# ATP-Binding Cassette Systems ofBrucella

Authors: Dominic C. Jenner; Elie Dassa; Adrian M. Whatmore; Helen S. Atkins
Journal: Comparative and Functional Genomics
 (2009)

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## Abstract

Brucellosis is a prevalent zoonotic disease and is endemic in the Middle East, South America, and other areas of the world. In this study, complete inventories of putative functional ABC systems of fiveBrucella species have been compiled and compared. ABC systems of Brucella melitensis 16M, Brucella abortus 9-941, Brucella canis RM6/66, Brucella suis 1330, and Brucella ovis 63/290 were identified and aligned. High numbers of ABC systems, particularly nutrient importers, were found in all Brucella species. However, differences in the total numbers of ABC systems were identified (B. melitensis, 79; B. suis, 72; B. abortus 64; B. canis, 74; B. ovis, 59) as well as specific differences in the functional ABC systems of the Brucella species. Since B. ovis is not known to cause human brucellosis, functional ABC systems absent in the B. ovis genome may represent virulence factors in human brucellosis.


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## Body

## 1. Introduction

Brucella species are the causative agents of brucellosis, the world’s most prevalent zoonotic disease, with high occurrences in endemic areas including the Middle East, Asia, Mexico, and the Mediterranean [1]. The bacteria are small nonmotile, Gram-negative, nonspore-forming  coccobacilli that reside within the subphylum α-proteobacteria, which also includes nitrogen-fixing bacteria of the genus Nitrobacter, Rhizobium, Agrobacterium, and Rickettsia[2]. They are considered facultative intracellular pathogens.There are six traditionally recognisedBrucellaspecies that have different host preferences: Brucella melitensis(which usually infects sheep and goats), Brucella abortus (cattle), Brucella suis(pigs), Brucella ovis (sheep), Brucella canis(dogs), and Brucella neotomae(desert wood rats). Furthermore, there are three newly identified Brucellaspecies isolated from marine mammals: Brucella pinnipedialis (seals) [3],Brucella ceti(dolphins and porpoises) [3], and Brucella microti (voles) [4]. Although Brucellaare primarily animal pathogens causing infectious abortions in females and orchitis in males [5], four of the nine species may infect humans (B. melitensis, B. abortus, B. suis, and occasionally B. canis, in order of disease severity) causing a range of flu-like symptoms including fever, sweats, malaise, and nausea [6]. Transmission to humans takes place via three recognised channels: (i) the consumption of infected animal products, (ii) direct contact with infected animal birth products, and (iii) the inhalation of aerosolised Brucella. Due to the nature of the human disease and the ability to be infectious via aerosol, Brucella species have been classified as category B threat agents by the US Centre for Disease Control and Prevention (CDC) [7].Genome sequence analysis ofB. melitensis16M [8], B. suis 1330 [9], B. abortus 9-941 [10], B. canisRM6/66 (NCBI: NC_009504 and NC_009505, unpublished), and B. ovis 63/290 (NCBI: NC_010103 and NC_0010104, unpublished) has demonstrated the close relatedness of these organisms [11, 12]. The genomic DNA of each strain comprises two chromosomes of approximately 2.1 Mb and 1.2 Mb. DNA-DNA hybridisations between the species had previously revealed over 90% similarity between the species, leading to the suggestion that all Brucellaspecies should be classified as B. melitensis[13, 14]. However, it is widely believed that the differences in host specificity and pathogenicity are related to Brucella genetics; although there is currently little experimental evidence to support this, a few studies have found differences between the Brucella species genomes that may support this hypothesis [10, 15, 16]. A significant proportion of the Brucellagenomes appear to code for ATP-binding cassette (ABC) systems.ABC transporters are responsible for the import and export of many different substances across cellular membranes [17]. Although ABC transporters are extremely versatile, they all contain one defining feature, the ability to hydrolyse ATP to ADP, providing the energy needed for active transport. ABCs have three main conserved motifs known as Walker A (G-X-X-G-X-G-K-S/T, where X represents any amino acid residue), Walker B (ø-ø-ø-ø-D, where ø designates a hydrophobic residue), and a signature sequence (LSGGQ) [18]. The Walker A and Walker B motifs form tertiary structure enabling ATP-binding and can be found in all ATP-binding molecules. The signature sequence is well conserved in all ABC proteins and is also known as the linker peptide or C motif [19]. Although the configuration of ABC systems varies, the majority of ABC systems comprise of two hydrophilic ABC domains associated with two hydrophobic membrane-spanning domains (inner membrane (IM) proteins). Import systems are only found in prokaryotic organisms and contain both ABC domains and IM domains, along with extra-cytoplasmic binding proteins (BPs) designed to bind the specific allocrite of that ABC system. In Gram-negative bacteria the BPs are located in the periplasm whereas, in Gram-positive bacteria, they are anchored to the outer membrane of the cell via N-terminal lipid groups [20]. ABC systems import a diverse range of substrates into the bacterial cell including peptides [21], polyamines [22], metal ions [23], amino acids [24], iron [25], and sulphates [26]. In comparison, ABC systems involved in export functions usually contain only IM and ABC domains fused together via either the N-terminus (IM-ABC) or the C-terminus (ABC-IM), which homodimerise to create a functional system [27]. Substances exported by ABC transporters include antibiotics in both producing and resistant bacteria [28, 29], fatty acids in Gram-negative bacteria [27], and toxins [30]. In addition to transporters, many ABC proteins have roles in house-keeping functions, such as regulation of gene expression [31] and DNA repair [27, 32]. These proteins do not contain IM domains but are constituted of two fused ABC domains (ABC2) [27]. There is now increasing evidence that ABC systems can play roles in bacterial virulence [33–36] and can be used as targets for vaccine development [37].The recent sequencing of the genomes ofB. melitensis16M [8], B. abortus 9-941 [10], B. suis 1330 [9], B. ovis 63/290 (NCBI: NC_009504 and NC_009505, unpublished), and B. canis RM6/66 (NCBI: NC_010103 and NC_0010104, unpublished) has enabled the genomic comparison of different Brucella species. We report the creation and comparison of reannotated inventories of the functional ABC systems in Brucella. This improved annotation has assisted in understanding Brucella lifestyles and the identification of ABC systems that may be involved in virulence.


