Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray

lable at ScienceDirect

Microbial Pathogenesis 47 (2009) 189ndash195

Contents lists avai

Microbial Pathogenesis

journal homepage wwwelsevier comlocatemicpath

Construction and evaluation of an ORFeome-based Brucella whole-genomeDNA microarray

C Viadas a MC Rodrıguez b JM Garcıa-Lobo b FJ Sangari b I Lopez-Goni a

a Departamento de Microbiologıa y Parasitologıa Universidad de Navarra Irunlarrea N 1 31008 Pamplona Navarra Spainb Departamento de Biologıa Molecular Universidad de Cantabria and Instituto de Biomedicina y Biotecnologıa de Cantabria (IBBTEC) UCCSICIDICANAvda de los Castros sn E-39005 Santander Cantabria Spain

a r t i c l e i n f o

Article historyReceived 30 April 2009Received in revised form28 May 2009Accepted 4 June 2009Available online 12 June 2009

KeywordsBrucellaMicroarrayORFeome

Corresponding author Tel thorn34 948 425600 620E-mail addresses cviadasgmailcom cviadas

cruzrodriguezhumves (MC Rodrıguez) jmglobosangarifunicanes (FJ Sangari) ilgoniunaves (I Lo

0882-4010$ ndash see front matter 2009 Elsevier Ltddoi101016jmicpath200906002

a b s t r a c t

The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact Thecomplete nucleotide sequence of eight Brucella isolates is currently available This information can beused for high throughput approaches to the biology of this genus such as the construction of compre-hensive collections of ORF clones or ORFeomes The ORFeome of Brucella melitensis was a first contri-bution to this goal Using the Brucella ORFeome as starting material we have amplified each ORF andprinted them in duplicate onto coated glass slides along with the appropriate positive and negativecontrols Quality control of the microarray was performed by image analysis after ethidium bromidestaining This Brucella DNA microarray was used to determine the global transcriptional profile of Brucellaabortus grown under laboratory conditions Two sets of genes representing strongly and poorlyexpressed genes have been defined The occurrence of several genes of the same operon in the same dataset has been taken as additional proof of the significance of the results The two sets have been validatedby RT-PCR of retrotranscribed RNA Among the more abundant transcripts we found ribosomal proteinsKrebs cycle and oxidative phosphorylation enzymes virB flagellar components and other genes relatedwith virulence and intracellular growth were in the poorly transcribed set

This report demonstrated the usefulness of the ORFeome for the construction of a PCR productmicroarray for the analysis of global gene expression in Brucella and also applicable to other microor-ganisms The results provided here represent a comprehensive description of the global transcriptionalprofile of B abortus grown under laboratory conditions and at the same time validate the use of thisBrucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of geneexpression under any experimental conditions

2009 Elsevier Ltd All rights reserved

1 Introduction

Brucella is the etiological agent of brucellosis one of the mostcommon bacterial zoonosis worldwide with significant impact onlivestock and human health Brucella infection causes abortions andsterility in domestic animals and insidious syndromes in humansThe disease is a major cause of direct economical losses and animpediment for trade and exportations While brucellosis is largelyunder control in Europe and the USA human and animal brucel-losis are increasing in certain parts of the world especially indeveloping areas of the Mediterranean region Middle East

5 fax thorn34 948425649alumniunaves (C Viadas)

unicanes (JM Garcıa-Lobo)pez-Goni)

All rights reserved

western Asia and parts of Africa and Latin America and has beenclassified as a neglected zoonosis [1] Brucella is also considered asa potential agent of biological warfare or terrorist threat Brucellaeare mainly intracellular pathogens and the molecular mechanismsof their virulence are still poorly understood However severalsystems have been implicated in the virulence of this bacteriumsuch as the VirB type IV secretion system (T4SS) the two compo-nent regulatory system BvrSR the composition of the LPS theproduction of cyclic glucans etc The complete genome sequencesof eight different isolates belonging to five Brucella species (Brucellaabortus Brucella melitensis Brucella suis Brucella ovis and Brucellacanis) are now available and confirm the high degree of similarity ofthe genomic sequences and synteny among them [2]

An ORFeome is a comprehensive collection of the predictedcoding sequences or open reading frames (ORFs) of a givenorganism The ORFs are usually individually PCR amplified and thencloned into a suitable vector The Gateway plasmid system

Kegg Categories

Nu

mb

er o

f g

en

es

0

20

40

120

140

160Strongly expressedPoorly expressed

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Fig 1 Strongly and poorly expressed B abortus genes grouped by functional classifi-cation according to the KEGG pathway categories Columns 1 membrane transport 2xenobiotics biodegradation and metabolism 3 metabolism of amino acid and carbo-hydrate 4 metabolism of cofactors and vitamins 5 replication and repair 6 trans-lation and transcription 7 folding sorting and degradation 8 energy metabolism 9nucleotide metabolism 10 lipid metabolism 11 signal transduction 12 cellularprocesses 13 biosynthesis of polyketides and non-ribosomal peptides 14 glycanbiosynthesis and metabolism 15 unassigned

C Viadas et al Microbial Pathogenesis 47 (2009) 189ndash195190

(Invitrogen) provides a flexible platform for ORFeome constructionallowing easy insert exchange between different vectors Thisapproach was first used to verify the genome annotation and tocreate a resource to functionally characterise the proteome ofCaenorhabditis elegans [3] ORFeomes can be used for multiplepurposes such as protein over expression mutant constructioninteraction mapping etc ORFeomes are also easily amenable toautomatizable procedures [4] Using the complete genomesequence of B melitensis Dricot et al [5] generated a database ofprotein coding ORFs and constructed an ORFeome library of 3091Gateway entry clones each containing a defined ORF Prior to highthroughput ORFeome cloning the overall quality of the B melitensisgenome annotation was validated [5] The new annotatedsequences are available at the lsquolsquoAlpha-proteobacterial genomeannotation databasersquorsquo [6] Due to the reported homogeneity ofBrucella genomes the B melitensis ORFeome can be used for anal-ysis of any of the Brucella species sequenced so far This first versionof the Brucella ORFeome (version 11) provides the codingsequences in a user-friendly format amenable to high throughputfunctional genomic and proteomic experiments The availability ofall ORFs in the same vector (pDONR201) facilitates and reduces thecosts of producing DNA microarrays by PCR since a single pair ofoligonucleotides is sufficient to amplify all the ORFs This BrucellaDNA microarray will be used to analyse gene expression and shouldhelp to provide a better understanding of the molecular mecha-nisms of virulence including the identification of bacterial regu-latory networks

