Open Access

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

  • Julie Lamontagne1,
  • Maxime Béland1,
  • Anik Forest1,
  • Alexandra Côté-Martin1,
  • Najib Nassif1,
  • Fadi Tomaki1,
  • Ignacio Moriyón2,
  • Edgardo Moreno3 and
  • Eustache Paramithiotis1Email author
BMC Genomics201011:300

DOI: 10.1186/1471-2164-11-300

Received: 18 November 2009

Accepted: 12 May 2010

Published: 12 May 2010

Abstract

Background

Brucellosis 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.

Results

The 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.

Conclusions

An 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.

Background

Brucella species bacteria are gram negative alpha proteobacteria superbly adapted for survival in intracellular environments. They infect a wide range of mammals, including essentially all economically important domestic mammals, many wild species, and humans. Brucellosis is the largest bacterial zoonosis in the world [13]. In humans, untreated brucellosis is a long lasting disease characterized by recurrent fever episodes and clinical manifestations that include spondylitis, severe headaches, joint or abdominal pain, endocarditis, and meningoencephalitis. In severe non-treated cases brucellosis can cause death [13].

Seven terrestrial Brucella species have been defined: Brucella melitensis, Brucella abortus, Brucella suis, Brucella ovis, Brucella canis, Brucella neotomae and Brucella microti which infect goats, cattle, pigs, sheep, dogs, desert wood rats and common voles, respectively [1, 4]. Two Brucella species infecting marine mammals such as dolphins, whales, seals, sea lions and walrus have also been defined as Brucella ceti and Brucella pinnipedialis [57]. With the exception of B. suis biovar 3, the Brucella genome is encoded on two chromosomes, containing in total approximately 3,500 genes. Genome sequences from 32 different Brucella strains, representing all species, have been published either as complete genomes (10 strains) or as draft assemblies in NCBI (22 strains) [814]. The raw genome sequencing data of 78 other strains is also available in the Sequence Read Archive of NCBI. The genome sequences were very highly homologous, although regions of unique genetic material were also observed. It is possible that these regions are involved in establishing the distinct host preferences and biological behavior of the different Brucella species sequenced to date [15].

Unlike other pathogenic bacteria, Brucella virulence does not appear to be the result of relatively few virulence genes that can be transferred horizontally via plasmids, phages, or assembled in pathogenicity islands. Brucella also lack typical virulence factors such as exotoxins, flagella, capsules, and type III secretion systems. Rather, the pathogen's virulence appears to be an integrated aspect of its physiology. Therefore, to better understand Brucella virulence, we will need to better understand the Brucella proteome, including how it changes during the different stages of the intracellular and extracellular Brucella lifecycles, and how it interacts with host proteins and processes. Indeed, we have previously demonstrated that Brucella bacteria are capable of extensive, reversible, remodeling of their cell envelopes [16]. Furthermore, during the establishment of an intracellular infection, Brucella bacteria also appear able to carry out extensive, and reversible, modifications to their biosynthetic pathways and respiration in order to adapt to the changing microenvironments encountered in infected host cells [17]. This suggests that the Brucella proteome is considerably more dynamic than previously suspected, and that in depth proteomic analysis of the pathogen, as well as integration of these data with the available genomic information, will result in novel mechanistic and possibly therapeutic insights.

In this work we have generated a synthesis of the proteomic datasets we produced from multiple independent comparisons of Brucella strains either grown in media or retrieved from infected host cells. Some of this data is currently publicly available [[16, 17];http://proteomicsresource.org/Default.aspx] with the remainder becoming available as part of this work. These studies were originally designed to identify experimental condition-specific differences in the Brucella proteome. We compiled the experimental evidence for any Brucella protein detected and compared the proteomic data to the available genomic data. We provide the first direct experimental evidence for the expression of 305 Brucella proteins, but also identified experimental evidence for the expression of five genes previously annotated as pseudogenes, and of start site errors in two other genes.