## 2. Methods

The prediction of ABC systems in sequenced bacterial genomes is based on annotation- and similarity-based homology assessment of identified or predicted ABC proteins from heterologous bacterial systems. The Artemis viewer (available fromhttp://www.sanger.ac.uk) was used to visualise the sequenced genomes of B. melitensis16 M, B. suis1330, B. abortus9-941, B. canisRM6/66, and B. ovis63/290 [8–10]. Using the annotated genomes, ABC proteins were searched for using an array of related words, specifically “ATP-binding cassettes,” “binding protein”, or “outer membrane protein.” For completeness all proteins that were labelled as hypothetical or conserved hypothetical proteins were also checked. Hits from this search were compiled and then genes upstream and downstream were also checked to ensure that all genes from one system were found. After the genome searches were completed, protein sequences were aligned using the basic local alignment search tool (BlastP) against other ABC proteins using the ABC systems: Information on Sequence Structure and Evolution (ABCISSE) database [27, 38]. The ABCISSE database comprises 24000 proteins from 9500 annotated systems over 795 different organisms. Proteins searched against ABCISSE that scored a threshold e-value of 10-6 were assigned to an ABC family and subfamily based on the hits from the ABCISSE database. Where searches on ABCISSE were unclear or hits for multiple families were produced, proteins were aligned using BlastP searches against the Genbank protein database. Use of this larger database increased the number of positive hits and functions that could be assigned. An ABC system was defined as a series of contiguous ORFs that shared the same family, subfamily, and substrate. A complete signal sequence (LSGGQ) was identified in the majority of the ABC proteins identified, and all of the other ABC proteins contained remnants of a complete signal sequence. Walker A and Walker B sequences were not sought during these searches.The ABC system inventories compiled in this study include systems that contain genes with predicted frame shift mutations and premature stop codons. For example, theB. melitensis 16M gene BMEII0099 is a known pseudogene with multiple premature stop codons. However, this gene is part of an ABC system that is encoded by another four genes (BMEII0098, BMEII00101, BMEII102, and BMEII0103), all of which are predicted to be functional; the mutation in BMEII0099 might render the whole system nonfunctional or it is possible that the other four genes could create a partially functional system. Due to the inability to determine the functionality of ABC systems using bioinformatic techniques, the ABC systems where one or more components were predicted to be nonfunctional were excluded from the total ABC system numbers and functions of the ABC systems. Within the genomes of all Brucella species single components of ABC systems (mainly BP) not attached to individual systems were located. These were included in ABC system inventories and termed lone components but were not included in total functional ABC system counts.