2 Results and discussion

To construct the Brucella-specific whole-genome microarraythe B melitensis ORFeome (version 11) was used to generate PCRamplicons for every ORF as described in 51 The microarray alsoincluded several normalization controls that represented consti-tutively expressed Brucella genes [7] or negative controls withArabidopsis thaliana genes The rationale behind designing andutilizing PCR amplicons as probes was chosen for the availability ofall ORFs in the same vector (pDONR201) facilitating and reducingthe costs of producing microarrays since a single pair of oligonu-cleotides was required to amplify all the ORFs The microarray hada total of 7680 spots and represented over 964 of the completecoding sequences assigned to B melitensis A quality controlregarding the position intensity and morphology of the spots thebackground and the reproducibility of the arrays was performed byethidium bromide staining (not shown)

To test the usefulness of the newly constructed PCR Brucellamicroarray a direct analysis of the global transcriptional profile ofB abortus cells grown in laboratory conditions was made Thusthree independent biological replicas were made and following theRNA isolation samples were converted to aRNA and labelled withCy3 fluorescent dye The use of a single dye labelling approachallowed us an estimation of the genes more abundantly expressedin Brucella under laboratory conditions The evaluation experimentwas composed of three slides yielding six measurements per gene(representing three biological replicates since each gene is presenttwice on each slide) to confirm the reproducibility of the geneexpression data

The complete data set was sorted by the normalized and cor-rected fluorescence intensity values Table S1 (supplementalmaterial) represents the combined results of all experiments andshows the 10 of the total Brucella genome with the highest and thelowest signal values Highly expressed genes showed a Log2 signalintensity above 97 and the genes considered to show poorexpression showed a Log2 signal intensity below 59 The moreexpressed gene was an outer membrane protein (BAB1_0989)

described as Omp25b [8] with a Log2 signal intensity of 1359 andthe less expressed gene was an outer membrane autotransporter(BAB2_1107) with a Log2 signal intensity of 287 The gene for IF-1(BAB1_0282) used as marker for constituvely expression [7] in themicroarray showed a Log2 signal intensity of 102 indicating that itwas well expressed in B abortus

Fig 1 provides a summary of the strongly and poorly expressedgene sets grouped by KEGG pathway categories according to the Babortus genome annotation Almost 47 of the genes selected cor-responded to unassigned categories For genes of known or anno-tated function those encoding proteins involved in membranetransport amino acid and carbohydrate metabolism and cellularprocess were among the less expressed However genes encodingproteins involved in translation and transcription replication andrepair and metabolism of cofactors and vitamins were among themore expressed

An interesting observation from the data in Table S1 was thatgenes from the chromosome II were under represented among themore strongly transcribed genes while 35 of the B abortus genomecorrespond to chromosome II only 13 of the strongly expressedgenes were localised in this chromosome (Fig 2) On the other hand62 of the poorly expressed genes were localised in chromosome IILower expression of genes in chromosome II may simply reflect anasymmetry on the chromosome distribution of genes relevant forgrowth in laboratory conditions It is well known that the twochromosomes of the Brucellae differ in important ways ChromosomeII is enriched in genes encoding nutrient transport and metabolismgenes while most of the essential genes encoding for biosynthesis ofstructural components and replicative transcription and translationmachinery reside on chromosome I [9] In addition it has beendescribed that the origin of replication of the chromosome I (thelarge chromosome) is typical of bacterial chromosomes while that ofthe chromosome II (the small chromosome) is plasmid like In thisregard a hypothesis suggesting that the Brucella small chromosomeevolved from an ancestral megaplasmid has been proposed [10]

Data generated in the array were further validated by real-timequantitative reverse transcription-PCR (RT-PCR) as described in 44To analyze the correlation between the results obtained with thearray and RT-PCR the fluorescence intensity and threshold ampli-fication cycles were made relative to the values obtained for thereference constitutive gene IF-1 and compared with a Spearmanconcordance test A high level of correlation (rfrac14 082 plt 0001)was observed between array and RT-PCR data (Fig 3)

Table 1Representative B abortus putative operons strongly and poorly expressed in labo-ratory conditions (TSB) identified by microarray analysis

Function ORFa Product

Strongly expressedTCA cycle BAB1_1922 2-oxoglutarate dehydrogenase E2 component

BAB1_1923 2-oxoglutarate dehydrogenase E1 componentBAB1_1924 pseudogeneBAB1_1925 SucD succinyl-CoA synthetase alpha chainBAB1_1926 SucC succinyl-CoA synthetase beta chainBAB1_1927 Malate dehydrogenase

Oxidativephosphorylation

BAB1_0410 ATPasa synthase F0 subunit IBAB1_0411 ATPasa synthase F0 subunit ABAB1_0412 AtpE ATPasa synthase F0 subunit CBAB1_0413 ATPasa synthase F0 subunit B0

BAB1_0414 AtpF ATPasa synthase F0 subunit BBAB1_1806 AtpC ATPasa synthase F1 subunit epsilonBAB1_1807 AtpD ATPasa synthase F1 subunit betaBAB1_1808 AtpG ATPasa synthase F1 subunit gammaBAB1_1809 AtpA ATPasa synthase F1 subunit alphaBAB1_1810 AtpH ATPasa synthase F1 subunit deltaBAB1_1900 SdhB succinate dehydrogenaseBAB1_1901 SdhA succinate dehydrogenaseBAB1_1902 SdhD succinate dehydrogenaseBAB1_1903 SdhC succinate dehydrogenaseBAB1_0492 Cytochrome C oxidase polypeptide IIBAB1_0493 Cytochrome C oxidase polypeptide IBAB1_1557 Cytochrome c1 reductaseBAB1_1558 Cytochrome bBAB1_1559 Cytochrome c reductaseBAB1_0822 NuoA NADH dehydrogenase I subunit ABAB1_0823 NuoB NADH dehydrogenase I subunit BBAB1_0824 NuoC NADH dehydrogenase I subunit CBAB1_0825 NuoD NADH dehydrogenase I subunit DBAB1_0826 NuoE NADH dehydrogenase I subunit EBAB1_0827 NuoF NADH dehydrogenase I subunit FBAB1_0828 NuoG NADH dehydrogenase I subunit GBAB1_0829 NuoH NADH dehydrogenase I subunit HBAB1_0830 NuoI NADH dehydrogenase I subunit IBAB1_0831 NuoJ NADH dehydrogenase I subunit JBAB1_0832 NuoK NADH dehydrogenase I subunit KBAB1_0833 NuoL NADH dehydrogenase I subunit LBAB1_0834 NuoM NADH dehydrogenase I subunit MBAB1_0835 NuoN NADH dehydrogenase I subunit NBAB1_0836 Biotin operon repressor

Ribosome BAB1_1230 RplQ ribosomal protein L17BAB1_1231 RpoA RNA polymerase subunit alphaBAB1_1232 RpsK ribosomal protein S11