Results and Discussion

First experimental evidence of the expression of 305 proteins in B. abortus 2308

Samples used for the proteomic analysis came from B. abortus either grown extracellularly in media or isolated from infected RAW264.7 macrophages. The extracellular samples included whole bacteria grown directly in tryptic soy broth, outer membrane preparations (blebs) [16] and cytosols. Intracellular samples consisted of viable B. abortus isolated at different time points post-infection from RAW264.7 macrophages [17] and of phagosomes isolated from infected murine phagocytic cells. We obtained 1704 peptides representing 621 different proteins, corresponding to approximately 20% of the predicted proteome. For 305 proteins, we are reporting the first experimental evidence of their expression in B. abortus 2308 (Table 1). We also report genome annotation errors for two proteins, expression of ORFs annotated pseudogenes for four proteins and one correction to the sequence of another previously annotated pseudogene which allows for its full length expression. Peptide sequences corresponding to these 312 proteins are listed in Additional File 1. The peptide coverage for the 305 newly demonstrated proteins varied from 1 to 20, with an average of three peptides per protein. In order to confirm the expression of proteins identified by a single peptide, we manually validated all MSMS spectra that had a sequence assignment score smaller than 45. Forty-four of the 305 proteins were described previously as hypothetical with no putative function. When subcellular localizations were predicted using three publicly available tools [1820], 226 proteins were predicted to be cytosolic, ten were inner membrane proteins, 25 were periplasmic, three were outer membrane proteins and the localization of 48 proteins could not be predicted (Table 1). Experimental evidence for the expression of the other 309 of the 620 proteins has been demonstrated previously by our group [16, 17] and others [2131]. It is important to note that we are reporting an analysis of the combined results of several independent experiments using the same bacterial strain and technology to acquire the data. However, each experiment was a separate comparative study designed to identify differentially expressed bacterial proteins under specific conditions per experiment. Proteins that were not sufficiently differentially expressed under the experimental conditions used would have not been identified. Thus, while our results can be used to confirm that the proteins reported were expressed, they may underestimate under what conditions they can become expressed.
Table 1

B. abortus 2308 proteins for which the expression was demonstrated for the first time

Cytoplasm

BAB1_0002

DnaN

BAB1_0855

GRX family

BAB1_1449

UDP-N-

BAB1_2149

PepS

BAB1_0022

Unknown

BAB1_0856

BolA-related

 

acetylmuramate

BAB1_2168

RpsO; S15

BAB1_0023

AroA

BAB1_0857

FGAM synthase II

 

L-alanine ligase

BAB1_2173

FabB

BAB1_0035

KdsB

BAB1_0861

PurS

BAB1_1508

CarB

BAB2_0083

Eda2

BAB1_0063

Unknown

BAB1_0864

HpcH/HpaI

BAB1_1512

CspA

BAB2_0090

GCN5-related

BAB1_0071

ArgG

BAB1_0874

AcpP

BAB1_1523

GreA

 

N-acetyltransferase

BAB1_0100

Putative AsnC family

BAB1_0880

HAD-like

BAB1_1528

SseA-1

BAB2_0109

Gnd

BAB1_0107

Trs-ABC (P-loop)

BAB1_0886

NN:DBI PRT

BAB1_1538

OmpR

BAB2_0160

Unknown

BAB1_0118

Unknown

BAB1_0896

ArgS

BAB1_1547

PepQ

BAB2_0162

L-carnitine

BAB1_0122

GyrB

BAB1_0898

NagZ

BAB1_1549

PrsA

 

dehydratase

BAB1_0139

NifU

BAB1_0918

GatB/Yqey

BAB1_1553

YchF

BAB2_0177

YafB

BAB1_0159

S30EA

BAB1_0924

AccC

BAB1_1613

Unknown

BAB2_0186

Fumarate hydratase

BAB1_0160

PtsN-like

BAB1_0933

PCRF 2

BAB1_1645

DhaK-1

BAB2_0187

Unknown

BAB1_0191

GABAtrnsam

BAB1_0943

TyrS

BAB1_1646

DhaK-2

BAB2_0191

HAD-like,

BAB1_0204

AdhP

BAB1_0949

SufC

BAB1_1655

GabD

 

subfamily IIA

BAB1_0215

ThiE

BAB1_0955

DeaD

BAB1_1669

PAS domain

BAB2_0198

Pseudouridine

BAB1_0216

ThiG

BAB1_0960

Trs heavy metal

BAB1_1671

TcaR

 

synthase

BAB1_0242

ManR

BAB1_1014

MetG

BAB1_1687

Dut

BAB2_0216

3-hydroxybutyryl-CoA

BAB1_0285

HisD

BAB1_1030

Gor

BAB1_1695

PurA

 

dehydrogenase

BAB1_0317

Trs arginine/ornithine

BAB1_1037

Mandelate racemase;