## 3. Results and Discussion

The genome structures ofBrucellaspecies are very similar [10–12], and although it is widely believed that the differences in Brucella species virulence and host preferences are related to their genetic composition,  there is little experimental evidence to support this belief. However, there are a few studies that have uncovered differences between the genomes [10, 15, 16]. In this study we have compared the presence of putative functional ABC systems in the genomes of B. melitensis16M (BM), B. suis1330 (BS), B. abortus 9-941(BS), B. canis RM6/66 (BC), and B. ovis63/290 (BO). In the original annotations of these genomes, a uniform nomenclature was not used and functional assignment of the systems varied considerably. Here we describe a reannotation of the ABC systems of these bacterial strains, leading to new predicted functions of the systems and predictions about how the individual components combine to form functional systems. Complete inventories of the ABC systems of BM, BS, BA, BC, and BO are shown in Table 1.Table 1
Reconstruction and comparative inventories ofBrucella ABC systems.
NumberFamilySubfamilySubstrate/FunctionTypeB. melitensisB. abortusB.suisB. ovisB. canis1ARTREGInvolved in gene expression regulationABC2BMEI0288BruAb11738BR1753BOV_1692BCAN_A17912ARTREGInvolved in gene expression regulationABC2BMEI0553BruAb11451BR1456BOV_1411BCAN_A14913ARTREGInvolved in gene expression regulationABC2BMEI1258BruAb10711BR0692BOV_0683BCAN_A07044CBYCBUCobalt importABCBMEI0635BruAb11365BR1368BOV_1324BCAN_A1395CBYCBUCobalt importIMBMEI0637BruAb11364BR1367BOV_1323BCAN_A1394, CbiQ5CCMPossibly heme exportIMBMEI1851BR0096, ccmCBOV_0094BCAN_A0098, ccmCCCMPossibly heme exportIMBMEI1852BR0095, ccmBBOV_0093BCAN_A0097, ccmBCCMPossibly heme exportABCBMEI1853BR0094, ccmABOV_0092BCAN_A0096, ccmA6CDIInvolved in cell divisionIMBMEI0073, ftsXBruAb11971BR1996BCAN_A2042CDIInvolved in cell divisionABCBMEI0072, ftsEBruAb11972, ftsEBR1997, ftsEBCAN_A2043, ftsE7CLSO antigen export systemABCBMEI1416, rfbBBR0519, rfbEBOV_0523BCAN_A0531, rfbBCLSO antigen export systemIMBMEI1415, rfbDBR0520, rfbDBOV_0524BCAN_A0532, rfbD8DLM (ABCY)D-L-Methionine and derivatives importLPPBMEI19549DLM (ABCY)D-L-Methionine and derivatives importIMBMEII0336BruAb20271BRA0962BOV_A0903BCAN_B0983DLM (ABCY)D-L-Methionine and derivatives importABCBMEII0337BruAb20272BRA0961BOV_A0902BCAN_B0982DLM (ABCY)D-L-Methionine and derivatives importLPPBMEII0338BruAb20273BRA096010DPLCYDCytochrome bd biogenesis and cysteine exportIM-ABCBMEII0761, cydCBruAb20713BRA0509BOV_A0443BCAN_B0508DPLCYDCytochrome bd biogenesis and cysteine exportIM-ABCBMEII0762, cydDBruAb20714, cydDBRA0508, cydDBOV_A0442BCAN_B0507, CydD11DPLMDLInvolved in mitochondrial export systemsIM-ABCBMEI0323, msbABruAb11700BR1715BOV_1657BCAN_A175312DPLHMTInvolved in mitochondrial export systemsIM-ABCBMEI0472BruAb11533BR1545BOV_1493BCAN_A1581DPLHMTInvolved in mitochondrial export systemsIM-ABCBMEI0471BruAb11534BR1544BOV_1494BCAN_A158213DPLPRTProteases, lipase, S-layer protein exportOMPBMEI1029, TolCBruAb10954BCAN_A095714DPLCHVBeta-(1–>2) glucan exportIM-ABCBMEI0984BruAb11004BR0998BCAN_A101515DPLHMTHeavy metal tolerance proteinIM-ABCBMEI1492BruAb10321BR0442BOV_0449BCAN_A044616DPLHMTInvolved in mitochondrial export systemsIM-ABCBMEI1743DPLHMTInvolved in mitochondrial export systemsIM-ABCBMEI1742BOV_019817DPLLIPInvolved in lipid A or polysaccharide exportIM-ABCBMEII0250BruAb20990BRA1050BOV_A0988BCAN_B107118DRIYHIHUnknownIMBMEI0656BruAb11347BR1349BOV_1307BCAN_A1377DRIYHIHUnknownIMBMEI0655DRIYHIHUnknownABC2BMEI0654BruAb11348BR1350 (ABC2-IM)BOV_1308BCAN_A1378DRIYHIHUnknownMFPBMEI0653BruAb11349BR1351BOV_1309BCAN_A137919DRIYHIHUnknownIMBMEII0801BruAb20757BRA0465BCAN_B0467DRIYHIHUnknownABCBMEII0802, drrABruAb20758BRA0464BOV_A0403BCAN_B0466DRIYHIHUnknownMFPBMEII0803BruAb20759BRA0463BOV_A0404BCAN_B046520DRINOSNitrous oxide reductionIMBMEII0970, nosYBruAb20902, nosYBRA0278, nosYBOV_A0254BCAN_B0280DRINOSNitrous oxide reductionABCBMEII0971, nosFBruAb20903, nosFBRA0277, nosFBOV_A0253BCAN_B0279DRINOSNitrous oxide reductionSSBMEII0972BruAb20904, nosDBRA0276, nosDBOV_A0252BCAN_B027821FAEFatty acid exportIM-ABCBMEI0520BruAb11484BR1490BCAN_A152822FAEFatty acid exportIM-ABCBMEII0976BruAb20908BOV_A0247BCAN_B027323HAABranched-chain amino acidsIMBMIE0258, LivHBruAb11771BR1790BOV_1725BCAN_A1829HAABranched-chain amino acidsIMBMIE0259, LivMBruAb11772BR1791BOV_1724BCAN_A1828HAABranched-chain amino acidsABCBMEI0260, braFBruAb11770BR1788BOV_1723BCAN_A1827HAABranched-chain amino acidsABCBMEI0261, braGBruAb11769BR1789BOV_1722BCAN_A1826HAABranched-chain amino acidsBPBMEI0263BruAb11765BR1785BOV_1720BCAN_A1823HAABranched-chain amino acidsBPBMEI0264BruAb11767BR1782BCAN_A1820HAABranched-chain amino acidsBPBMEI0265BOV_171924HAABranched-chain amino acidsBPBMEI1930BR0014BOV_0012BCAN_A001425HAABranched-chain amino acidsABCBMEII0065, livFBruAb20027BRA0028BOV_A0025BCAN_B0030HAABranched-chain amino acidsABCBMEII0066, livGBruAb20028BRA0027BOV_A0024BCAN_B0029HAABranched-chain amino acidsIMBMEII0067, livMBruAb20025BRA0026BOV_A0023BCAN_B0028HAABranched-chain amino acidsIMBMEII0068, livHBruAb20026BRA0025BOV_A0022BCAN_B0027HAABranched-chain amino acidsBPBMEII0069BruAb20024BRA0024BOV_A0021BCAN_B002626HAABranched-chain amino acidsABCBMEII0098BruAb21132BRA1197BOV_A1099BCAN_B1227HAABranched-chain amino acidsABCBMEII0099BruAb21133BRA1196BOV_A1098BCAN_B1226HAABranched-chain amino acidsIMBMEII0101BruAb21131BRA1194BOV_A1097BCAN_B1225HAABranched-chain amino acidsIMBMEII0102BruAb21130BRA1195BOV_A1096BCAN_B1224HAABranched-chain amino acidsBPBMEII0103BruAb21129BRA1193BOV_A0195BCAN_B122327HAABranched-chain amino acidsABCBMEII0119BruAb21111BRA1176BOV_A1079BCAN_B1207HAABranched-chain amino acidsIM-ABCBMEII0120BruAb21112BRA1175BOV_A1078BCAN_B1206HAABranched-chain amino acidsIMBMEII0121BruAb21110BRA1174BCAN_B1205HAABranched-chain amino acidsBPBMEII0122BruAb21109BRA1173BOV_A1076BCAN_B120428HAABranched-chain amino acidsIMBMEII0340BruAb20276BRA0957BCAN_B0977HAABranched-chain amino acidsIMBMEII0341BruAb20277BRA0956BOV_A0897BCAN_B0978HAABranched-chain amino acidsABCBMEII0342BruAb20278BRA0955BOV_A0896BCAN_B0976HAABranched-chain amino acidsABCBMEII0343BruAb20279BRA0954BOV_A0895BCAN_B0975HAABranched-chain amino acidsBPBMEII0344BruAb20280BRA0953BOV_A0894BCAN_B097429HAABranched-chain amino acidsABCBMEII0628BruAb20574BRA0652BOV_A0613BCAN_B0652HAABranched-chain amino acidsABCBMEII0629BruAb20575BRA0651BOV_A0614BCAN_B0651HAABranched-chain amino acidsIMBMEII0630BruAb20577BRA0650BOV_A0611BCAN_B0649HAABranched-chain amino acidsIMBMEII0632BruAb20576BRA0649BOV_A0612BCAN_B0650HAABranched-chain amino acidsBPBMEII0633BruAb20578BRA0648BOV_A0610BCAN_B064830HAABranched-chain amino acidsBPBMEII0875BruAb20801BRA0392BCAN_B0398HAABranched-chain