Ge

ne

s (

)

0

20

40

60

80

100

Chromosome II

Chromosome I

Genome Stronglyexpressed

Poorlyexpresses

Fig 2 Distribution on chromosomes I and II of the whole genome and of the stronglyand poorly expressed genes

C Viadas et al Microbial Pathogenesis 47 (2009) 189ndash195 191

In the two gene sets many genes have been observed to becontiguous and probably form part of the same operon suggestingthat the whole operon could be included in the set (Table 1)Interestingly putative operons involved in housekeeping functionsas synthesis of ribosomes Krebs cycle (malate dehydrogenasesuccinyl-CoA synthase) electron chain transport and oxidativephosphorylation (ATP synthases succinate dehydrogenase NAHDdehydrogenase and cytochrome oxidases) and lipopolysaccharidebiosynthesis were included in the genes highly expressed in labo-ratory conditions It was noteworthy the presence in the stronglyexpressed genes of 35 genes from the same gene cluster relatedwith ribosomal biosynthesis translation and transcription

By the contrary putative operons involved in amino acid andsugar ABC transport systems virB T4SS flagellar biosynthesis andassembly nitrogen metabolism and inositol metabolism were in

ORFs

BAB1

_124

6

BAB1

_083

2

BAB1

_126

4

BAB1

_024

6

BAB1

_054

4

BAB1

_023

9

BAB1

_117

4

BAB2

_052

7

BAB2

_006

6

BAB2

_006

1

BAB2

_092

8

BAB2

_052

8

BAB2

_052

2

BAB2

_014

8

BAB2

_095

4

BAB2

_013

0

Levels o

f exp

ressio

n

-8

-6

-4

-2

0

2

4RT-PCRMicroaray

Fig 3 Concordance between the expression data obtained in the microarray and withRT-PCR The level of expression represents the Log2 signal intensity and the Ct valuesobtained in the microarray and RT-PCR adjusted to the values obtained for the refer-ence gene IF-1 The concordance between both methods was strongly significant(rfrac14 082 plt 0001) The genes chosen for confirmation represented the putativeoperons strongly and poorly expressed in the array (see Table 1)

BAB1_1233 RpsM ribosomal protein S13BAB1_1234 AdK adenylate kinaseBAB1_1235 SecY preprotein translocase subunitBAB1_1236 RplO ribosomal protein L15BAB1_1237 RpmD ribosomal protein L30BAB1_1238 RpsE ribosomal protein S5BAB1_1239 RplR ribosomal protein L18BAB1_1240 LSU ribosomal protein L6PBAB1_1241 SSU ribosomal protein S8PBAB1_1242 RpsN ribosomal protein S14BAB1_1243 RplE ribosomal protein L5BAB1_1244 RplX ribosomal protein L24BAB1_1245 Ribosomal protein L14BAB1_1246 RpsQ ribosomal protein s17BAB1_1247 RpmC ribosomal protein L29BAB1_1248 RplP ribosomal protein L16BAB1_1249 SSU ribosomal protein S3PBAB1_1250 RplV ribosomal protein L22BAB1_1251 RpsS ribosomal protein S19BAB1_1252 LSU ribosomal protein L2PBAB1_1253 LSU ribosomal protein L23PBAB1_1254 LSU ribosomal protein L1E (frac14 L4P)BAB1_1255 RplC ribosomal protein L3BAB1_1256 RpsJ ribosomal protein S10BAB1_1257 Tuf-2 translation elongation factor TuBAB1_1258 EF-G protein translation elongation factor GBAB1_1259 RpsG ribosomal protein S7

(continued on next page)

BAB1_1260 RpsL ribosomal protein S12BAB1_1261 Hypothetical proteinBAB1_1262 Hypothetical proteinBAB1_1263 RpoC RNA polymerase beta0 subunitBAB1_1264 RpoB RNA polymeraseBAB1_1265 RplL ribosomal protein L7L12BAB1_1266 LSU ribosomal protein L10PBAB1_1267 LSU ribosomal protein L1PBAB1_1268 RplK ribosomal protein L11BAB1_1269 NusG transcription antitermination protein

NusGBAB1_1270 SecE preprotein translocaseBAB1_2124 RplT ribosomal protein L20

LPS biosynthesis(O-antigen)

BAB1_0540 WbkC mannose formyl transferaseBAB1_0541 WbkB transferaseBAB1_0542 Wzt ABC transporter ATP-bindingBAB1_0543 Wzm ABC transporter permeaseBAB1_0544 per perosamine synthetaseBAB1_0545 Gmd GDP-mannose dehydrataseBAB1_0561 ManB mannose-1-phosphate

guanylyltransferaseBAB1_0562 ManA mannose-6-phosphate isomerase

LPS biosynthesis(lipid-A)

BAB1_1171 LpxB lipid-A-disaccharide synthaseBAB1_1172 Hypothetical proteinBAB1_1173 LpxA UDP-acetylglucosamine

o-acyltransferaseBAB1_1174 3R-hydroxymyristoylBAB1_1175 LpxD UDP-3-OH-myristoyl glucosamine

N-acyltransferaseBAB1_1176 Outer membrane protein

Maltose BAB1_0236 Phosphoribosyl transferaseBAB1_0237 Transcriptional regulator IclR familyBAB1_0238 MalE maltose ABC transporter periplasmic

proteinBAB1_0239 MalF maltose ABC transporter permease sugarBAB1_0240 MalG maltose ABC transporter permease sugarBAB1_0241 MalK maltose ABC transporterBAB1_0242 Mandelate racemaseBAB1_0243 Hypothetical proteinBAB1_0244 OxidoreductaseBAB1_0245 Hypothetical proteinBAB1_0246 OxidoreductaseBAB1_0247 Fumaryl acetate hidrolaseBAB1_0248 Mandelate racemase

Poorly expressedABC transporters BAB2_1132 LivK branched-chain amino acid transport

systemBAB2_1133 LivH branched-chain amino acid transport

systemBAB2_1134 LivM branched-chain amino acid transport

systemBAB2_1135 LivF branched-chain amino acid transport

systemBAB2_0023 LivK branched-chain amino acid transport


systemBAB2_0025 Branched-chain amino acid transport systemBAB2_0026 LivG branched-chain amino acid transport


systemBAB2_1037 OppF oligopeptide transport systemBAB2_1049 OppA oligopeptide transport systemBAB2_1051 OppC oligopeptide transport systemBAB2_1052 OppD oligopeptide transport systemBAB2_1056 OppB oligopeptide transport systemBAB2_1057 OppC oligopeptide transport systemBAB2_1058 OppD oligopeptide transport systemBAB1_1955 Amino acid ABC transporterBAB1_1956 Amino acid ABC transporterBAB1_1957 Amino acid ABC transporterBAB1_1958 Arginase family protein