BAB1_1702

Phosphoglucosamine

BAB2_0246

P47K

BAB1_0331

ArgD

 

muconate lactonizing

 

mutase

BAB2_0293

Gal

BAB1_0344

Pip

BAB1_1043

Unknown

BAB1_1719

ThiE

BAB2_0295

DgoK

BAB1_0353

Unknown

BAB1_1050

FolB

BAB1_1722

Efp

BAB2_0296

KdgA

 

dehydrogenase

BAB1_1077

Ach1p

BAB1_1751

Unknown

BAB2_0335

NADH:flavin oxidore-

BAB1_0416

DUF85

BAB1_1096

NifU-like

BAB1_1761

PyK

 

ductase/NADH oxidase

BAB1_0429

Polyprenyl synthetase

BAB1_1098

PRA-CH

BAB1_1778

FdxA

BAB2_0337

RocF

BAB1_0446

DnaJ

BAB1_1121

DNA gyrase subunit A

BAB1_1781

Unknown

BAB2_0343

Trx-2

BAB1_0447

FabI-1

BAB1_1130

ClpA/B

BAB1_1804

MarR family

BAB2_0358

Dcp

BAB1_0482

FabD

BAB1_1132

ClpP

BAB1_1810

AtpH

BAB2_0361

TypA

BAB1_0484

AcpP

BAB1_1156

KdsA

BAB1_1813

Transaldolase

BAB2_0365

FbaA

BAB1_0489

Guanylate kinase

BAB1_1157

PyrG

BAB1_1815

LeuS

BAB2_0366

RpiB/LacA/LacB

BAB1_0510

ThrC

BAB1_1161

TpiA

BAB1_1819

ACAT

BAB2_0367

TIM 2

BAB1_0525

PpdK

BAB1_1164

TrpC

BAB1_1824

PurH

BAB2_0370

EryC

BAB1_0532

Transthyretin

BAB1_1169

GltX

BAB1_1837

CynT

BAB2_0448

Unknown

BAB1_0540

Formyl transferase,

BAB1_1170

GltA

BAB1_1840

MmsA

BAB2_0457

FolD

 

N-terminal

BAB1_1174

FabZ

BAB1_1872

PrfA

BAB2_0459

Pgl

BAB1_0544

DegT/DnrJ/EryC1/StrS

BAB1_1187

Endoribonuclease

BAB1_1874

LysC

BAB2_0460

Zwf

BAB1_0561

Man-6-P isomerase

 

L-PSP

BAB1_1879

GrxC

BAB2_0483

ShuT

 

type II

BAB1_1188

GDPD

BAB1_1887

HemC

BAB2_0513

GcvT

BAB1_0570

XylA

BAB1_1205

ElaB-domain

BAB1_1895

FtsK-gamma

BAB2_0518

PutA

BAB1_0587

Unknown

BAB1_1212

BhbA

BAB1_1918

LpdA-2

BAB2_0566

AldA

BAB1_0588

ATP/GTP-binding

BAB1_1213

Unknown; conserved

BAB1_1926

SucC

BAB2_0568

Unknown

BAB1_0641

Alanine aminopep-

BAB1_1223

AlaS

BAB1_1936

GloB

BAB2_0572

IlvE

 

tidase: Neutral zinc

BAB1_1224

RecA

BAB1_1946

SecA

BAB2_0620

Unknown

 

metallopeptidase,

BAB1_1233

RpsM; S13

BAB1_1970

FadB

BAB2_0642

Acyl-CoA

 

zinc-binding region

BAB1_1234

Adk

BAB1_1971

EtfA

 

dehydrogenase

BAB1_0666

DapA

BAB1_1241

RpsH; S8

BAB1_1988

HisC

BAB2_0644

Metal-dependent

BAB1_0671

RpoZ

BAB1_1242

RpsN; S14

BAB1_1993

Ppa

 