amino acidsBPBMEII0868BruAb20809BRA0400BOV_A0343BCAN_B0395HAABranched-chain amino acidsABCBMEII0874BruAb20806BRA0395BOV_A0338BCAN_B0389HAABranched-chain amino acidsABCBMEII0873BruAb20807BRA0394BOV_A0337BCAN_B0396HAABranched-chain amino acidsIMBruAb20808BRA0393BOV_A0336BCAN_B039731ISB (ABCX)Iron/sulphur centre biogenesisCYTPBMEI1040BruAb10941BR0931ISB (ABCX)Iron/sulphur centre biogenesisCYTPBMEI1042BruAb10940BR0933ISB (ABCX)Iron/sulphur centre biogenesisABCBMEI1041BruAb10942BR093232ISVHIron-siderophores, VB12 and Hemin importABCBMEI0660BruAb11342BR1344BOV_1302BCAN_A1371ISVHIron-siderophores, VB12 and Hemin importIMBMEI0659BruAb11343BR1345BOV_1304BCAN_A1372ISVHIron-siderophores, VB12 and Hemin importOMRBMEI0657BruAb11344BR1347BOV_1306BCAN_A1374ISVHIron-siderophores, VB12 and Hemin importBPBMEI0658BruAb11345BR1346BOV_1305BCAN_A137333ISVHIron(III) dicitrate importBPBMEII0535BruAb20476BRA0756BOV_A0705BCAN_B0763ISVHIron(III) dicitrate importIMBMEII0536, fecDBruAb20477BRA0755BOV_A0704BCAN_B0764ISVHIron(III) dicitrate importABCBMEII0537, fecEBruAb20478BRA0754BOV_A0703BCAN_B076234ISVHIron(III) importABCBMEII0604BruAb20550BRA0678BOV_A0635BCAN_B0677ISVHIron(III) importIMBMEII0605, fatCBruAb20551BRA0676BOV_A0634BCAN_B0675ISVHIron(III) importIMBMEII0606, fatDBruAb20552BRA0677BOV_A0633BCAN_B0676ISVHIron(III) importBPBMEII0607BruAb20553BRA0675BOV_A0632BCAN_B067435METZinc importIMBMEII0176, ZnuBBruAb21061, ZnuBBRA1124, ZnuBBOV_A1029BCAN_B1152METZinc importABCBMEII0177, ZnuCBruAb21060, ZnuCBRA1123, ZnuCBOV_A1028BCAN_B1151METZinc importBPBMEII0178, ZnuABruAb21059, ZnuABRA1122, ZnuABOV_A1027BCAN_B115036MKLInvolved in toluene toleranceABCBMEI0964BruAb11025BR1020BOV_0987MKLInvolved in toluene toleranceIMBMEI0965, ttg2BBruAb1024BR1019BOV_0986MKLInvolved in toluene toleranceSSBMEI0963, ttg2CBruAb11026BR1021BOV_098837MOIThiamine importABCBMEI0283, thiQBruAb11744BR1759BOV_1698BCAN_A1798MOIThiamine importIMBMEI0284, thiPBruAb11743, thiPBR1758, thiPBOV_1696thiP, BCAN_A1797MOIThiamine importBPBMEI0285BruAb11744, thiBBR1757, thiBBOV_1695thiB, BCAN_A179638MOIPutrescine importBPBMEI0411, potFBruAb11599BR1612BOV_1556BCAN_A1649MOIPutrescine importABCBMEI0412BruAb11598BR1611BOV_1555BCAN_A1648MOIPutrescine importIMBMEI0413BruAb11596BR1609BOV_1554BCAN_A1647MOIPutrescine importIMBMEI0414BruAb11597BR1610BOV_1553BCAN_A164639MOISulphate importIMBMEI0675, cysWBruAb11328, cysW2BR1328, cysW2BOV_1288CysW, BCAN_A1353MOISulphate importIMBMEI0674, cysTBruAb11329BR1329BOV_1289CysT, BCAN_A1354MOISulphate importBPBMEI0673BruAb11330BR1330BOV_1290BCAN_A135540MOISulphate importABCBMEI1838 cysABruAb10107BR0110BOV_0107CysA, BCAN_A0113MOISulphate importIMBMEI1839, cysWBruAb10106BR0109, cysW1BOV_0106CysW, BCAN_A0112MOISulphate importIMBMEI1840, cystBruAb10105, cysTBR0108BOV_0105CysT, BCAN_A0111MOISulphate importBPBMEI1841BruAb10104BR0107BOV_0104BCAN_A011041MOIPhosphate importABCBMEI1986, pstBBruAb12116, pstBBR2141, pstBBOV_2056BCAN_A2185, pstBMOIPhosphate importIMBMEI1987, pstABruAb12114, pstCBR2139, pstCBOV_2055BCAN_A2184, pstAMOIPhosphate importIMBMEI1988, pstCBruAb12115, pstABR2140BOV_2054BCAN_A2183, pstCMOIPhosphate importBPBMEI1989BruAb12113BR2138BOV_2053BCAN_A212842MOIMolybdenum importABCBMEII0003, modCBruAb20090BRA0090, modCBOV_A0084BCAN_B0093, ModCMOIMolybdenum importIMBMEII004, modBBruAb20089BRA0089, modBBOV_A0083BCAN_B0092, ModBMOIMolybdenum importBPBMEII0005BruAb20088BRA0088, modABOV_A0082BCAN_B009143MOISpermidine/putrescine importABCBMEII0193, potABruAb21046BRA1107BCAN_B1129MOISpermidine/putrescine importIMBMEII0194, potBBruAb21044BRA1106BCAN_B1128MOISpermidine/putrescine importIMBMEII0195, potCBruAb21045BRA1105BCAN_B1127MOISpermidine/putrescine importBPBMEII0196BruAb21043BRA1104BCAN_B112644MOIUnknownBPBMEII0479BruAb20422BRA0810BOV_A0760BCAN_B0824MOIUnknownABCBMEII0481BruAb20423BRA0809BOV_A0759BCAN_B0823MOIUnknownIMBMEII0483BruAb20424BRA0807BOV_A0758BCAN_B0822MOIUnknownIMBMEII0484BruAb20425BRA0808BOV_A0757BCAN_B082145MOIIron(III) importBPBMEII0565BruAb20510BRA0720BOV_A0676BCAN_B0726MOIIron(III) importIM2BMEII0566BruAb20511BRA0719BOV_A0675BCAN_B0274MOIIron(III) importABCBMEII0567BruAb20512BRA0718BOV_A0674BCAN_B072546MOIIron(III) importABCBMEII0583BruAb20529BRA0701BOV_A0656BCAN_B0702MOIIron(III) importBPBMEII0584BruAb20530BRA0700BOV_A0655BCAN_B0703MOIIron(III) importIM2BMEII0585BruAb20531BRA0699BOV_A0654BCAN_B070147MOISpermidine/putrescine importIMBMEII0920, potCBruAb20852BRA0328BOV_A0303BCAN_B0331MOISpermidine/putrescine importIMBMEII0921, potBBruAb20853BRA0329BOV_A0302BCAN_B0330MOISpermidine/putrescine importABCBMEII0922, potABruAb20855BRA0327BOV_A0301BCAN_B0329MOISpermidine/putrescine importBPBMEII0923, potDBruAb20854BRA0326BOV_A0300BCAN_B032848MOIIron(III) importBPBMEII1120BruAb20113BRA0115BOV_A0105BCAN_B0119MOIIron(III) importIMBMEII1121, sufBBruAb20111BRA0114BOV_A0104BCAN_B0118MOIIron(III) importIMBMEII1122, sufBBruAb20112BRA0113BOV_A0103BCAN_B0117MOIIron(III) importABCBMEII1123, sufCBruAb20110BRA0112BOV_A0102BCAN_B011649New1UnknownIMBMEI0013BruAb12030BR2055BOV_1975BCAN_A2101New1UnknownABCBMEI0012BruAb12031BR2056BCAN_A2102New1UnknownBPBMEI0014New1UnknownBPBMEI001550MOSRibose importABC2BMEI0391BruAB11620, rbsA-2BR1632, rbsA-2BOV_1576BCAN_A1669MOSRibose importIMBMEI0392BruAB11619, rbsC-2BR1631, rbsC-2BOV_1575, rbsC2BCAN_A1668MOSRibose importBPBMEI0393BruAB11618BR1630BOV_1574BCAN_A166751MOSRibose ImportABCBMEI0665BruAb11337BR1339BOV_1299BCAN_A1367MOSRibose ImportIMBMEI0664BruAb11338BR1340BOV_1300BCAN_A1368MOSRibose ImportBPBMEI0663BruAb11340BR1342BOV_1301BCAN_A1369MOSRibose ImportBPBMEI0662BruAb1133552MOSRibose importBPBMEI1390BruAb10566, rbsB1BR0544, rbsB1BOV_0546 rbsB1BCAN_A0557MOSRibose importIMBMEI1391, rbsCBruAb10565, rbsC1BR0543, rbsC1BOV_0545 rbsC1BCAN_A0555MOSRibose importABC2BMEI1392, rbsABruAb10564, rbsA1BR0542, rbsA1BOV_0544 rbsA1BCAN_A0554, rsbA53MOSPossibly galactosideBPBMEII0083BruAb20010BRA0010BOV_A0007MOSPossibly galactosideABC2BMEII0085, mglABruAb20009BRA0009BOV_A0006MOSPossibly galactosideIMBMEII0086, mglCBruAb20007BRA0007BOV_A0005MOSPossibly galactosideIMBMEII0087BruAb20008BRA0008BOV_A000454MOSXylose importIMBMEII0144, xylHBruAb21089, xylHBRA1152, xylHBOV_A1057BCAN_B1181MOSXylose importABC2BMEII0145, xylGBruAb21088, xylGBRA1151, xylGBOV_A1056BCAN_B1180, xylGMOSXylose importBPBMEII0146, xylFBruAb21087, xylFBRA1150, xylFBOV_A1055BCAN_B1179, xylF55MOSRibose importABC2BMEII0300, rbsABruAb20239rbsA4BRA0995, rbsA4BOV_A0937BCAN_B1014MOSRibose importIMBMEII0301 rbsCBruAb20240,rbsC5BRA0993, rbsC5BCAN_B1013MOSRibose importIMBMEII0302 rbsCBruAb20239, rbsC4BRA0994, rbsC5BOV_A0935BCAN_B1012MOSRibose importBPBruAb20238BRA0996, rbsB3BOV_A0938BCAN_B101556MOSMonosaccharide importBPBMEII0360, chvEBruAb20296BRA0937BOV_A0879BCAN_B0957MOSMonosaccharide importABC2BMEII0361BruAb20297BRA0936BOV_A0878BCAN_B0956MOSMonosaccharide importIMBMEII0362BruAb20298BRA0935BOV_A0877BCAN_B095557MOSErythritol importABC2BMEII0432, rbsABruAb20371, rbsA3BRA0860, rbsA3BOV_A0807, rsbA3BCAN_B0877MOSErythritol importIMBMEII0433, rbsCBruAb20372, rbsC3BRA0859, rbsC3BCAN_B0876MOSErythritol importBPBMEII0435BruAb20373, rbsB2BRA0858, rbsB2BOV_A0805BCAN_B087558MOSGalactoside/Ribose importABC2BMEII0698BruAb20654BRA0570BOV_A0533BCAN_B0570MOSGalactoside/Ribose importIMBOV_A0534BCAN_B0567MOSGalactoside/Ribose importIMBMEII0700BruAb20655BRA0568BOV_A0535MOSGalactoside/Ribose importIMBMEII0701BruAb20656BRA0569BCAN_B0568MOSGalactoside/Ribose importBPBMEII0702BRA0567BOV_A0532BCAN_B056759MOSMonosaccharide importIMBMEII0981BruAb20913BRA0267BOV_A0242BCAN_B0268MOSMonosaccharide importABC2BMEII0982BruAb20914BRA0266BOV_A0241BCAN_B0267MOSMonosaccharide importBPBMEII0983BruAb20916BRA0265BOV_A0240BCAN_B026660o228UnknownIMBMEI0361O228UnknownMFPBMEI0359o228UnknownABCBMEI036061o228UnknownIMBruAb10085BR0087BOV_0085O228UnknownMFPBCAN_A1712o228UnknownABCBruAb10084BR0086BOV_0084BCAN_A171162o228UnknownMFPBruA11658BR1671BOV_1617o228UnknownIM-ABCBruA11657BR1670BOV_1616BCAN_A008763o228Lipoprotein release systemABCBMEI1138, LolDBruAb10838,LolDBR0824, LolDBOV_0818BCAN_A0839o228Lipoprotein release systemIMBMEI1139, LolEBruAb10837, LolEBR0823, LolEBOV_0817BCAN_A083864OPNDipeptide importABCBMEI0438, dppFBruAb11569BR1582BOV_1527BCAN_A1617OPNDipeptide importABCBMEI0437, dppDBruAb11570BR1583BOV_1528BCAN_A1618OPNDipeptide importIMBMEI0435, dppCBruAb11571BR1584BOV_1530BCAN_A1620OPNDipeptide importIMBMEI0436, dppCBruAb11572BR1585BOV_1529BCAN_A1619OPNDipeptide importBPBMEI0433, dppABruAb11573BR1586BOV_1531BCAN_A162165OPNOligopeptide importABC2BMEI1938, oppDBruAb10006BR0006BOV_0006BCAN_A0006OPNOligopeptide importBPBMEI1934BruAb10007BR0007BOV_0009BCAN_A0010OPNOligopeptide importBPBMEI1935BruAb10008BR0008BOV_0010BCAN_A0009OPNOligopeptide importIMBMEI1936, oppBBruAb10009BR0009BOV_0008BCAN_A0008OPNOligopeptide importIMBMEI1937, oppCBruAb10010BR0010BOV_0007BCAN_A000766OPNOligopeptide importABCBMEII0199, oppFBruAb21039BRA1100OPNOligopeptide importABCBMEII0200, oppDBruAb21040BRA1101BCAN_B1123OPNOligopeptide importIMBMEII0201, oppCBruAb21037BRA0099BCAN_B1122OPNOligopeptide importIMBMEII0202, oppBBruAb21038BRA0098BCAN_B1121OPNOligopeptide importBPBMEII01203BruAb21036BRA0097BCAN_B111967OPNDipeptide importABCBMEII0205, dppFBruAb21033BRA1095BOV_A0950BCAN_B1117OPNDipeptide importABCBMEII0206, dppDBruAb21034BRA1094BOV_A0951BCAN_B1116OPNDipeptide importIMBMEII0207, dppCBruAb21031BRA1093BCAN_B1115, dppCOPNDipeptide importIMBruAb21032BRA1092BOV_A0952OPNDipeptide importIMBMEII0209, dppBBOV_A0953BCAN_B1114OPNDipeptide importBPBMEII0210BruAb21030BRA1090BOV_A0954BCAN_B111368OPNDipeptide/ Oligopeptide importBPBMEII0217BruAb21024BRA1084BCAN_B1107OPNDipeptide/ Oligopeptide importIMBMEII0220BruAb21020BRA1081BCAN_B1104OPNDipeptide/ Oligopeptide importIMBMEII0221BruAb21021BRA1080BCAN_B1103OPNDipeptide/ Oligopeptide importABCBMEII0222BruAb21018BRA1079BCAN_B1102OPNDipeptide/ Oligopeptide importABCBMEII0223BruAb21019BRA1078BCAN_B110169OPNDipeptide importBPBMEII0284BruAb20952BRA1012BOV_A0504BCAN_B1032OPNDipeptide importIMBMEII0285BruAb20950BRA1009BOV_A0501BCAN_B1031OPNDipeptide importIMBMEII0286BruAb20951BRA1008BOV_A0502BCAN_B1030OPNDipeptide importABCBMEII0287BruAb20948BRA1011BOV_A0500BCAN_B1029OPNDipeptide importABCBMEII0288BruAb20949BRA1010BOV_A0501BCAN_B102870OPNNickel importBPBMEII0487BruAb20428BRA0804BOV_A0754BCAN_B0818, NikAOPNNickel importIMBMEII0488, nikBBruAb20429, nikBBRA0802, nikCBOV_A0752BCAN_B0817, NikBOPNNickel importIMBMEII0489, nikCBruAb20430, nikVBRA0803, nikBBOV_A0753BCAN_B0816, NikCOPNNickel importABCBMEII0490, nikDBruAb20431, nikDBRA0800, nikEBOV_A0751BCAN_B0815, NikDOPNNickel importABCBMEII0491, nikEBruAb20432, nikEBRA0801, nikDBCAN_B0814, NikE71OPNOligopeptide importBPBMEII0504BruAb20446BRA0783BOV_A0737BCAN_B0800OPNOligopeptide importIMBMEII0505BruAb20447BRA0788BOV_A0736BCAN_B0799OPNOligopeptide importIMBMEII0506BruAb20448BRA0787BOV_A0735BCAN_B0798OPNOligopeptide importABCBMEII0507BruAb20449BRA0786BOV_A0734BCAN_B0797OPNOligopeptide importABCBMEII0508BOV_A0733BCAN_B079672OPNOligopeptide importBPBMEII0691BruAb20648BRA0576BOV_A054273OPNOligopeptide importBPBMEII0734BruAb20684BRA0538BOV_A0468BCAN_B0538OPNOligopeptide importBPBMEII0735, oppABruAb20685BRA0537BOV_A0467BCAN_B0537OPNOligopeptide importIMBMEII0736BruAb20686BRA0536BOV_A0466BCAN_B0535OPNOligopeptide importIMBMEII0737BruAb20687BRA0535BOV_A0465BCAN_B0536OPNOligopeptide importABC2BMEII0738BruAb20688BRA0534BOV_A0464BCAN_B053474OPNOligopeptide importBPBMEII0859BruAb20792BRA0409BOV_A0352BCAN_B0412OPNOligopeptide importIMBMEII0860BRA0408BOV_A0351BCAN_B0411OPNOligopeptide importIMBMEII0861BruAb20794BRA0407BOV_A0350BCAN_B0410OPNOligopeptide importABCBMEII0863BruAb20796BRA0405BOV_A0347BCAN_B0408OPNOligopeptide importABCBMEII0864BruAb20797BRA0404BOV_A0348BCAN_B040775OSPMaltose importABCBMEI1713, malKBruAb10233BR0238BOV_0231BCAN_A0241OSPMaltose importIMBMEI1714, malGBruAb10231BR0237BOV_0230BCAN_A0240OSPMaltose importIMBMEI1715, malFBruAb10232BR0236BOV_0229BCAN_A0239OSPMaltose importBPBMEI1716BruAb10230BR0235BOV_0228BCAN_A023876OSPOligosaccharide or polyol importABCBMEII0112, ugpCBruAb21119BRA1183BOV_A1086BCAN_B1214OSPOligosaccharide or polyol importIMBMEII0113, ugpABruAb21118BRA1181BOV_A1085BCAN_B1213OSPOligosaccharide or polyol importIMBMEII0114, ugpEBruAb21117BRA1182BOV_A1084BCAN_B1212OSPOligosaccharide or polyol importBPBMEII0115BruAb21116BRA1180BCAN_B121177OSPOligosaccharide or polyol importIMBMEII0541BruAb20483BRA0749BOV_A0700BCAN_B0757OSPOligosaccharide or polyol importIMBruAb20482BRA0750BOV_A0699BCAN_B0756OSPOligosaccharide or polyol importBPBMEII0542BruAb20484BRA0748BOV_A0698BCAN_B0755OSPOligosaccharide or polyol importABCBMEII0544BruAb20487BRA0745BOV_A0696BCAN_B075378OSPOligosaccharide or polyol importBPBMEII0590BruAb20537BRA0693BOV_A0648BCAN_B0691OSPOligosaccharide or polyol importIMBMEII0591BruAb20538BRA0691BOV_A0647BCAN_B0690OSPOligosaccharide or polyol importIMBMEII0592BruAb20539BRA0692BOV_A0646BCAN_B0689OSPOligosaccharide or polyol importABCBMEII0593BruAb20540BRA0690BOV_A0645BCAN_B068879OSPSN-glycerol-3-phosphate importABCBMEII0621, ugpCBruAb20568, ugpCBRA0658, ugpCBOV_A0620BCAN_B0658OSPSN-glycerol-3-phosphate importIMBMEII0622, ugpEBruAb20569, ugpEBRA0657, ugpEBOV_A0619BCAN_B0657OSPSN-glycerol-3-phosphate importIMBMEII0623, ugpEBruAb20570, ugpABRA0656, ugpABOV_A0618BCAN_B0656OSPSN-glycerol-3-phosphate importIMBMEII0624, ugpAOSPSN-glycerol-3-phosphate importBPBMEII0625BruAb20571, ugpBBRA0655, ugpABOV_A0617BCAN_B065580OSPOligosaccharide or polyol importABCBMEII0750BruAb20702BRA0521BOV_A0454BCAN_B0520OSPOligosaccharide or polyol importIMBMEII0752BruAb20704BRA0519BOV_A0452BCAN_B0518OSPOligosaccharide or polyol importIMBMEII0753BruAb20705BRA0518BOV_A0451BCAN_B0517OSPOligosaccharide or polyol importBPBMEII0754BruAb20706BRA0516BOV_A0449BCAN_B0516OSPOligosaccharide or polyol importBPBMEII075581OSPMaltose importABCBMEII0940BruAb20874BRA0307BOV_A0282BCAN_B0308OSPMaltose importIMBMEII0942BruAb20875BRA0306BOV_A0281BCAN_B0307OSPMaltose importIMBMEII0943BruAb20876BRA0305BOV_A0280BCAN_B0306OSPMaltose importBPBMEII0944BOV_A0279OSPMaltose importBPBMEII0945BruAb20877BRA0304BCAN_B030582OTCNGlycine betaine/L-proline importABCBMEI0439, proVBruAb11568BR1581BOV_1526BCAN_A1616OTCNGlycine betaine/L-proline importIMBMEI0440, proWBruAb11567BR1580BOV_1525BCAN_A1615OTCNGlycine betaine/L-proline importBPBMEI0441, proXBruAb11566BR1579BOV_1524BCAN_A161483OTCNCholine SS-dependent regulation of yehZYXWBPBMEI1725BruAb10220BR0225BOV_0216BCAN_A0228OTCNCholine SS-dependent regulation of yehZYXWIMBMEI1726, proWBruAb10217BR1222BOV_0215BCAN_A0227OTCNCholine SS-dependent regulation of yehZYXWIMBMEI1728, proWBruAb10219BR0224BOV_0213BCAN_A0225OTCNCholine SS-dependent regulation of yehZYXWABCBMEI1727, proVBruAb10218BR0223BOV_0214BCAN_A022684OTCNOsmoprotectants, Taurine, Cyanante & NitrateBPBMEI1737BruAb10207BR0211BOV_0204BCAN_A0215OTCNOsmoprotectants, Taurine, Cyanante & NitrateIMBMEI1739BruAb10206BR0213BOV_0202BCAN_A0213OTCNOsmoprotectants, Taurine, Cyanante & NitrateABCBruAb1020885OTCNTaurine importBPBMEII0109BruAb21122BRA1186BOV_A1089BCAN_B1218OTCNTaurine importIMBMEII0107, tauCBruAb21124BRA1188BOV_A1091BCAN_B1219OTCNTaurine importABCBMEII0108, tauBBruAb21123BRA1187BOV_A1090BCAN_B121786OTCNGlycine betaine/L-proline importABCBMEII0548BruAb20492BRA0740BOV_A0692BCAN_B0748OTCNGlycine betaine/L-proline importIMBMEII0549BruAb20493BRA0739BOV_A0691BCAN_B0747OTCNGlycine betaine/L-proline importBPBMEII0550BruAb20494BRA0738BOV_A0690BCAN_B074687OTCNNitrate importBPBMEII0797BruAb20753BRA0469BOV_A0406BCAN_B0471OTCNNitrate importABCBMEII0798, nrtCBruAb20755BRA0467BOV_A0407BCAN_B0470OTCNNitrate importIMBMEII0799, nrtBBruAb20755BRA0468BOV_A0408BCAN_B046988OTCNTaurine importABCBMEII0961BruAb10894BRA0286BOV_A0262BCAN_B0288OTCNTaurine importIMBMEII0962BruAb10895BRA0285BOV_A0261BCAN_B0287OTCNTaurine importBPBMEII0963BruAb10896BRA0284BOV_A0260BCAN_B028689PAOPolar amino acid importABCBMEI0108BruAb11932BR1959BOV_A0336BCAN_A2004PAOPolar amino acid importABCBMEI0111BruAb11935BR1956BOV_1885BCAN_A2001PAOPolar amino acid importIMBMEI0112BruAb11931BR1955BOV_1882BCAN_A2000PAOPolar amino acid importIMBMEI0113BruAb11930BR1954BOV_1081BCAN_A1999PAOPolar amino acid importBPBMEI0114BruAb11929BR1953BOV_1880BCAN_A1998PAOPolar amino acid importBPBOV_187990PAOArginine/Ornithine biding protein precursorBPBruAb20594BOV_A0594PAOArginine/Ornithine biding protein precursorBPBMEI1022BruAb20595BRA0632BOV_A0593PAOArginine/Ornithine biding protein precursorBPBruAb10874BRA0631BOV_0945BCAN_A096791PAOGeneral L-amino acid importABCBMEI1208, appPBruAb10762BR0745BOV_A0890BCAN_A0760PAOGeneral L-amino acid importIMBMEI1209, appMBruAb10758BR0744BOV_0739BCAN_A0759PAOGeneral L-amino acid importIMBMEI1210, appQBruAb10760BR0743BOV_0737BCAN_A0758PAOGeneral L-amino acid importBPBMEI1211, appJBruAb10761BR0741BOV_0738BCAN_A0756PAOGeneral L-amino acid importBPBMEII0349, appJBruAb20285BRA0948BOV_0736BCAN_B096992PAOArginineBPBMEI1627BruAb10321BR0295BOV_030893PAOCystine importABCBMEII0599BruAb20545BRA0684BOV_A0640BCAN_B0682PAOCystine importIMBMEII0600BruAb20546BRA0683BOV_A0639BCAN_B0681PAOCystine importBPBMEII0601BruAb20547, fliYBRA0682, fliYBOV_A0638, fliYBCAN_B068094PAOPolar amino acid importIMBR0952BCAN_A0964PAOPolar amino acid importIMBR0953BCAN_A0965PAOPolar amino acid importBPBMEI1104BR0955BOV_085495PAOPolar amino acid importBPBR0862BOV_A090396UVRDNA repairABC2BMEI0878BruAb1110, UvrAUvrABOV_1063BCAN_A112497YHBGPossible LPS transport to outer membraneABCBMEI1790BruAb10153BR157BOV_0152BCAN_A0162YHBGPossible LPS transport to outer membraneSSBMEI1791BruAb10152BR156BOV_0151BCAN_A0161ABC: ATP-Binding Cassette; IM: Inner membrane protein; BP: Binding protein; IM-ABC: Inner membrane protein-ATP binding cassette fusion; ABC2: 2 ABC proteins fused together; OMP: Outer membrane protein; MFP: Membrane fusion protein; SS: Signal sequence; LPP: Extracytoplasmic protein with a lipoprotein type signal sequence; BM:Brucella melitensis;BA: Brucella abortus;BS: Brucella suis;Bold Text: Indicates a frame shift mutation or premature stop codon in these genes.TheBrucellastrains investigated in this study all have approximately 3.3 Mb genomes comprising two chromosomes of approximately 2.1 Mb and 1.2 Mb. The total number of predicted functional ABC systems encoded by the genomes of the Brucella strains is similar but does show some variability (BM = 79, BS = 72, BA = 64, BC = 74, BO = 59). Our evaluation of the Brucella genomes confirms that these species encode a relatively high proportion of ABC system genes when compared to other bacteria [39], with an average of 8.8% of their genomes dedicated to predicted functional ABC system genes (if lone components and mutated genes are included this figure increases to 9.3%). This may reflect their relatedness to environmental α-proteobacteria such as Nitrobacterand Agrobacterium which also encode high numbers of ABC systems [39] that may assist in their survival in diverse conditions.This work reports the first full inventories of ABC systems within five genome-sequencedBrucella strains. There are a number of specific ABC systems/genes that have previously been identified in the published literature. For example, Paulsen et al. describe two ABC systems that are present in B. suis and absent in B. melitensis. The first of these is an ABC importer encoded by BR0952 (IM), BR0953 (IM), and BR0955 (BP) [9]. Although this particular system is listed in the inventory, the ABC protein component of the system was not located in the BS genome and so this system was deemed incomplete and unlikely to be functional. The system was almost completely missing from the BM genome which is consistent with the findings of Paulsen et al. [9].  The second reported system is encoded by BRA0630, BRA0631, BRA0632, BRA0633, BRA0634, and BRA0635. However, when these genes were assessed using ABCISSE, only two of the five genes were predicted to be ABC transporter binding proteins (BRA0631 and BRA0632) and no other ABC components were located. Thus we deem this system also likely to be nonfunctional. Other genes that have been identified in the literature are BRA1080 (a dipeptide ABC transporter protein indentified in BS), BMEI1742 (a mitochondrial export ABC transporter identified in BM), and BRA0749-BRA0750 (involved in oligopeptide import) [10], all of which are present in our inventories.


## 4. ABC System Functions

In this study, we have classified the ABC systems of BM, BS, BA, BC, and BO into classes, families, and subfamilies according to the functional classification system described by Dassa and Bouige [27] (Table 2). The Brucellastrains encode 8–12 class 1 systems, characterised by an ABC-IM domain fusion and comprising predicted export systems, and 5 class 2 systems, characterised by a duplicated fused ABC and with predicted functions in antibiotic resistance and house-keeping functions. However, we have found that most of the ABC systems of Brucella species belong to class 3 with roles predicted in import processes. The further classification of BrucellaABC systems into families and subfamilies shows that there are a high number of ABC systems of specific importer families, particularly the MOI (minerals and organic ions), MOS (monosaccharide), OPN (oligopeptides and nickel), OSP (oligosaccharides and polyols), and OTCN (osmoprotectants taurine cyanate and nitrate) families, all of which primarily function to acquire nutrients.Table 2
ABC system families/subfamilies.
NameDescription and FunctionFamilySubfamilyExporters (predicted and experimental)DPL,Drugs, Peptides,HMTMitochondrial and bacterial transporters IILipidsCHVBeta(1–2) Glucan exportMDLMitochondrial and bacterial transporters ILIPLipid A or glycerophospholipid exportPRTProteases, Lipases, S-Layer protein exportCYDCytochrome bd biogenesisCCMCytochrome C biogenesisCLSCapsular polysaccharide, lipopolysaccharide and teichoic acidsFAEFatty Acid ExportImportersDLMD- L-Methionine and derivativesCBYCBUCobalt uptake, putativeMKLRelated to MOI family but unknown substrateYHBGRelated to HAA family, but unknown substrateCDICell divisionMETMetalsMOSMonosaccharidesMOIMineral and Organic ionsPAOPolar amino acids and OpinesHAAHydrophobic amino acids and amidesOSPOligosaccharides and polyolsOPNOligopeptides and NickelOTCNOsmoprotectants Taurine Cyanate and NitrateISVHIron-Siderophores VitaminB-12 and Hemincellular process (experimental)ISBIron-sulphur centre biogenesisART,Antibiotic resistance and translation regulationREGTranslation regulationUVRDNA repair and drug resistanceUnknownDRI,Drug resistance, bacteriocin, and lantibiotic immunityYHIHDrug resistance, putativeNOSPossible nitrous oxide reductionNOUnclassified Systemso228UnknownThe predicted functionality of the ABC systems within theBrucella genomes is dominated by ABC systems involved in the import of nutrients (Figure 1), and although this is not uncommon amongst bacteria, it is probable that Brucella species utilise ABC transporters to provide most of the nutrients they require [8, 39]. In support of the findings of Paulsen et al. [9], the 2.1 Mb chromosome encodes a large proportion of the ABC systems involved in molecular export and cellular process whereas the ABC systems located on the smaller chromosome are largely biased toward nutrient acquisition, leading to the idea that this second chromosome is important in the acquisition and processing of nutrients in Brucella.Figure 1
ABC system class/family-subfamilySince the ABC systems were identified by homology searches, it is possible to assign each ABC importer with a predicted substrate that it imports, providing an overview of the ABC system-based import ability of theBrucella species. Table 3 shows the range of predicted substrates imported via ABC transporters within the Brucella genomes. Overall, our results show that there is little difference in the import ability between strains of the four species of Brucella that are pathogenic to humans (BM, BS, BA, and BC). However, BO lacks the ability to import 8 of the 26 listed nutrients via ABC systems. In fact, all of the 29 pseudogenes that are present within the BO ABC system inventory occur within nutrient importers. The nutrients that BO appears to be unable to import using ABC systems include polyamines (specifically spermidine and putrescine), nickel, thiamine, glycine betaine, erythritol, xylose, and molybdenum. It is possible that the defective uptake of one or more of these substrates by B. ovismay contribute to its likely lack of virulence in humans. For example, polyamines have recently been associated with bacterial virulence and pathogenicity in human pathogens [40] and polyamine transporters have therefore been targeted as novel vaccine candidate targets for human pathogens [41, 42].Table 3
Brucella ABC import ability.
SubstrateB. melitensisB. abortusB. suisB. ovisB. canisBranch chain amino acids***************Iron (III)********************Cobalt—****Zinc*****Thiamine***—*Putrescine*******—**Sulphate**********Phosphate*****Molybdenum***—*Spermidine*****—*Ribose***************Galactoside—*******Xylose***—*Erythritol***—*Dipeptides***********Oligopeptides******************Nickel*—*—*Maltose*****Oligosaccharide or polyol***********SN-glycerol-3-phosphate****—Taurine*************Glycine betaine*—*—*Nitrate*****Polar amino acids———**Cystine*****General L amino acids*—***This table does not include any ABC system with pseudogenes present. ****>5 functional systems, ***3 or 4 functional systems, **2 functional systems, *1 functional system, — No functional systems.Two predicted erythritol transport systems have been reported that have yet to be confirmed by experimental data [8, 43]. Although the erythritol transporter identified in this study has also been identified by Crasta et al. [43], it should be noted that B. abortus S19 has this transport system inactivated by pseudogenes and yet it is still able to incorporate erythritol [43], indicating that this ABC system might not be wholly responsible for erythritol transport. Another study has demonstrated that B. ovis does not utilise erythritol as readily as other sugars [44].In this study we have identified one ABC system in BM that we have categorised within a new ABC system family (currently labelled NEW1; See Table1). This system includes BP and IM proteins related to those of the MOS family and ABC proteins that are different to those from the MOS family. We previously identified a similar ABC system in the genomes of Burkholderia pseudomalleiand Burkholderia malleistrains [45]. Clearly, experimental data is required to define the function of this system.


## 5. Differences betweenBrucella Species

Although there is similarity between the ABC system inventories of theBrucella strains studied in this work, we have identified systems that are absent in one or several Brucella species (Table 4). The systems that are absent from species are not critical for bacterial survival but could contribute to differences in the lifestyles or virulence of the Brucella species. Our data shows that there are ABC systems absent from all of the Brucella strains studied. In particular, BO (5 systems), BC (4 systems), and BA (4 systems) lack systems that are present in BM and/or BS. The absence of the ISB (formally ABCX) system from BO and BC is an interesting observation since the ISB systems are soluble complexes involved in labile [Fe-S] biogenesis, which is important in resistance to oxidative stresses. This could indicate that B. ovis and B. canis reside in environments that are low in oxygen or high in oxygen reducatants, or that they lack enzymes that need labile [Fe-S] centres [46, 47]. Furthermore, this difference may be a factor contributing to the reduced virulence for humans of B. ovis and B. canis when compared to B. melitensis, B. suis,and B. abortus. The CDI system absent from B.ovis is comprised of two proteins, FtsE (ABC protein) and FtsX (IM protein) [48], and has been studied in E. coli and other bacteria including Bacillus subtilis [49] and Mycobacterium tuberculosis [50]. This CDI system is involved in cell division. E. coli mutants of ftsE show a reduced growth capacity [51]. The MKL system absent from BC may play a role in toluene tolerance, since Tn5 insertions within the ttgA2gene coding for the MKL ABC protein in Pseudomonas putida elicited a toluene-sensitive phenotype [52].Table 4
ABC system genes absent in at least one species when compared toB. melitensis.
NumberFamilySubfamilySubstrate/ FunctionTypeB. melitensisB. abortusB. suisB. ovisB. canisIMBMEI1851-+++5CCMPossibly heme exportIMBMEI1852-+++ABCBMEI1853-+++6CDIInvolved in cell divisionIMBMEI0073, ftsX++-+ABCBMEI0072, ftsE++-+7CLSO antigen export systemABCBMEI1416, rfbB-+++IMBMEI1415, rfbD-+++13DPLPRTProteases, lipase, S-layer protein exportOMPBMEI1029+--+14DPLCHVBeta-(1→2) glucan exportIM-ABCBMEI0984++-+16DPLHMTInvolved in mitochondrial export systemsIM-ABCBMEI1743----IM-ABCBMEI1742--+-22FAEFatty acid exportIM-ABCBMEII0976+-++CYTPBMEI1040++--31ISB (ABCX)Iron/sulphur centre biogenesisCYTPBMEI1042++--ABCBMEI1041++--ABCBMEI0964+++-36MKLInvolved in toluene toleranceIMBMEI0965, ttg2B+++-SSBMEI0963, ttg2C+++-IMBMEII0087+++-IMBMEI0361----60o228UnknownMFPBMEI0359----ABCBMEI0360----IM-BruAb10085++-61o228UnknownMFP----BCAN_A1712ABC-BruAb10084+++62o228UnknownMFP-££BOV_1617-IM-ABC-££+BCAN_A0087Excludes ABC systems involved in import;-: gene absent in the Brucella species; +: gene present in the Brucellaspecies; £: pseudogene present in the Brucellaspecies; Number: refers to ABC system number in the full inventories/alignments of Brucella ABC systems


## 6. Conclusions

In this study the ABC systems ofB. melitensisstrain 16 M, B. suisstrain 1330, B. abortus9-941, B. canisstrain RM6/66, and B. ovisstrain 63/290 have been reannotated using the ABCISSE database in order to provide a new and improved set of annotated Brucella ABC systems for the strains studied. The information obtained and the uniform annotation and classification of ABC systems in these closely related species has enabled a more detailed analysis of the roles of ABC systems in Brucella species, contributing to an improved understanding of Brucella lifestyle and pathogenicity. Previous analysis of the Brucella genomes has shown that there is over 90% genome similarity between the Brucella species [13, 14]. Similarly, the ABC system inventory compiled in this work reflects the close similarities of the Brucella species. However, despite the high genetic homology of Brucella, this work highlighted differences in the predicted numbers and functions of the ABC systems encoded by each Brucella species. It is widely accepted that the three species that may cause the most human brucellosis are B. melitensis, B. suis, and B. abortus(and occasionally B. canis). This study has shown that these four species of Brucella have a larger set of ABC systems encoded within their genomes than B. ovis, which is not known to cause human disease. Although it is difficult to ascertain the exact effect of the loss of these ABC systems on B. ovis, it is possible to hypothesise that, along with other genetic differences observed [15], they contribute to its overall reduced virulence in humans. It should also be noted there that four further Brucella strains have been genome sequenced since this work was completed: B. melitensis63/9, B.  abortus2308, B. abortusS19, and B. suis Thomsen. Compiling ABC systems inventories of these strains may identify further differences between strains that may have biological relevance. Among the newly sequenced strains are B. suis Thomsen, a strain which is not known to cause disease in humans, and B. abortus S19, a vaccine strain. ABC system inventories of these strains would be of particular interest since they are considered less pathogenic than the wild-type strains and yet the reasons for this lack of pathogenicity are currently unknown. Overall, the identified differences observed in the ABC system inventory of the Brucellastrains studied should contribute to a greater understanding of differences in the lifestyles of the Brucella species.



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*Source: 354649-2010-02-11.xml*