Table 1 (continued)


BAB1_1959 Aspartate ammonia-lyaseBAB1_1960 Amino acid ABC transporterBAB1_0767 AapJ general L-amino acid transport systemBAB1_0768 AapQM general L-amino acid transport

systemBAB1_0769 AapP general L-amino acid transport systemBAB2_0286 AapJ general L-amino acid transport systemBAB2_0428 PotA multiple sugar-binding transport

systemBAB2_1064 PotB multiple sugar-binding transport

systemBAB2_1063 PotC multiple sugar-binding transport systemBAB2_1062 PotD multiple sugar-binding transport

systemBAB2_1142 UpgA multiple sugar transport systemBAB2_1143 UpgC multiple sugar transport systemBAB2_0583 UpgE multiple sugar transport systemBAB2_0491 UpgB multiple sugar transport system

Type IV secretionsystem (VirB)

BAB2_0057 VirB12 (outer membrane protein OprF)BAB2_0058 VirB11 (ATPase)BAB2_0059 VirB10BAB2_0060 VirB9BAB2_0061 VirB8BAB2_0062 VirB7BAB2_0063 VirB6BAB2_0064 VirB5BAB2_0065 VirB4 (ATPase)BAB2_0066 VirB3BAB2_0067 VirB2BAB2_0068 VirB1

Flagellar apparatus BAB2_1086 FlgJ flagelar proteinBAB2_1087 TropomyosinBAB2_1088 FliR export apparatusBAB2_1089-90 FlhA export apparatusBAB2_1091 FlhA export apparatusBAB2_1092 FliQ export apparatusBAB2_1093 FlgD flagellar hook capping proteinBAB2_1094 FlbT flagellin biosynthesis repressorBAB2_1095 FlaF flagellin biosynthesis regulatorBAB2_1096 FlgL flagellar hook-associated proteinBAB2_1097 FlgK flagellar hook-associated proteinBAB2_1098 FlgE flagellar hook monomerBAB2_1099 FtcR transcriptional regulatorBAB2_1100 hypothetical proteinBAB2_1101 MotD flagellar motor proteinBAB2_1102 MotC flagellar motor proteinBAB2_1103 MotB flagellar motor proteinBAB2_1104 hypothetical proteinBAB2_1105 FliF MS-ring monomerBAB2_1106 FliC flagellinBAB2_0148 FlgB flagellar basal-body rod proteinBAB2_0149 FlgG flagellar basal-body rod proteinBAB2_0150 FliE flagellar hook-basal complex proteinBAB2_0151 FlgG flagellar basal-body rod proteinBAB2_0152 FlgA flagella basal-body P-ring biosynthesis

proteinBAB2_153-54 FlgL P-ring monomerBAB2_0155 MotE flagellar motor switch proteinBAB2_0156 FlgH L-ring monomerBAB2_0157 FliL flagellar basal-body-associated proteinBAB2_0158 FliP export apparatusBAB2_0120 FlhB export apparatusBAB2_0122 FliG flagellar motor switch proteinBAB2_0123 hypothetical proteinBAB2_124-25 FliNM flagellar motor switch proteinBAB2_0126 MotA flagellar motor proteinBAB2_0127 FlgF flagellar basal-body rod proteinBAB2_0128 FlgF flagellar basal-body rod proteinBAB2_0129 FliL flagellum-specific ATP synthaseBAB2_0130 hypothetical protein

Denitrification BAB2_0941 NnrA Crp transcription regulatorBAB2_0942 NirV nitrite reductaseBAB2_0943 NirK copper-contaning nitrite reductaseBAB2_0952 NorD

Table 1 (continued)



BAB2_0953 NorQBAB2_0954 NorB nitric oxide reductase large subunitBAB2_0955 NorC nitric oxide reductase small subunitBAB2_0956 NorF nitric oxide reductaseBAB2_0957 NorEBAB2_0922 NnrB Crp transcription regulatorBAB2_0923 NosX thiamine biosynthesis lipoproteinBAB2_0924 NosL hypothetical protein disulfide

isomeraseBAB2_0925 NosY membrane protein precursorBAB2_0926 NosF copper ABC transporter ATP-binding

proteinBAB2_0927 NosD copper ABC transporter periplasmic

proteinBAB2_0928 NosZ nitrous oxide reductaseBAB2_0929 NosR regulatory protein

Inositolmetabolism

BAB2_0521 SfuC iron(iii)-transport atp-binding proteinBAB2_0522 Inositol monophosphataseBAB2_0523 IolB inositol catabolismBAB2_0524 IolE (MocC) inositol catabolismBAB2_0525 IolD inositol catabolismBAB2_0526 IolC inositol catabolismBAB2_0527 Transcriptional regulator RpiRBAB2_0528 MocA inositol 2-dehydrogenase

a Designation is based on the B abortus 2308 genome sequences ORF over orunder expressed are mark in bold (see Table S1)

Table 1 (continued)



the poorly expressed genes set Some of these operons have beenrelated with Brucella virulence and their expression is tightlyregulated and probably these functions are turned off duringgrowth in laboratory conditions For instance the Brucella T4SS isencoded by the virB locus containing genes virB1 to virB12 that aretranscribed as an operon [11 12] This T4SS is essential for intra-cellular survival and multiplication in B suis B melitensis and Babortus and virB mutants are highly attenuated in the mouse modeland in the natural host [12-17] Recently it was demonstrated thatone of the VirB proteins is expressed during infection of bothexperimental and natural host of Brucella species [18] Transcrip-tion of the virB operon is induced specifically within macrophages[12 19] suggesting that the expression of this T4SS is tightlyregulated In fact in vitro studies have shown that expression ofVirB operon was also dependent on the phase of the growth curvepH temperature and carbon source [11] Six out of the 12 virBgenes appeared in the poorly expressed data set corroborating theexpected down regulation of these genes under laboratoryconditions

Another example is the thirty-one ORFs encoding flagellar andmotor proteins distributed in three clusters on the chromosome II [920 21] The expression of Brucella flagellar genes was growth phasedependent and at least one of the promoters was expressed intra-cellularly [20] In addition Brucella flagellar mutants were attenu-ated in mice [20] Twenty-six flagellar genes distributed in the threegene clusters were also found among the poorly expressed genes

The expression and assembly of both multimolecular surfacestructures (T4SS and flagellum) are energy intensive processesrequiring an intricate regulatory control to allow their expression atthe very precise steps of the infection where they are needed Inthis regard it has been shown that the quorum-sensing regulatorsVjbR and BlxR control expression of both the T4SS and the flagellarapparatus [22 23]