hydrolase

BAB1_0688

PyrC-1

BAB1_1244

RplX; L24

BAB1_2006

RegA

BAB2_0645

GatC

BAB1_0697

CysS

BAB1_1245

RplN; L14

BAB1_2016

RpmB; L28

BAB2_0646

GatA

BAB1_0718

MoaD

BAB1_1248

RplP; L16

BAB1_2023

ClpA/clpB

BAB2_0851

GuaB

BAB1_0740

Unknown

BAB1_1249

RpsC; S3

BAB1_2059

ParB

BAB2_0961

DapA

BAB1_0775

AspS

BAB1_1256

RpsJ; S10

BAB1_2080

HslU

BAB2_0976

AldB

BAB1_0780

HemB

BAB1_1266

RplJ; L10

BAB1_2081

HslV

BAB2_0988

ArgB

BAB1_0787

GlyA

BAB1_1280

Unknown

BAB1_2087

HisE

BAB2_0990

Unknown

BAB1_0789

RibD

BAB1_1286

GloA

BAB1_2096

PTS system IIA

BAB2_0991

DapD

BAB1_0790

RibE

BAB1_1294

Aminotransferase

 

subunit

BAB2_0993

DapE

BAB1_0813

CysD

BAB1_1297

Unknown

BAB1_2109

AccD

BAB2_1009

MgsA

BAB1_0817

Unknown; conserved

BAB1_1376

UreA

BAB1_2133

Unknown

BAB2_1012

DapB

BAB1_0826

NuoE

BAB1_1408

IlvB

BAB1_2134

SMP-30

BAB2_1013

Gpm

BAB1_0842

ProS

  

BAB1_2135

Glutathione synthetase

  

Inner membrane

BAB1_0400

Unknown

BAB1_1283

DUF192

BAB2_0261

RecA

BAB2_0877

Binding-protein-

BAB1_0425

NhaA

BAB1_1703

FtsH

BAB2_0709

FtsK-alpha

 

dependent transport

BAB1_0542

WbkC

BAB1_1712

MotA; TolQ; ExbB

BAB2_0728

CydA

 

system inner

       

membrane component

Periplasm

BAB1_0010

Trs-ABC oligopeptide

BAB1_1118

PpiB-1

BAB2_0427

Trs-ABC spermidine/putrescine

BAB2_0697

Unknown; conserved

BAB1_0155

OstA-like

BAB1_1362

LacI

  

BAB2_0812

Trs-ABC oligopeptide

BAB1_0404

Unknown

BAB1_1413

DegP

BAB2_0451

Trs-ABC oligopeptide

 

AppA family

BAB1_0444

PdxH

BAB1_1890

YciI-like protein

 

AppA family

BAB2_0879

Trs-ABC spermidine/putrescine

BAB1_0739

ETC complex I

BAB1_1919

Unknown

BAB2_0593

Trs-ABC amino acid

  

BAB1_0776

Unknown

BAB1_1981

TlpA

BAB2_0611

Trs-ABC amino acid

BAB2_0880

Unknown

BAB1_0881

Trs-ABC amino acid

BAB2_0374

Unknown

BAB2_0664

Trs-ABC peptide

BAB2_1109

XylF

BAB1_1117

PpiB-2

      

Outer membrane

BAB1_0659

Omp2a

BAB1_0707

OstA

BAB1_0963

TolC

  

Unknown localization

BAB1_0030

Unknown

BAB1_0991

Unknown

BAB1_1543

DUF526

BAB1_2123

RpmI; L35

BAB1_0170

GrpE

BAB1_1070

WrbA

BAB1_1559

FbcF

BAB1_2176

YaeC/NLPA lipoprotein

BAB1_0389

CcoP

BAB1_1113

Unknown; conserved

BAB1_1641

Unknown

BAB1_2186

RpsT; S20

BAB1_0413

AtpB

BAB1_1152

PdhA

BAB1_1647

FabG domain

BAB2_0207

Unknown

BAB1_0418

Unknown

BAB1_1230

RplQ; L17

BAB1_1693

bZIP

BAB2_0243

YedY

BAB1_0420

Unknown

BAB1_1232

RpsK; S11

BAB1_1728

RpmE; L31

BAB2_0269

RpsU; S21

BAB1_0453

Unknown

BAB1_1240

PplF; L6

BAB1_1749

Unknown

BAB2_0351

OsmC-like protein

BAB1_0479

RpsR, S18

BAB1_1260

RpsL; S12

BAB1_1768

Unknown

BAB2_0356

Unknown

BAB1_0627

Unknown

BAB1_1270

SecE

BAB1_1784

DUF336

BAB2_0677

Unknown

BAB1_0650

Unknown

BAB1_1341

Unknown

BAB1_1814

Unknown

BAB2_0726

YbgT

BAB1_0810

RpsI; S9

BAB1_1384

Cibk

BAB1_1858

RplU; L21

BAB2_0869

HlyD

BAB1_0830

NDH-1 subunit I

BAB1_1514

AspC

BAB1_1984

LysA

BAB2_1002

NqoB

Locus tags and descriptions of proteins are indicated and proteins are organized by predicted subcellular localization.

