genome Sequence of Brucella abortus Vaccine Strain S19 Compared to Virulent Strains Yields Candidate Virulence Genes , 2008, PloS one.

[37]  Z. Hubálek,et al.  Brucella microti sp. nov., isolated from the common vole Microtus arvalis. , 2008, International journal of systematic and evolutionary microbiology.

[38]  B. Mantur,et al.  Brucellosis in India — a review , 2008, Journal of Biosciences.

[39]  Michael J MacCoss,et al.  Use of shotgun proteomics for the identification, confirmation, and correction of C. elegans gene annotations. , 2008, Genome research.

[40]  Maulik Shukla,et al.  Analysis of Ten Brucella Genomes Reveals Evidence for Horizontal Gene Transfer Despite a Preferred Intracellular Lifestyle , 2009, Journal of bacteriology.

[41]  F. Denis,et al.  Intracellular adaptation of Brucella abortus. , 2009, Journal of proteome research.

[42]  J. Claverie,et al.  Brucella microti: the genome sequence of an emerging pathogen , 2009, BMC Genomics.