Other putative operons poorly expressed in laboratory condi-tions included denitrification genes (nir nor and nos operons)Brucella posses an anaerobic electron transfer system able to reducenitrate into dinitrogen gas under low-oxygen conditions (Narnitrate reductase Nir nitrite reductase Nor nitric oxide reductase

Nos nitrous oxide reductase) Since Brucella is an intracellularfacultative pathogen during the infectious process the bacteriacould use denitrification to survive using nitrogen oxides asterminal electron receptors and limiting the production of reactivenitrogen intermediates by the host In fact some of these denitri-fication genes have been related with the virulence in mice [24-26]As expected these genes were turned off in the aerobic conditionsused in our experiment Finally other operon poorly expressed wasinvolved in the metabolism of inositol homologous to the mocoperon of Rhizobium leguminosarum [27] Interestingly iolE (mocCBAB2_0524) was described as one of the genes that was expressedintracellularly at 24 h following macrophage invasion [7] Insummary it appears that strongly expressed genes were thoserequired for high-speed aerobic growth while virulence relatedgenes and other genes known to be expressed intracellularlyappeared poorly expressed in the microarray

A global proteomic analysis of cell envelope of B abortus grownunder laboratory conditions indentified 163 proteins [28] Some ofthe major protein components include Omp25 (BAB1_0722)Omp25c (BAB1_0116) Omp2b porin (BAB1_0660) several ribo-somal proteins (BAB1_1230-1256 BAB1_1263-1270) ATP synthasesubunits (BAB1_0413-0414 BAB1_1806-1810) NADH-dehydroge-nase subunits (BAB1_0824-08828) 2-oxoglutarate dehydrogenasecomponents (BAB1_1922-1923) and malate (BAB1_1927) andsuccinate dehydrogenases (BAB1_1900) Interestingly similarresults were obtained by our microarray analysis and the corre-sponding genes that coded for these proteins are also among thegenes strongly expressed (Table 1) A total of fifty-six of the 163ORFs detected by this proteomic analysis were also detected asgenes strongly expressed in our microarray assay Similarly Wagneret al [29] performed a global analysis of the B melitensis proteomeand identified 187 ORFs expressed in laboratory grown culture Inspite of the different experimental conditions and that twodifferent Brucella species were used we found that 37 ORFs fromour highly expressed data set were also present among the proteinsidentified in the B melitensis proteome Also in B melitensis growthin vitro Teixeira-Gomes et al [30] detected by two-dimensionalelectrophoresis and microsequencing several proteins which geneswere among the highly expressed in the microarray bacter-ioferritin (BA12_0675) Cu-Zn superoxide dismutase (BAB2_ 0535)succinyl-CoA synthetase alpha subunit (BAB1_1925) and Clpprotein (BAB1_1131) Comparison of all these proteomic studieswithin our microarray analysis demonstrates a good correlationbetween the highly expressed genes with the most abundantcellular proteins

Finally to validate the used of our ORFeome microarraya comparison with cells growth under two different conditions(Tryptic soy broth versus Brucella broth see 42) were performedInterestingly the IF-1 gene used as control in our assays wasexpressed similarly in both conditions (with Log2 signal intensity of102 in TSB and 104 in BB) demonstrating that this gene wasexpressed constitutive in broth cultures (not shown) Most of therepresentative B abortus putative operons strongly and poorlyexpressed were similar in both conditions (result not shown) TCAcycle oxidative phosphorylation ribosomal genes O-antigen andlipid-A biosynthesis type IV secretion system flagellar apparatusdenitrification or inositol metabolism (Table 1) However themaltose operon (BAB1_0236-0248) was strongly expressed only inTSB and some ABC transporters were also differently expressed forexample LivF-K (BAB2_0023-0027) and PotA-D (BAB2_0428 andBAB2_1062-1064) transporters were among the most poorlyexpressed in TSB but not in BB The different chemical compositionin both mediums could explain these differences These resultsstrongly suggest that our Brucella ORFeome microarray can be usedto compare global gene expression under different conditions


3 Conclusions

This report demonstrated the usefulness of the ORFeome asstarting material for the construction of a PCR product microarrayfor the analysis of global gene expression in Brucella Moreover thisprocess could be applied to any organism with and availableORFeome The hybridization of this microarray with a probe derivedfrom mRNA obtained from a B abortus culture in TSB in the midlogarithmic phase allowed the identification of two sets of stronglyand poorly expressed genes The two categories have beenconfirmed by RT-PCR and some concordance has also been foundwith the proteome of B melitensis Products related with high-speedgrowth in aerobic conditions (ribosomal proteins Krebs cycleenzymes oxidative phosphorylation) were among the highlyexpressed genes while genes related with virulence and intracel-lular growth were in the poorly expressed data set A strong asym-metry in chromosome distribution has also been detected withstrongly expressed genes more abundant in chromosome I andpoorly expressed genes specially represented in chromosome II

4 Materials and methods

41 Construction of the Brucella DNA microarray

The custom microarray and the experimental design were madeaccording to the MIAME recommendations [31] The completeBrucella ORFeome library [5] was isolated and purified by thePlasmid Miniprep 96 System (Millipore) following the manu-facturerrsquos instructions Each Brucella ORF was amplified by PCR withthe iQ SuperMix (Bio-Rad) using pDONR201 specific primers (attl150-CAAGTTTGTACAAAAAAGCAGGC-30 and attl2 50-CCACTTTGTACAAGAAAGCTGG-30) The thermal cycling conditions were asfollows after an initial denaturation at 95 C for 5 min 35 s oftemplate denaturation at 95 C 45 s of primer annealing at 65 Cand 60 s per Kb of primer extension at 72 C for a total of 30 cycleswith a final extension at 72 C for 7 min PCR products were puri-fied using the Montage PCRm96 Cleanup System (Millipore) andwere visually scored for presence purity and size after 08ndash12agarose gel electrophoresis (120 V for 1 h) Subsequently PCRproducts were dried resuspended in 50 dimethylsulfoxide (vv)and arrayed into 384-well plates For the construction of the DNAmicroarray PCR products were printed in duplicated onto2575 mm UltraGAPs Coated Slides (Corning Life Sciences) usingMicroGrid II 610 Robotic System (Genomic Solutions) DNA wascross-linked to the surface by UV and slides were baked at 80 C for1 h The spots 150 mm in diameter were separated from each otherby 265 mm and arranged in 32 subarrays (16 columns and 15 rowseach) The spotted area was w1717 mm The PCR-amplifiedconstitutively expressed Brucella translation initiation factor IF-1gene (BMEI1671) [7] was spotted at 128 positions distributedthroughout the printed area as positive and homogeneity controlsNegative controls containing spotting buffer (50 dimethylsulf-oxide) or PCR-amplified A thaliana gene (porB protochlorophyllideoxidoreductase B) were also spotted at 256 different positions