Correction of five pseudogene annotations

In previous studies using B. abortus 2308, we used the genome databases available on NCBI for B. abortus, B. melitensis and B. suis for protein identification. More than once, we obtained peptides which matched proteins supposedly expressed only by the latter two species. Upon verification, those peptides were manually assigned to ORFs of previously annotated pseudogenes of B. abortus strain 2308 (NCBI taxonomy ID 359391). We therefore assembled a custom protein database which included the predicted translation sequence of all B. abortus 2308 ORFs annotated as pseudogenes. Using this database, we were able to confirm the protein expression of five of these ORFs (Figure 1): BAB1_1205, BAB1_1645, BAB1_1646, BAB1_1768 and BAB2_0216. The MSMS spectra of the 18 peptides representing these former pseudogenes were manually validated. We thus investigated the reasons for which these genes had been annotated as pseudogenes. The genomic sequence of the cytoplasmic protein with a conserved DUF 883 domain BAB1_1205 was found to be identical to BMEI0805, its B. melitensis counterpart. Apart from the short length of this protein, there was no apparent reason for its pseudogene annotation (Figure 1). For BAB1_1645 and BAB1_1646 (Figure 1), the nucleotidic sequence was 99% identical to their BMEI0397 and BMEI0396 counterparts, leading to two cytoplasmic B. abortus 2308 dihydroxyacetone kinases involved in glycerolipid metabolism that are 98% and 100% identical to the B. melitensis proteins, respectively. The case of BAB2_0216, which seems to be a 3-hydroxybutyryl-CoA dehydrogenase, was more complex and confusing, having a single nucleotide deletion when compared to B. melitensis. This deletion would lead to the silencing of the stop codon which creates two separate proteins in B. melitensis, BMEII1020 and BMEII1021. In B. abortus 2308, a fusion of the two genes would generate a much larger protein. However, the start codon in the corresponding ORF of vaccine B. abortus S19 (BAbS19_II02060) is different from BMEII1020, and even more different from the start codon and carboxyl terminal sequence of the counterparts in B. suis (BSUIS_B0227), B. ovis (BOV_A0203), B. canis (BCAN_B0224) and B. ceti (BCETI_6000534). As a consequence, the lengths of B. abortus and B. melitensis proteins differ considerably from those of other Brucella. Since the BAB2_0216 peptide that we found is located in the N-terminal section of the protein (Figure 1), we are able to confirm the expression of this originally annotated pseudogene, but were unable to confirm the expression of the full length protein.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-300/MediaObjects/12864_2009_Article_2894_Fig1_HTML.jpg
Figure 1

B. abortus 2308 former pseudogenes. Peptide sequences identified by mass spectrometry are highlighted in grey. Corresponding B. melitensis 16 M locus tags are indicated between parentheses.

The sequence of the BAB1_1768 pseudogene was found to be misannotated in B. abortus 2308. The peptide sequence "TAGYGVGGAALGALAGGAIGGNGR" could not be found in the B. abortus 2308 nucleotide-derived proteome but matched the B. melitensis locus tag BMEI0287. In fact, except for 1 nucleotide, the corresponding 2308 genomic sequence is identical to that of BMEI0287 (Figure 2C). In B. abortus 2308, a single nucleotide insertion in BAB1_1768 modifies the reading frame, hence its original annotation as a pseudogene. The manually validated peptide matches B. abortus 2308 only when the additional nucleotide is removed, indicating that the sequence for locus BAB1_1768 should be corrected (Figure 1). Also to note is the earlier start site in B. abortus 2308, and all other species sequenced to date, when compared to B. melitensis 16 M. We believe that the B. abortus 2308 start site was correctly assigned in the publicly available genome given the clear presence of a ribosome binding site in position -8 of the B. abortus sequence.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-300/MediaObjects/12864_2009_Article_2894_Fig2_HTML.jpg
Figure 2

Annotation errors in the B. abortus 2308 genome. (A, B) The original start codon annotation in the publicly available genome (NCBI taxonomy ID 359391) of the succinyl-CoA synthetase subunit beta (BAB1_1926, panel A) and of the KHG aldolase (BAB2_0083, panel B) are indicated by double asterisks whereas the corrected start site is indicated by a single asterisk (BAB1_1926 only). The peptides sequenced by mass spectrometry are highlighted in grey. The 5'-end of the CDS, as currently annotated, are underlined. The predicted sequence of the RBS found in proximity of the corrected start site of BAB1_1926 is boxed. Numbers next to the nucleotide sequence and the schematic gene representation indicate the position in the genome sequence (NC_007618 or NC_007624). (C) Genomic and amino acid sequences of BAB1_1768, as currently found in the publicly available genome, were aligned to their counterparts in B. melitensis 16 M (BMEI0287). The sequence of the peptide detected by mass spectrometry is highlighted in grey. Matching nucleotides are indicated by vertical bars and matching amino acids are indicated by asterisks. The predicted sequence of the RBS found in proximity of the B. abortus start site is boxed.

Correction of two start site annotations errors

Another type of annotation error identified in our studies was the erroneous assignment of gene translation start sites. For 2 proteins of B. abortus 2308, we report the expression of manually validated peptides corresponding to the sequence found upstream of their currently annotated start sites (Figure 2). The peptide sequence "MNIHEYQAK" was first found to match the cytoplasmic B. melitensis succinyl-CoA synthetase subunit beta protein (BMEI0138) and then assigned manually to BAB1_1926. Sequence comparison with other Brucella species and strains shows that the B. abortus 2308 protein start site is not shared with any of the subject sequences (Figure 2A). In fact, all homologues of this protein in other Brucella strains or species share the same start site, which is found 22 amino acids upstream of the B. abortus 2308 site. Moreover, a ribosome binding site can clearly be mapped to position -8 of the proposed new translation start site. We therefore believe this new start site to be accurate.

The second peptide, "TDLLPIMK", was found to match the cytoplasmic B. melitensis keto-hydroxyglutarate-aldolase (BMEII0009) and then assigned to BAB2_0083 in B. abortus 2308. This peptide overlaps the region upstream to the currently annotated translation start site and the first three amino acids based on the annotated translation start site (Figure 2B). Alignment of the current B. abortus 2308 protein sequence with its counterparts in other Brucella strains and species indicates that the 2308 protein sequence is falsely truncated. Other start sites lead to proteins having N-terminals longer by 11, 26 or 44 amino acids. Although we cannot clearly indicate the actual start site of BAB1_1926 or BAB2_0083, we can confirm that their N-terminals are longer than currently annotated. Based on the homology of the B. abortus 2308 genome being highest with that of other B. abortus strains, one can speculate that the start sites would be identical to those mapped in these strains.

Operons

Since genes that are part of an operon are usually co-transcribed, it is possible that these genes might also be co-translated [32]. Considering all proteins identified by our studies, we were able to almost fully reconstitute one of the two ribosomal RNA operons, with all but BAB1_1237 found. Additionally, the previously mentioned BAB1_1645 and BAB1_1646 genes are predicted to be part of an operon containing 6 genes, BAB1_1645 to BAB1_1650 http://www.microbesonline.org/operons/gnc359391.html. Four of these proteins were detected in our studies, although only BAB1_1645, -46 and -48 were found in the same experimental condition.

Conclusions

Mass spectrometry has proven to be a valuable tool to identify and correct genomic annotation errors in the study of microorganisms [3337]. We performed a proteomics analysis of B. abortus 2308 proteins expressed upon extracellular and intracellular growth conditions to validate existing gene predictions at the protein level, to acquire useful information on B. abortus 2308 expressed proteins and to identify and correct inaccurately annotated ORFs. We were able to confirm the expression of over 300 previously unreported proteins and five pseudogenes, and corrected two wrongly assigned translation start sites. Taken together, these findings further demonstrate that computational genomic annotation errors can be corrected using proteomics. This will lead to improved databases and thus better protein identification and functional annotation.