42 Brucella culture and RNA isolation and labelling

B abortus strain 2308 was grown in 10 mL of Tryptic Soy Broth(TSB Biomerieux Trypticase 17 gL Soyase 3 gL NaCl 5 gL K2PO4

25 gL glucose 25 gL final pHfrac14 73) into a 100-mL flask on anorbital shaker (200 rpm) at 37 C until mid log phase (OD600frac14 06ndash07) Alternatively Brucella broth was used (BB Pronadisa Meatpeptone 10 gL Casein peptone 10 gL Yeast extract 2 gL NaCl 5 gLNaHSO3 01 gL glucose 1 gL final pHfrac14 73) Brucella RNA formicroarray analysis was purified and amplified by the MessageAmp

II-Bacteria RNA Amplification Kit (Ambion) which enablesprokaryotic RNA amplification for whole genome expression anal-ysis from bacterial samples Briefly the bacterial culture wasstabilized with the Protect Bacteria Reagent (Ambion) and total RNAwas extracted with the RNeasy Mini System (Qiagen) in combinationwith the RNase-Free DNase Set (Qiagen) RNA preparations weretested for the lack of genomic DNA contamination by PCR BrucellamRNA was enriched using MICROBExpress Kit (Ambion) andantisense amino-allyl dUTP marked RNA (aRNA) was obtained byamplification with the MessageAmp II-Bacteria kit (Ambion) andlabelled with Cy3 fluorescent dye (Amersham Bioscience) followingthe manufacturerrsquos instructions

43 Microarray hybridization scanning image quantification anddata analysis

Previous to the hybridization process the microarray slideswere blocked by washing with 5 SSC 01 (wv) SDS and 1 (wv)bovine serum albumin pre-heated to 42 C After 45 min at 42 Cthe microarray slides were washed with water at room tempera-ture and then with isopropanol The slide was then allowed to drySamples containing 10 mg of Cy3 labelled aRNA were dissolved in25 mL of a solution containing 50 (vv) deionized formamide5 SSC and 02 (wv) SDS pre-heated to 42 C After 2 min at95 C to denature the aRNA the solution was applied to themicroarray slide covered with a 24 60 mm cover glass andincubated into a hybridization chamber at 42 C for 18 h Afterremoving the cover glass the microarray was washed twice with1 SSC 02 (wv) SDS at 42 C and then successively with02 SSC 01 (wv) SDS 02 SSC 005 SSC and water at roomtemperature The microarray was allowed to dry and fluorescentimages were generated by scanning the slides using a GenePix4100A microarray scanner (Amersham Bioscience) at 600 PMT Gainand with filter 670DF40 Spot intensity was determined using thesoftware packages Genepix Pro 50 (Axon) Six measurements pergene were made representing three independent RNA extractionsof Brucella cells growth in standard conditions since each gene ispresent twice on each slide Data were normalized and statisticallyanalysed using the BRB array tools v 36 [32] Raw fluorescenceintensity data from each array were background corrected andnormalized by the lsquolsquomedian normalizationrsquorsquo procedure using themedian array as reference After this normalization intensity datawere corrected for the size of the PCR product in the spot (Log2Isize-correctedfrac14 Log2I Log2 size in kb)

44 Quantitative real-time PCR (RT-PCR)

Determination of gene expression levels was made by RT-PCRBriefly 24 mg of total RNA were reverse transcribed into cDNAusing random oligonucleotide hexamers and SuperScript III RT(Invitrogen) according to manufacturerrsquos protocol Then 1 mL of theresulting cDNA was used in quantitative real-time PCR reactionsusing Power SYBR Green PCR Master Mix (Applied Biosystems)and a 7500 Real Time PCR System (Applied Biosystems) Primers(supplementary Table S2) were designed using Primer Express 30software (Applied Biosystems) To confirm the lack of DNAcontamination reactions without reverse transcriptase were per-formed Dissociation curve analysis was performed for verificationof product homogeneity Threshold fluorescence was establishedwithin the geometric phase of the exponential amplification andthe cycle of threshold (Ct) was determined for each reaction Thereactions were made by triplicate and the constitutively expressedgene IF-1 of Brucella [7] was used as internal control for datanormalization


45 Microarray accession number

The microarray data has been deposited in the EMBL-EBIArrayExpress repository (httpwwwebiacukmicroarray-asae)with the accession number E-MEXP-1887

Acknowledgements

This work was supported by the Ministerio de Ciencia y Tec-nologıa of Spain (BIO2005-04985 and AGL2008-04514 to ILG andBIO2007-63656 to FJS) and Instituto de Salud Carlos III (PI050894 toJMG-L) Fellowships support to CV for the Gobierno Vasco and toMCR for Fundacion Marques de Valdecilla-IFIMAV are gratefullyacknowledged We are thankful to Progenika Biopharma (httpwwwprogenikacom) for their helpful assistance in printing themicroarray

Appendix Supplementary data

Supplementary data associated with this article can be found inthe online version at doi101016jmicpath200906002

References

[1] World Health Organization The control of neglected zoonotic disease a routeto poverty alleviation Report of a joint WHODFID-AHP meeting with theparticipation of FAO and OIE Geneva WHO 2006

[2] Chain PS Comerci DJ Tolmasky ME Larimer FW Malfatti SA Vergez LM et alWhole-genome analyses of speciation events in pathogenic Brucellae InfectImmun 2005738353ndash61

[3] Reboul J Vaglio P Rual JF Lamesch P Martinez M Armstrong CM et alC elegans ORFeome version 11 experimental verification of the genomeannotation and resource for proteome-scale protein expression Nat Genet20033435ndash41

[4] Rual JF Hill DE Vidal M ORFeome projects gateway between genomics andomics Curr Opin Chem Biol 2004820ndash5

[5] Dricot A Rual JF Lamesch P Bertin N Dupuy D Hao T et al Generation of theBrucella melitensis ORFeome version 11 Genome Res 2004142201ndash6

[6] Alpha-proteobacterial genome annotation database httpurbm59urbmfundpacbe7EdharbiaPAGe

[7] Eskra L Canavessi A Carey M Splitter G Brucella abortus genes identifiedfollowing constitutive growth and macrophage infection Infect Immun2001697736ndash42

[8] Salhi I Boigegrain RA Machold J Weise C Cloeckaert A Rouot B Character-ization of new members of the group 3 outer membrane protein family ofBrucella spp Infect Immun 2003714326ndash32

[9] Paulsen IT Seshadri R Nelson KE Eisen JA Heidelberg JF Read TD et al TheBrucella suis genome reveals fundamental similarities between animal andplant pathogens and symbionts Proc Natl Acad Sci U S A 20029913148ndash53