Methods

Brucella abortus protein preparation for mass spectrometry analysis

Four types of B. abortus 2308 samples were prepared: outer membranes, cytosols, intracellular bacteria isolated from infected RAW264.7 macrophages and extracellular bacteria from overnight cultures. Outer membrane samples were prepared and processed for mass spectrometry analysis as previously described [16]. Cytoplasmic fractions were prepared as described previously [38]. Briefly, bacteria grown in tryptic soy broth (Difco) in 2-liter flasks on an orbital shaker and harvested by centrifugation in sealed cups at 7,000 × g for 20 min. The thick slurry of bacteria were suspended in 10 mM phosphate-buffered saline (pH 7.2) was passed twice through a French press (Pressure Cell 40 K, Aminco; SLM Instruments Inc., Urbana, Ill.) at an internal pressure of 35,000 lb/in2. The homogenate was digested with 50 mg of DNase II type V and RNase A per ml (Sigma) for 18 h at 37°C and fractionated by ultracentrifugation. The cell envelopes in the bottom of the tube removed and the cytoplasmic fractions in the supernatant, filtered, lyophilized and characterized as described previously [39]. Intracellular bacteria were isolated from RAW264.7 macrophages 3, 20 and 44 hours post-infection as previously described [17]. Proteins were extracted from intracellular and extracellular bacteria using the same method and digested for mass spectrometry as previously described [17].

Liquid Chromatography - Mass Spectrometry (LC-MS)

Peptide digests were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS) as described [40]. Briefly, the samples were injected onto a reversed-phase column (Jupiter C18, Phenomenex, Torrance, CA) for HPLC separation. For LC-MS survey scans, the mass spectra were acquired over 400-1600 Da at a rate of 1 spectrum/second. Peptide sequencing was achieved by targeted and shotgun LC-MS/MS. For MS/MS scans, the mass range was 50-2000 Da, and each spectrum was acquired in 2 seconds. For LC-MS/MS, the duty cycle was one survey scan followed by one product ion scan (MS/MS).

Protein identification

Protein identification was done by submitting LC-MS/MS spectra to Mascot software (MatrixScience, Boston, MA) and searching against custom protein databases (see below). The parameters used for the Mascot search and protein homology clustering were previously detailed [16]. No multidimensional fingerprinting method was used. Annotation for each protein was performed using ExPASy Proteomics tools http://us.expasy.org/tools/#proteome, Kegg GenomeNet Database Service http://www.genome.jp/ and literature mining of orthologous genes and proteins.

Protein databases

The databases were composed of protein sequences obtained from the National Center for Biotechnology Information (NCBI) protein database (for B. abortus 2308, NC_007618 and NC_007624; for B. melitensis 16 M, NC_003317 and NC_003318; for Mus musculus, all protein sequences contained under taxonomy ID 10090) and of B. abortus 2308 "pseudoproteins" corresponding to the custom translation of pseudogenes. Genomic regions corresponding to the 316 entries annotated as pseudogenes in NCBI were directly translated and added to the database. Additionally, the ORF Finder tool from NCBI was used to determine other possible protein sequences corresponding to the pseudogenes. The ORF search was done by including 0 to 200 bp upstream or downstream from these regions. All resulting ORFs spanning the entire pseudogene sequence were kept. Ribosome binding sites were mapped when possible according to the sequence described in reference [41]. A total of 471 translated protein sequences were added to the NCBI databases.

Validation of mass spectrometry results

Sequences assigned to MS/MS spectra of peptides, which were mapped to pseudogenes or to genomic regions annotated as untranslated regions, were manually validated. For proteins identified by a single peptide, manual validation of the spectra was performed for peptide sequences having a Mascot score below 45.

Prediction of protein localization

The localization of newly demonstrated proteins was predicted using PSORTb version 2.0.4 http://www.psort.org/psortb/index.html, CELLO version 2.5 http://cello.life.nctu.edu.tw/ and PSLpred http://www.imtech.res.in/raghava/pslpred/index.html. For a localization to be assigned, a minimum of 2 of the 3 predictions had to match.

Declarations

Acknowledgements

This work was funded by the NIAID/NIH contract HHSN266200400056C.

Authors’ Affiliations

(1)
Caprion Proteomics Inc.
(2)
Depto. Microbiología - Edificio de Investigación, Universidad de Navarra
(3)
Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional

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