[10] Moreno E Moriyon I Brucella melitensis a nasty bug with hidden credentialsfor virulence Proc Natl Acad Sci U S A 2002991ndash3

[11] Boschiroli ML Ouahrani-Bettache S Foulongne V Michaux-Charachon SBourg G Allardet-Servent A et al Type IV secretion and Brucella virulence VetMicrobiol 200290341ndash8

[12] Sieira R Comerci DJ Sanchez DO Ugalde RA A homologue of an operonrequired for DNA transfer in Agrobacterium is required in Brucella abortus forvirulence and intracellular multiplication J Bacteriol 20001824849ndash55

[13] Comerci DJ Martınez-Lorenzo MJ Sieira R Gorvel JP Ugalde RA Essential roleof the VirB machinery in the maturation of the Brucella abortus-containingvacuole Cell Microbiol 20013159ndash68

[14] Hong PC Tsolis RM Ficht TA Identification of genes required for chronicpersistence of Brucella abortus in mice Infect Immun 2000684102ndash7

[15] Kahl-McDonagh MM Elzer PH Hagius SD Walker JV Perry QL Seabury CMet al Evaluation of novel Brucella melitensis unmarked deletion mutants forsafety and efficacy in the goat model of brucellosis Vaccine 2006245169ndash77

[16] OrsquoCallaghan D Cazevieille C Allardet-Servent A Boschiroli ML Bourg GFoulongne V et al A homologue of the Agrobacterium tumefaciens VirB andBordetella pertussis Ptl type IV secretion systems is essential for intracellularsurvival of Brucella suis Mol Microbiol 199961210ndash20

[17] Sun YH den Hartigh AB Santos RL Adams LG Tsolis RM virB-Mediatedsurvival of Brucella abortus in mice and macrophages is independent ofa functional inducible nitric oxide synthase or NADPH oxidase in macro-phages Infect Immun 2002704826ndash32

[18] Rolan HG den Hartigh AB Kahl-McDonagh M Ficht T Adams LG Tsolis RMVirB12 is a serological marker of Brucella infection in experimental and naturalhosts Clin Vaccine Immunol 200815208ndash14

[19] Boschiroli ML Ouahrani-Bettache S Foulongne V Michaux-Charachon SBourg G Allardet-Servent A et al The Brucella suis virB operon is inducedintracellularly in macrophages Proc Natl Acad Sci U S A 2002991544ndash9

[20] Fretin D Fauconnier A Kohler S Halling S Leonard S Nijskens C et al Thesheathed flagellum of Brucella melitensis is involved in persistence in a murinemodel of infection Cell Microbiol 20057687ndash98

[21] DelVecchio VG Kapatral V Redkar RJ Patra G Mujer C Los T et al Thegenome sequence of the facultative intracellular pathogen Brucella melitensisProc Natl Acad Sci U S A 200299443ndash8

[22] Delrue RM Deschamps C Leonard S Nijskens C Danese I Schaus JM et al Aquorum-sensing regulator controls expression of both the type IV secretionsystem and the flagellar apparatus of Brucella melitensis Cell Microbiol200571151ndash61

[23] Rambow-Larsen AA Rajashekara G Petersen E Splitter G Putative quorum-sensing regulator BlxR of Brucella melitensis regulates virulence factors includingthe type IV secretion system and flagella J Bacteriol 20081903274ndash82

[24] Baek SH Rajashekara G Splitter GA Shapleigh JP Denitrification genesregulate Brucella virulence in mice J Bacteriol 20041866025ndash31

[25] Haine V Dozot M Dornand J Letesson JJ De Bolle X NnrA is required for fullvirulence and regulates several Brucella melitensis denitrification genesJ Bacteriol 20061881615ndash9

[26] Loisel-Meyer S Jimenez de Bagues MP Basseres E Dornand J Kohler SLiautard JP et al Requirement of norD for Brucella suis virulence in a murinemodel of in vitro and in vivo infection Infect Immun 2006741973ndash6

[27] Kim KS Chilton WS Farrand SK A Ti plasmid-encoded enzyme required fordegradation of mannopine is functionally homologous to the T-region-enco-ded enzyme required for synthesis of this opine in crown gall tumors J Bac-teriol 19961783285ndash92

[28] Connolly JP Connolly JP Comerci D Alefantis TG Walz A Quan M et al Proteomicanalysis of Brucella abortus cell envelope and identification of immunogeniccandidate proteins for vaccine development Proteomics 200663767ndash80

[29] Wagner MA Eschenbrenner M Horn TA Kraycer JA Mujer CV Hagius S et alGlobal analysis of the Brucella melitensis proteome Identification of proteinsexpressed in laboratory-grown culture Proteomics 200221047ndash60

[30] Teixeira-Gomes AP Cloeckaert A Bezard G Dubray G Zygmunt MS Mappingand identification of Brucella melitensis proteins by two-dimensional elec-trophoresis and microsequencing Electrophoresi 199718(1)156ndash62

[31] Brazma A Hingamp P Quackenbush J Sherlock G Spellman P Stoeckert Cet al Minimum information about a microarray experiment (MIAME)-towardstandards for microarray data Nat Genet 200129365ndash71

[32] BRB array tools v 36 httplinusncinihgovBRB-ArrayToolshtml

Kegg Categories

Nu

mb

er o

f g

en

es

0

20

40

120

140

160Strongly expressedPoorly expressed

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Fig 1 Strongly and poorly expressed B abortus genes grouped by functional classifi-cation according to the KEGG pathway categories Columns 1 membrane transport 2xenobiotics biodegradation and metabolism 3 metabolism of amino acid and carbo-hydrate 4 metabolism of cofactors and vitamins 5 replication and repair 6 trans-lation and transcription 7 folding sorting and degradation 8 energy metabolism 9nucleotide metabolism 10 lipid metabolism 11 signal transduction 12 cellularprocesses 13 biosynthesis of polyketides and non-ribosomal peptides 14 glycanbiosynthesis and metabolism 15 unassigned


(Invitrogen) provides a flexible platform for ORFeome constructionallowing easy insert exchange between different vectors Thisapproach was first used to verify the genome annotation and tocreate a resource to functionally characterise the proteome ofCaenorhabditis elegans [3] ORFeomes can be used for multiplepurposes such as protein over expression mutant constructioninteraction mapping etc ORFeomes are also easily amenable toautomatizable procedures [4] Using the complete genomesequence of B melitensis Dricot et al [5] generated a database ofprotein coding ORFs and constructed an ORFeome library of 3091Gateway entry clones each containing a defined ORF Prior to highthroughput ORFeome cloning the overall quality of the B melitensisgenome annotation was validated [5] The new annotatedsequences are available at the lsquolsquoAlpha-proteobacterial genomeannotation databasersquorsquo [6] Due to the reported homogeneity ofBrucella genomes the B melitensis ORFeome can be used for anal-ysis of any of the Brucella species sequenced so far This first versionof the Brucella ORFeome (version 11) provides the codingsequences in a user-friendly format amenable to high throughputfunctional genomic and proteomic experiments The availability ofall ORFs in the same vector (pDONR201) facilitates and reduces thecosts of producing DNA microarrays by PCR since a single pair ofoligonucleotides is sufficient to amplify all the ORFs This BrucellaDNA microarray will be used to analyse gene expression and shouldhelp to provide a better understanding of the molecular mecha-nisms of virulence including the identification of bacterial regu-latory networks

2 Results and discussion

To construct the Brucella-specific whole-genome microarraythe B melitensis ORFeome (version 11) was used to generate PCRamplicons for every ORF as described in 51 The microarray alsoincluded several normalization controls that represented consti-tutively expressed Brucella genes [7] or negative controls withArabidopsis thaliana genes The rationale behind designing andutilizing PCR amplicons as probes was chosen for the availability ofall ORFs in the same vector (pDONR201) facilitating and reducingthe costs of producing microarrays since a single pair of oligonu-cleotides was required to amplify all the ORFs The microarray hada total of 7680 spots and represented over 964 of the completecoding sequences assigned to B melitensis A quality controlregarding the position intensity and morphology of the spots thebackground and the reproducibility of the arrays was performed byethidium bromide staining (not shown)

To test the usefulness of the newly constructed PCR Brucellamicroarray a direct analysis of the global transcriptional profile ofB abortus cells grown in laboratory conditions was made Thusthree independent biological replicas were made and following theRNA isolation samples were converted to aRNA and labelled withCy3 fluorescent dye The use of a single dye labelling approachallowed us an estimation of the genes more abundantly expressedin Brucella under laboratory conditions The evaluation experimentwas composed of three slides yielding six measurements per gene(representing three biological replicates since each gene is presenttwice on each slide) to confirm the reproducibility of the geneexpression data

The complete data set was sorted by the normalized and cor-rected fluorescence intensity values Table S1 (supplementalmaterial) represents the combined results of all experiments andshows the 10 of the total Brucella genome with the highest and thelowest signal values Highly expressed genes showed a Log2 signalintensity above 97 and the genes considered to show poorexpression showed a Log2 signal intensity below 59 The moreexpressed gene was an outer membrane protein (BAB1_0989)

described as Omp25b [8] with a Log2 signal intensity of 1359 andthe less expressed gene was an outer membrane autotransporter(BAB2_1107) with a Log2 signal intensity of 287 The gene for IF-1(BAB1_0282) used as marker for constituvely expression [7] in themicroarray showed a Log2 signal intensity of 102 indicating that itwas well expressed in B abortus

Fig 1 provides a summary of the strongly and poorly expressedgene sets grouped by KEGG pathway categories according to the Babortus genome annotation Almost 47 of the genes selected cor-responded to unassigned categories For genes of known or anno-tated function those encoding proteins involved in membranetransport amino acid and carbohydrate metabolism and cellularprocess were among the less expressed However genes encodingproteins involved in translation and transcription replication andrepair and metabolism of cofactors and vitamins were among themore expressed

An interesting observation from the data in Table S1 was thatgenes from the chromosome II were under represented among themore strongly transcribed genes while 35 of the B abortus genomecorrespond to chromosome II only 13 of the strongly expressedgenes were localised in this chromosome (Fig 2) On the other hand62 of the poorly expressed genes were localised in chromosome IILower expression of genes in chromosome II may simply reflect anasymmetry on the chromosome distribution of genes relevant forgrowth in laboratory conditions It is well known that the twochromosomes of the Brucellae differ in important ways ChromosomeII is enriched in genes encoding nutrient transport and metabolismgenes while most of the essential genes encoding for biosynthesis ofstructural components and replicative transcription and translationmachinery reside on chromosome I [9] In addition it has beendescribed that the origin of replication of the chromosome I (thelarge chromosome) is typical of bacterial chromosomes while that ofthe chromosome II (the small chromosome) is plasmid like In thisregard a hypothesis suggesting that the Brucella small chromosomeevolved from an ancestral megaplasmid has been proposed [10]

Data generated in the array were further validated by real-timequantitative reverse transcription-PCR (RT-PCR) as described in 44To analyze the correlation between the results obtained with thearray and RT-PCR the fluorescence intensity and threshold ampli-fication cycles were made relative to the values obtained for thereference constitutive gene IF-1 and compared with a Spearmanconcordance test A high level of correlation (rfrac14 082 plt 0001)was observed between array and RT-PCR data (Fig 3)









Ge

ne

s (

)

0

20

40

60

80

100

Chromosome II

Chromosome I


Poorlyexpresses





ORFs

BAB1

_124

6

BAB1

_083

2

BAB1

_126

4

BAB1

_024

6

BAB1

_054

4

BAB1

_023

9

BAB1

_117

4

BAB2

_052

7

BAB2

_006

6

BAB2

_006

1

BAB2

_092

8

BAB2

_052

8

BAB2

_052

2

BAB2

_014

8

BAB2

_095

4

BAB2

_013

0

Levels o

f exp

ressio

n

-8

-6

-4

-2

0

2

4RT-PCRMicroaray
























Table 1 (continued)












Table 1 (continued)







Inositolmetabolism



Table 1 (continued)











3 Conclusions
















Acknowledgements




References









































Ge

ne

s (

)

0

20

40

60

80

100

Chromosome II

Chromosome I


Poorlyexpresses





ORFs

BAB1

_124

6

BAB1

_083

2

BAB1

_126

4

BAB1

_024

6

BAB1

_054

4

BAB1

_023

9

BAB1

_117

4

BAB2

_052

7

BAB2

_006

6

BAB2

_006

1

BAB2

_092

8

BAB2

_052

8

BAB2

_052

2

BAB2

_014

8

BAB2

_095

4

BAB2

_013

0

Levels o

f exp

ressio

n

-8

-6

-4

-2

0

2

4RT-PCRMicroaray
























Table 1 (continued)












Table 1 (continued)







Inositolmetabolism



Table 1 (continued)











3 Conclusions
















Acknowledgements




References





















































Table 1 (continued)












Table 1 (continued)







Inositolmetabolism



Table 1 (continued)











3 Conclusions
















Acknowledgements




References





































Inositolmetabolism



Table 1 (continued)











3 Conclusions
















Acknowledgements




References


































3 Conclusions
















Acknowledgements




References




































Acknowledgements




References

































Documents

Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray