Transport Capabilities of Eleven Gram-positive Bacteria:Comparative Genomic Analyses

Graciela L. Lorca†,1, Ravi D. Barabote1, Vladimir Zlotopolski, Can Tran, Brit Winnen, RikkiN. Hvorup, Aaron J. Stonestrom, Elizabeth Nguyen, Li-Wen Huang, David S. Kim, and MiltonH. Saier Jr.*Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116

AbstractThe genomes of eleven Gram-positive bacteria that are important for human health and the foodindustry, nine low G+C lactic acid bacteria and two high G+C Gram-positive organisms, wereanalyzed for their complement of genes encoding transport proteins. Thirteen to eighteen percent oftheir genes encode transport proteins, larger percentages than observed for most other bacteria. Allof these bacteria possess channel proteins, some of which probably function to relieve osmotic stress.Amino acid uptake systems predominate over sugar and peptide cation symporters, and of the sugaruptake porters, those specific for oligosaccharides and glycosides often outnumber those for freesugars. About 10% of the total transport proteins are constituents of putative multidrug efflux pumpswith Major Facilitator Superfamily (MFS)-type pumps (55%) being more prevalent than ATP-binding cassette (ABC)-type pumps (33%), which, however, usually greatly outnumber all othertypes. An exception to this generalization is Streptococcus thermophilus with 54% of its drug effluxpumps belonging to the ABC superfamily and 23% belonging each to the Multidrug/Oligosaccharide/Polysaccharide (MOP) superfamily and the MFS. These bacteria also display peptide efflux pumpsthat may function in intercellular signalling, and macromolecular efflux pumps, many of predictablespecificities. Most of the bacteria analyzed have no pmf-coupled or transmembrane flow electroncarriers. The one exception is Brevibacterium linens, which in addition to these carriers, also hastransporters of several families not represented in the other ten bacteria examined. Comparisons withthe genomes of organisms from other bacterial kingdoms revealed that lactic acid bacteria possessdistinctive proportions of recognized transporter types (e.g., more porters specific for glycosides thanreducing sugars). Some homologues of transporters identified had previously been identified onlyin Gram-negative bacteria or in eukaryotes. Our studies reveal unique characteristics of the lacticacid bacteria such as the universal presence of genes encoding mechanosensitive channels,competence systems and large numbers of sugar transporters of the phosphotransferase system. Theanalyses lead to important physiological predictions regarding the preferred signalling and metabolicactivities of these industrially important bacteria.

KeywordsLactic acid bacteria; transport proteins; genomic analyses; energetics

*Corresponding author: Phone: 858-534-4084, Fax: 858-534-7108, E-mail: msaier@ucsd.edu.†Current address: Department of Microbiology and Cell Science, University of Florida, Museum Road 1052, Gainesville, FL 326111These two authors contributed equally to the work reported.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptBiochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

Published in final edited form as:Biochim Biophys Acta. 2007 June ; 1768(6): 1342–1366. doi:10.1016/j.bbamem.2007.02.007.

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1. IntroductionMembrane transport systems catalyze the uptake of essential nutrients, ions and metabolites,as well as the expulsion of toxic compounds, cell envelope macromolecules, secondarymetabolites and the end products of metabolism. Transporters also participate in energygeneration and interconversion, and they allow communication between cells and theirenvironments. Transport is therefore essential to virtually every aspect of life.

Many Gram-positive bacteria are important in the food industry (see Table 1). These bacteriaare used for the fermentation of vegetables and fruits, for the conversion of dairy products intocheeses and yogurt, and for the preparation of beer and wine. A key feature of lactic acidbacteria (LABs) when grown in laboratory growth media that lack hematin and relatedcompounds is the inability to synthesize porphyrin (e.g., heme). Consequently, LABs grownunder these conditions are devoid of “true” catalase activity and cytochromes. They lackelectron transfer chains and rely on fermentation (i.e., substrate level phosphorylation) for thegeneration of energy. However if hematin is added to the growth medium, catalase andcytochromes may be formed, in some cases resulting in respiration [1–3].

Some LABs are both useful and harmful. For example, Lactobacillus brevis promotes both thepreparation and the spoilage of beer [4]. Other Gram-positive bacteria, such as Lactobacilluscasei, Lactibacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium longum, inaddition to being useful in the food industry, are natural inhabitants of the humangastrointestinal (GI) tract where they synthesize probiotic-like substances. These compoundsstimulate the immune system, provide key nutrients and promote the development of a healthyorganism [5].

The Lactic Acid Bacterial Genomics Consortium has recently carried out genome sequencingof eleven Gram-positive bacterial species [6–9]. The initial shotgun sequencing was conductedat the Joint Genome Institute in Walnut Creek, CA, and genome closure was achievedsubsequently [9]. Nine of the sequenced bacteria are low G+C Gram-positive lactic acidbacteria (LABs) (Pediococcus pentosaceus, Lactobacillus brevis, L. casei, L. delbruekii, L.gasseri, Lactococcus lactis, subspecies cremoris, Streptococcus thermophilus, Leuconostocmesenteroides and Oenococcus oeni) while two of them (Bifidobacterium longum andBrevibacterium linens) are high G+C Gram-positive bacteria. These bacteria exhibitconsiderable diversity with respect to their ecological habitats, being found in milk, meat,plants, the GI tracts of humans and other animals, and elsewhere (see Table 1). They also differwith respect to their carbohydrate metabolic pathways (Table 2). Little is known about thetransport capabilities of these important organisms.

In this review we report comprehensive analyses of the transport capabilities encoded withinthe eleven Gram-positive bacterial genomes noted above. Identification of all homologues ofrecognized transport proteins included in the transporter classification database (TCDB) [6,7] and topological prediction were achieved using established methods [10]. The identifiedtransporters were grouped according to topology, class, and function. The most importantconclusions are summarized in this report.

2. Computer MethodsGenomes of the eleven Gram-positive bacteria were screened for homologues of all proteinscontained in TCDB, a membrane transport protein classification database(http://tcdb.ucsd.edu) [6,11]. FASTA-formatted protein sequences of the completed genomeswere used. Each putative open reading frame was used as a query in the BLAST software[12,13] to search for homologues of proteins in the TCDB sequence library. In addition, each

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ORF was scanned with HMMTOP [14] to predict the number of putative transmembranesegments as reported in Table 3.

All of the potential transport proteins thus identified were subsequently examined in greaterdetail. Each transport protein identified was classified on the basis of sequence similarity intofamilies and subfamilies of homologous transporters based on the classification systempresented in TCDB. Each family/subfamily was noted with its standard abbreviation, its TCnumber and its typical substrates. The substrate specificities of particular homologuesidentified in the sequenced genomes have been predicted based on homology to functionallycharacterized genes and from their genomic context (see Table 1). Assignment to a family orsubfamily within the Transporter Classification System usually allows specification ofsubstrate type with high confidence [6,7,11]. The genome sequencing projects are describedin Klaenhammer et al. [8] and Makarova et al. [9]. Phylogenetic trees (Neighbor joining) werebased on multiple alignments generated with the Clustal X program [15] and drawn using theTreeView program [16].

Some readers may be interested in greater detail than provided in this paper regarding theidentified transporters and the families/superfamilies to which they belong. These readers areadvised to read this paper with the Transporter Classification Database (TCDB) at hand. Thespecificities, polarities of transport, mechanisms of transport, and transport protein and familydescriptions are provided in detail therein. Novel substrates are continually being discoveredfor many transporters, especially drug exporters, some of which function physiologically inthe absence of drugs. Moreover, some transport systems, initially characterized as narrowspecificity systems, prove to exhibit broad specificities when examined in greater detail. Whensuch substrates are known, they can be identified by searching TCDB, a database which iscontinually being updated as new experimental data become available. Table 5 provides ahelpful guide as to where in TCDB to look for specific information. Because of the continualupdating of TCDB, the interested reader who uses TCDB in conjunction with this paper willbe up to date for years to come.

Thousands of published papers have described the functional characterization of transportsystems and their constituent proteins. Several thousand such references can be found in TCDB.It is not possible to include them all in a review such as this one. We apologize to any researcherwhose work we have mentioned without providing the reference(s) to the experimental work.

3. Genome StatisticsBetween 89 and 95% of all predicted transport proteins for the various genomes had significantmatches (e-values <10−3) in TCDB. Remaining predicted transmembrane transport proteinsproved to be more distantly related to proteins included in TCDB. Some of these distantlyrelated proteins could not be classified into the established TC families/subfamilies. They weretherefore listed under the “Not Assigned” (N/A) category (see Table 4). The percentage ofrecognized genes that are predicted to encode transport proteins varies according to themicroorganism analyzed between 12.8% (Lactococcus lactis spp. cremoris) and 18.0%(Oenococcus oeni). Percent transporters encoded within the various genomes showed nocorrelation with genome size (Table 1).

4. Transporter ClassificationFive distinct well-defined types of functionally characterized transport systems, based uponmode of transport and energy coupling mechanism, are recognized by the TC system [6,7,11]. These classes are: (1) channels, (2) secondary carriers, (3) primary active transporters, (4)group translocators of the sugar transporting phosphoenolpyruvate-dependentphosphotransferase system, and (5) transmembrane electron flow carriers. Of these primary

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classes, classes 1, 3 and 5 are subdivided (see Table 4). In addition, class 8 is reserved foraccessory proteins involved in transport while class 9 specifies incompletely characterizedtransport systems. Class 9 is subdivided into classes 9A (recognized transporters of unknownmechanism) and 9B (putative transporters).

5. Organismal RepresentationTable 1 presents the eleven organisms included in this study. Two of these organisms,Bifidobacterium longum (Blo) and Brevibacterium linens (Bli) are high G+C Gram-positivebacteria and therefore in a bacterial phylum different from that of the other nine organisms,which are classified as low G+C Gram-positive lactic acid bacteria. As expected, thephylogenetic tree based on the 16S rRNAs of the eleven organisms (Fig. 1) shows Blo and Bliclustering loosely together, distant from all other organisms.

Two other organisms cluster together and separately from all others. These are Leuconostocmesenteroides (Lme) and Oenococcus oeni (Ooe) (Fig. 1). Moreover, situated in between thiscluster and the large cluster of remaining lactic acid bacteria is a cluster including Lactococcuslactic spp. cremoris (Lcr) and Streptococcus thermophilus (Sth). Of the five remaining closelyrelated organisms, Pediococcus pentosaceous (Ppe), Lactobacillus brevis (Lbr), andLactobacillus casei (Lca) cluster together as do L. delbruckii (Lde) and L. gasseri (Lga). Alltables included in this study will have the organisms presented according to their phylogeneticgroupings starting with Ppe and proceeding clockwise around the tree shown in Figure 1.

Table 1 presents the approximate genome sizes, G+C content, numbers of identified genes,and percent transport proteins (TP) available for analysis. As can be seen from Table 1, mostof the genomes sequenced are about 2 Mbp in size (1.7–2.8 Mbp), corresponding to 1788–2900 recognized genes, but that of Bli proved to be substantially larger (4.0 Mbp) with 4040genes. Between 12.8 and 18.0% of these genes encode recognizable transport proteins. Exceptfor the two high G+C Gram-positive bacteria, which have 60–62% G+C content, these bacteriahave G+C contents ranging from 35 to 49%.

Table 2 summarizes some of the key properties of these bacteria, particularly with respect tocarbohydrate utilization. This table summarizes the modes of sugar fermentation utilized andalso presents other key properties, particularly those of commercial significance.

6. Topological Distribution Among Transport Proteins (TPs)von Heijne and his associates have published several predictions and statistical analyses oftransmembrane protein topologies [17,18] in specific organisms such as Saccharomycescerevisiae [19], E. coli [20], and over 50,000 bacterial inner membrane proteins [21]. Theseand other studies have revealed that for certain classes and families of transporters, topologicalpredictions are surprisingly reliable although they are much less so for some other families oftransport proteins [22]. The topological results reported here for the 11 Gram-positive bacteriaexamined in this report are in general agreement with previous analyses for transport proteinsin other bacteria [6,23–30].

Table 3 and Figure 2 present the predicted topologies of the transport proteins (TPs) identifiedon the basis of their homology to entries in TCDB. The percentages of these proteins in generaldecrease as the numbers of TMSs increase from 0 to 3; then they increase as the numbers ofTMSs increase from 3 to 6; then they decrease again as the numbers of TMSs increase from 6to 9 before again increasing. There are consistently more proteins with even numbers of TMSsthan odd numbers of TMSs (Fig. 2). For example, there are more 10 TMS proteins than 9 or11 TMS proteins, far more 12 TMS proteins than 11 or 13 TMS proteins, and more 14 TMSproteins that 13 or 15 TMS proteins. These facts undoubtedly reflect the mechanisms by which

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these proteins arose through evolutionary history, mechanisms that involved intragenicduplication [28]. Only Bli has proteins predicted to have homologues of 17 and 19–25 TMSs(see Table 3), while only Ppe and Lca have proteins with 18 putative TMSs (one such proteinper organism).

The greatest variation between the organisms with respect to the numbers of TP homologueswith a single predicted topology are the 5 and 12 TMS proteins. Bli has the most predicted 12TMS TPs (18.8%) and the least 5 TMS TPs (2.4%) while Lde and Sth have the reciprocalrelationship: 10.0% and 7.4% 5 TMS proteins but only 4.1% and 4.9% 12 TMS proteins,respectively. This correlates with the percentages of integral membrane proteins of MFS-typesecondary active transporters versus ABC-type primary active transporters (see below). ABCuptake systems most commonly have integral membrane constituents of 5 TMSs perpolypeptide chain while MFS permeases usually have 12. It is noteworthy that proportions of5 or 12 TMS proteins in Bli and Lde relative to the other organisms do not correlate with therelative proportions of 4 and 6 TMS proteins, or 11 and 13 TMS proteins, respectively. Thisfact suggests that the TMS predictions are remarkably accurate, at least for the members of theMFS and ABC superfamilies. Since Lde (with 4.1% of its TPs having 12 TMSs) clusters inFig. 1 with Lga (with 11.7% of its TPs having 12 TMSs), it is clear that TP topologicaldistribution does not correlate with organismal phylogeny. The percentage of 12 TMS TPs forLga is the fifth largest of the eleven organisms examined (Table 3). Similarly, although Lbrand Lca cluster together in Figure 1, they exhibit about a 2-fold difference in numbers of 12TMS proteins. Based on these analyses, we conclude that transporter topology (Table 3) andmode of energy metabolism (Table 2) do not correlate well with organismal phylogeny (seebelow).

Three additional observations are worthy of note: (1) Blo has 16.7% 6 TMS proteins, morethan any other organism examined; (2) Lde, which has the greatest percentage of 5 TMSproteins but the lowest percentage of 12 TMS proteins, also has the lowest percentage of 8TMS proteins (Fig. 2), and (3) only Bli has proteins of greater than 18 TMSs. These facts mustall be explained in terms of the types of transporters employed by these organisms.

7. Classes of TPs in the 11 organismsTable 4 summarizes the distribution of TPs in the seven TC categories, and these values fordefined classes 1–4 are shown in bar graphs in Figure 3. Predicted transport proteins that couldnot be classified with certainty into specific TC families are indicated in the Not Assigned (N/A) category (Table 4). All organisms have only a small percentage of their TPs as channelproteins (5–14 proteins per organism or 1.7–4.1%). Almost all of these channel proteins areα-helical type channels of subclass 1.A. No β-type channels of class 1.B were detected, andvery few channel-forming toxins (class 1.C) were identified; none of the latter was found forLbr, Lca, Lde, Sth, Ooe, Blo and Bli. Ppe and Lcr each have 1, Lga has 2 and Lme has 6. Thepresence of class 1.A α-type channel proteins in these Gram-positive bacteria may relate to theneed of these organisms to respond to stress such as osmotic shock [31]. Channel-formingproteins will be analyzed in greater detail below.

Secondary carriers (such as major facilitator superfamily (MFS) carriers; class 2) usuallyconsist of single polypeptide chains, but primary active transporters (such as ABC carriers;class 3) usually consist of heterooligomeric complexes. The numbers of TPs listed in class 2therefore probably accurately reflect the numbers of secondary carriers, but the numbers ofTPs listed in class 3 are greatly in excess of the numbers of transport systems representedbecause of their multicomponent nature, probably by a factor of 3 or more. These facts mustbe taken into account when evaluating the data presented in Table 4 and Figure 3. Bli has byfar the highest class 2/3 ratio while Sth and Lde have by far the lowest, in agreement with the

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topological analyses discussed above. Excluding these three extreme organisms, this ratio forthe remaining eight organisms studied differs by less than a factor of two.

Almost all of the primary active transport proteins are ATP hydrolysis-driven systems of class3.A. Only one organism (Lca) has the transmembrane β-subunit of a decarboxylase-driventransporter, and consequently only Lca is predicted to have a decarboxylase-driven Na+ pumpof class 3.B. Only five organisms (Lbr, Lcr, Ooe, Blo and Bli) have subunits homologous tothose of some form of oxidoreductase-driven cation transporting systems (class 3.D). However,since most of these electron transfer complexes are multisubunit, it is possible that only Blipossesses complete proton pumping electron flow systems (see Table 5).

Class 4 proteins (PTS) show tremendous variation in representation among the elevenorganisms. Thus, Blo has only one recognized PTS permease, and this system is in the Glucose(Glc) Family (4.A.1). Lbr has just 7 recognized PTS protein homologues with four EnzymeIIC permeases, two of the Lac family, one of the Gat family (just the IIC constituent), and oneof the mannose (Man) family. Of these, only the Man family system is apparently a full EnzymeII complex with all requisite constituents. Lca has the most (63) PTS protein homologues withall 7 PTS families represented. This organism has between 1 and 8 intact PTS permeases perPTS family with 8 representatives in the Glc family and 1 in the Gut family. Lga and Ppe have36 and 37 PTS proteins, respectively. These two organisms possess complete glucose (Glc),fructose (Fru), lactose (Lac) and mannose (Man) family systems, and Lga also has a galactitol(Gat)-type system. Lcr and Ooe encode in their genomes 19 and 30 PTS proteins, respectively.Both have glucose, lactose, mannose and L-ascorbate (Asc)-type systems, and Lcr also has afructose-type system while Ooe additionally has a galactitol-type system. There is a reasonablecorrelation between prevalence of PTS permeases (Table 4) and organismal phylogeny (Fig.1).

Transmembrane electron carriers that transfer electrons from one side of the cytoplasmicmembrane to the other (TC class 5) are lacking in 10 of the 11 organisms. Bli is the onlyexception. It has 7 such proteins (1.3%) with representation in the DsbD and PMO families.

The eleven organisms have between 4 and 13 auxiliary transport proteins each (class 8). Allexcept Bli possess Enzyme I and HPr of the phosphotransferase system (PTS). Two organismseach possess a membrane fusion protein (MFP) family member, which in Gram-positivebacteria usually facilitates export of peptides via an ABC efflux pump [32]. Several organismshave homologues of the K+ transport/nucleotide binding regulatory protein (KTN) family, butthese homologues do not necessarily function to regulate transport and may serve an unrelatedfunction.

Of the poorly characterized class 9 transporters, the percentages are fairly constant in the 11organisms (3.6–7.4%). Within subclass 9A, we find putative transporters for heavy metals,including Fe3+ (OFeT and FeoB), Pb2+ (PbrT), Hg2+ (MerTP), and Mg2+ (MgtE). Threeorganisms also possess homologues of the nicotinamide mononucleotide (NMN) uptakepermease (PnuC) family.

Within subclass 9B, several potentially important families are represented. However, theevidence that many of these function in transport is marginal or nonexistent. For example, allor most members of the FAT family of acyl CoA synthetases may not play a role in fatty acidtransport [33]. The PerM, TEGT and YbbM families include 7 TMS protein members, and theYdjX–Z and YqiH families include 5 or 6 TMS members. The HCC family includes membersthat may be pore-forming hemolysins, and VGP family members may catalyze vectorialpolymerization of polysaccharides destined to be extracellular. Of greatest interest is thePutative Bacterial Murine Precursor Exporter (MPE) family (9.B.3) which is represented in all

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11 organisms, usually with 2 or 3 paralogues per organism. These putative transporters maybe required for synthesis of the bacterial cell wall.

8. Channel-forming ProteinsAs summarized in Table 5, all of the organisms examined contain recognized α-type channelproteins of TC class 1.A. These vary in number from 5 for Lde and Bli to 11 for Lcr.

Three channel protein families (Voltage-gated Ion Channel (VIC), Large ConductanceMechanosensitive Channel (MscL) and Small Conductance Mechanosensitive Channel(MscS)) as well as some aquaporins of the MIP family (see below) may function primarily toprotect the bacteria against osmotic stress [31,34–41]. All of the 11 bacteria examined have atleast one such channel suggesting that relief from osmotic stress is of general importance tothese bacteria as has been demonstrated for Lactococcus lactis [31]. However, the numbersand types of these channels vary. For example, five organisms (Ppe, Lbr, Lga, Ooe and Blo)have at least one of each of these three channel types. Lca, Lde and Lcr have two such channelproteins, one of the MscL family and one of the MscS family. In L. lactis, only the mscL geneis apparently expressed although both the MscL and MscS channel proteins from this organismare functional [31]. Lme is the only organism with just one of these channel proteins (MscL).In fact, Lme is the only bacterium that lacks an MscS channel homologue. Surprisingly, noorganism has more than one member of either the VIC, MscL or MscS family. Multiple MscSfamily paralogues are found in many bacteria although few bacteria have more than one MscLchannel [39].

The eleven organisms have from one to three chloride channel proteins of the ClC family (Table5; ref. 7). Thus, Ppe, Lbr, Lca, Lde and Ooe have just one of these anion-selective channels;Lga, Lcr, Lme, Blo have 2, and Sth has three. In E. coli, these channels are known to promoteresistance to extreme acid stress [42].

All eleven bacteria contain at least one heavy metal ion channel of the MIT (CorA) family[43,44], and five organisms have between two and five members. These divalent metal ionchannels are important for the uptake of Mg2+, Co2+ and Ni2+ in other bacteria in response tothe membrane potential, negative inside [45]. It can be presumed that these channels providethe same function in the organisms examined here.

Glycerol facilitators and aquaporins of the MIP family allow transmembrane passage of severalsmall neutral molecules including glycerol and other polyols, dihydroxyacetone, water, CO2,urea and ammonia [46–48]. In prokaryotes, most MIP family members transport glycerol astheir primary function. However, synthesis of aquaporins has been shown to be induced aftertransfer of certain bacteria to hyperosmotic media [49,50]. This fact suggests a role in osmoticshock protection.

Members of the MIP family of aquaporins and glycerol facilitators are found in all 11 bacteriaexamined here. Four of the LABs have a single putative glycerol facilitator, and the rest havetwo or more of them. Lactic acid bacteria in general cannot utilize glycerol as a sole carbonsource [51], but they can often co-metabolize glycerol and glucose [52]. The universaloccurrence of glycerol facilitators in these organisms may therefore reflect their abilities to useglycerol in the presence of a second carbon source. Five of the LABs (Ppe, Lbr, Lme, Blo andBli) have putative aquaporins that may play a role in osmotic shock responses. A singlebacterium (Sth) has a urea/short chain amide (UAC) family member. This protein may allowuptake of various amides as sources of nitrogen [53,54] (Table 5).

Hsp70 chaperone proteins are listed in Table 5 under class 1.A. This is because some eukaryoticHsp70 proteins have been shown to insert into membranes to form channels [55,56]. All of the

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organisms examined here have such a homologue. These proteins function primarily in proteinfolding as part of the general stress response system [57–60]. There is no evidence for channelformation by Hsp70 homologues in prokaryotes. However, merely because of thedocumentation of channel formation in eukaryotic homologues, we have included thesechaperones in this tabulation.

Several toxins of TC class 1.C were identified. In general, these are rare in the lactic acidbacteria examined. Six members of the large protein CCT toxin family were identified in Lme,but none was found in the other ten organisms analyzed. Members of this family are found inboth Gram-negative and Gram-positive bacteria [61,62]. Surprisingly, Lga has two colicinhomologues. Colicins are usually restricted to enteric bacteria. Finally, two of the organisms(Ppe and Lcr) encode peptide toxins (bacteriocins), Pediocin and Lactococcin A, respectively,which are exported to exert their toxic activities on other microorganisms [63,64].

Ppe, Lbr, Lcr and Sth each encode either one or two recognizable holin(s). These smalloligomeric proteins may promote cell death in response to stress stimuli by allowing export ofautolysins that digest the bacterial cell wall [65]. These proteins might therefore play a role inprogrammed cell death [66–68].

9. Secondary Carrier Superfamilies9.1. MFS (2.A.1)

There is tremendous variation in the numbers of secondary carriers in the different bacteriaexamined, and the same is true for the individual families of secondary carriers. The largestsuch family is the major facilitator superfamily (MFS) [69,70]. Sth has 5 such carriers whileBli has 70. All other bacteria have intermediate numbers (Table 5). Lde has 14, Ooe has 41and all other bacteria have between 21 and 35. Of equal interest are the probable substratespecificities of these transporters. In all but two of the bacteria analyzed, Lga and Bli, drugexporters comprise the majority of all MFS permeases. This is surprising since only 3 of the47 recognized MFS families are primarily concerned with export of hydrophobic andamphipathic substances. These three MFS families (called DHA1–3) are represented in alleleven bacteria with the exceptions of Ppe which lacks a DHA3 member. In Sth, 3 of the 5MFS permeases are putative drug exporters; in Lde, 8 of the 14 have this function, and in Lcr,20 of the 24 MFS homologues may export drugs. Of all the organisms analyzed, the high G+C Gram-positive bacterium, Bli, has the most non-drug-specific MFS transporters. Thus, inBli, 28 of the 70 MFS permeases probably act on drug-like substances, and more of thesesystems transport anionic compounds than drugs. It should be noted that DHA1–3 exporterscan act on a wide variety of compounds including hydrophilic metabolites (see TCDB).However, members of these three families always catalyze substrate efflux [69].

The sugar uptake systems of the MFS include members of the sugar porter (SP) family and theoligosaccharide:H+ symporter (OHS) family as well as the fucose:H+ symporter (FHS),sialate:H+ symporter (SHS) and polyol porter (PP) families. These families are poorlyrepresented in most of these Gram-positive bacteria. In fact, all but Lbr (5), Ooe (7), Blo (6)and Bli (5) have just 1–3 such systems.

Virtually all remaining MFS families represented (see Table 5) consist of permeases withspecificity for inorganic and organic anions. With the sole exception of Bli, these aniontransporting MFS permease families are poorly represented. The BST family is found only inLga which has 3 members and in Lca and Lde which have 1 member each. Multiple paraloguesof the OFA, ACS and AAHS families are found only in Bli, which has 2, 2 and 5 members ofthese three families, respectively, although Sth has three AAHS family paralogues. It is clear

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that Bli exhibits a diversity of MFS permeases that far exceeds that of the other organismsexamined.

9.2. GPH (2.A.2)In view of the paucity of MFS uptake systems for sugars, it is surprising that putative reducingoligosaccharide and non-reducing glycoside uptake systems of the GPH family (2.A.2) arerepresented in all of the eleven organisms except Bli, the one organism with by far the mostMFS permeases. The other bacteria have between 1 and 10 GPH permeases. Of the lactic acidbacteria, only Lca, Lde, Lga and Ooe have more MFS sugar permeases than GPH permeases.All functionally characterized members of the GPH family take up oligosaccharidespreferentially (hence the name Glycoside-Pentoside-Hexuronide:Cation Symporter Family(TC #2.A.2)). Some reports suggest that reducing sugars may also be substrates, but thissuggestion has been disputed for some GPH members [71,72]. This suggests that most lacticacid bacteria preferentially take up oligosaccharides and non-reducing glycosides over simplesugars via secondary carriers since almost all members of the GPH family take up glycosidesand oligosaccharides rather than reducing monosaccharides [71,73–75]. This observation mayreflect the fact that the former compounds are common plant products. They are also generatedin animals by the degradation of glycolipids and glycoproteins [76,77]. In lactic acid bacteria,simple hexoses may be taken up primarily via the PTS (see below).

9.3. APC (2.A.3)The Amino Acid/Polyamine/Organocation superfamily, the largest superfamily of amino acidand amine uptake transporters [78], is represented in all 11 Gram-positive bacteria studied here.Seven to 18 members of this superfamily are encode in each of the various genomes. Of theeleven currently recognized families within the APC superfamily, the AAT family (2.A.3.1)is most prevalent with 1–8 representatives per genome. However, ten of the eleven APCfamilies are found in these Gram-positive bacteria, a most surprising observation sincemembers of three of these families had not been identified in bacteria when this superfamilywas last described [78]. These three families are the ACT (2.A.3.4), LAT (2.A.3.8) and YAT(2.A.3.10) families. The ACT family is represented only in Bli (2 members), and the YATfamily is found only in Ppe (1 member), Lde (2) and Sth (1). However, members of the LATfamily are found in six of the eleven organisms (1–2 members each).

The glutamate:γ-aminobutyrate (GABA) antiporter (GGA) family (2.A.3.7) is found with from1 to 5 members in 7 of the 11 bacteria. All bacteria except Lde have 1–3 members of the cationicamino acid transporter (CAT) family. Two organisms (Ppe and Bli) have 1 member each ofthe ethanolamine transporter (EAT) family. One to 4 members of the archaeal/bacterialtransporter (ABT) family are found in 6 of the 11 organisms, the exceptions being Lbr, Lca,Lde, Ooe and Blo. Four organisms (Ppe, Lbr, Lca and Lga) have 1–2 members of the aspartate/glutamate transporter (AGT) family, while four organisms (Ppe, Lbr, Lde and Lcr) have 2members of the basic amino acid/polyamine antiporter (APA) family. It is clear that theseorganisms can take up a wide range of amino acids and their derivatives. The characteristicsof these families and primary references describing their members can be found in TCDB[78].

9.4. RND (2.A.6)The RND superfamily of efflux pumps is represented in 7 of the 11 organisms studied (1–7members per organism) (Table 5). This superfamily includes eight currently recognizedfamilies. Four of these families (HME, HAE1, NFE and ORF4) predominate in Gram-negativebacteria; a fifth (SecDF) is found in both Gram-negative and Gram-positive bacteria; a sixth(HAE2) is found only in Gram-positive bacteria; a seventh (HAE3) is found primarily inarchaea; and the eighth (EST) is restricted largely to eukaryotes [79].

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The HAE1 family of drug and lipid exporters is represented only in Bli (2 member), as is theSecDF family of protein secretion factors (1 member). However, the HAE2 family of drug andlipid exporters is found in 7 of the 11 organisms with 1–3 members each. No other RND familyis represented. It is clear that the RND superfamily is underrepresented in these bacteria.

9.5. DMT (2.A.7)The Drug Metabolite Transporter (DMT) superfamily consists of 18 currently recognizedfamilies [80], seven of which are represented in the Gram-positive bacteria under study. Infact, all eleven organisms have DMT representation (2–7 members/organism) (Table 5).

Of the 18 families, by far the greatest representation is in the Drug/Metabolite Efflux (DME)family, the largest of the prokaryotic families found in the DMT superfamily. Ten of the elevenorganisms have DME pumps, and each organism may have from 1 to 4 members. Six organisms(Ppe, Lbr, Lca, Lcr, Lme and Bli) have small multidrug resistance (SMR) export systems, andthe 9 lactic acid bacteria have representation in the Glucose/Ribose Uptake Porter (GRP) family(1–2 members). These proteins probably take up glucose via a proton symport mechanism[81]. Only the two high G+C Gram-positive bacteria (Blo and Bli) lack GRP family members.

Members of other DMT families are sparsely represented. For example, Lca has 1 member ofthe choline uptake transporter (LicB–T) family, Sth, Lme and Bli have 1, 1 and 2 members,respectively, of the chloramphenicol-sensitivity protein (RarD) family, while Blo has a singlemember of the paraquat (methyl viologen) exporter (PE) family. Surprisingly, Sth has amember of the plant drug/metabolite exporter (P-DME) family, which, however, is closelyrelated to the prokaryotic DME family.

9.6. SSS (2.A.21)The Sodium:Solute Symporter (SSS) superfamily [82] was not represented in the low G+CGram-positive bacteria studied but was found in the high G+C Gram-positive bacteria. Bli hasnine members while Blo has one. The single SSS protein in Blo may be a proline uptake porter,and the SSS proteins in Bli most closely resemble sodium iodide (3), proline (2), sugar (2),monocarboxylate (1), and phenylacetate (1) uptake permeases of other organisms.

9.7. MOP (2.A.66)The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily [83] is present in alleleven bacteria under study with 2–7 members each. The vast majority of these belong to thepolysaccharide transporter (PST) family within the MOP superfamily. Most organisms have1–7 such proteins, although Lbr and Blo lack these proteins. These exporters catalyze secretionof a variety of polysaccharides and their precursors using a proton antiport mechanism. All but2 of the organisms have at least one MATE-type multidrug resistance pump with 1–3 suchsystems each. Three organisms, a single low G+C Gram-positive bacterium (Lcr) and the twohigh G+C Gram-positive bacteria (Blo and Bli) have representation in the bacterial MouseVirulence Factor (MVF) family. The transport substrates of members of this family are notknown.

10. Smaller Families of Secondary Carriers10.1. Divalent cations

Monovalent and divalent cation concentrations must be strictly controlled for optimal growth.Bacteria therefore possess channels, carriers and primary active pumps that regulate theseconcentrations. Moreover, among the carriers, some are strict cation:cation antiporters, othersare electrogenic uniporters, and still others are cation:cation symporters as documentedparticularly well in E. coli [84–87].

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Five families of carriers for divalent cations are represented in these Gram-positive bacteria.The Calcium:Cation Antiporter (CaCA) family (2.A.19) is found only in Bli. These systemscatalyze Ca2+ efflux. The Cation Diffusion Facilitator (CDF) family (2.A.4) is found in 10 ofthe 11 organisms. Only Ppe lacks representation. These are likely to export heavy metal divalentcations such as Zn2+, Cd2+ and Co2+. The Ni2+-Co2+ Transporter (NiCoT) family (2.A.52) isfound only in three organisms (Ppe, Lbr and Lme). All functionally characterized members ofthis family function in the uptake of Ni2+ and/or Co2+. The Nramp family (2.A.55) isrepresented in all but two (Lde and Lga) of the eleven organisms examined, and these 9organisms have between 1 and 3 members. Nramp carriers take up a variety of heavy metals(Fe2+, Mn2+, Zn2+, Cu2+, Cd2+, Ni2+, Co2+, Pb2+) using a cation symport mechanism. Thepoorly characterized CadC family (2.A.77) is also represented in Table 5. Five organisms eachhave 1–3 members of this family of putative Cd2+ efflux pumps.

10.2. Monovalent cationsNine families of monovalent cation transporters are represented in Table 5. None of these isfound in all of the eleven organisms. NhaA family (2.A.33) members are found only in thehigh G+C Gram-positive bacteria, Blo and Bli, each having one member. The NhaC family(2.A.35) is found in 7 of the 11 organisms with 1 or 2 members each. The CPA1 family (2.A.36) was found in all organisms except Lde while the distantly related CPA2 family (2.A.37)was lacking only in Sth and Blo. A maximum of 4 members of each of these cation:protonantiporters is encoded in any one genome.

The Trk family (2.A.38) of monovalent cation symporters is represented in 6 of the 11organisms, but one bacterium (Sth) has three of these systems while another (Bli) has four. Asingle protein of the NhaD family (2.A.62) of Na+/H+ antiporters is found only in Lca.

Subunits of the CPA3 family of K+:H+ and Na+:H+ antiporters (2.A.63) are found only in Bli.Normally these systems have 7 subunits, but 10 are reported for this organism. Possibly thesesubunits are constituents of two distinct systems. Finally, the KUP family (2.A.72) of K+ uptakesystems is found in 7 of the 11 bacteria, and the Amt family (2.A.49) of NH3 channel proteins[88] is found in nine. This last family was previously thought to transport NH4

+ by a carrier-type mechanism, but high-resolution x-ray structures of the E. coli AmtB protein clearlysuggest that at least this member of the family deprotonates NH4

+ at the external surface ofthe pore and transports NH3 via a simple hydrophobic channel-type mechanism [88,89].

10.3. AnionsSeventeen families of secondary carriers represented in Table 5 include members that probablytransport anionic compounds. Most of these are specific for organic anions that can be used assources of carbon (gluconate, ketodeoxygluconate, tri-, di- and monovalent acids such ascitrate, succinate and lactate, respectively, and bile salts), but several can transport inorganicanions as well (formate, nitrate, arsenite, sulfate, phosphate). Perusal of Table 5 reveals thatthe gluconate porter (GntP) family (2.A.8) is represented in seven organisms. Three additionalwell-represented families (CCS, DASS and DAACS) primarily transport organic anions whilethree other well-represented families (PiT, FNT and SulP) transport inorganic anions. Otheranion transporting families are represented to a lesser degree.

Interestingly, the poorly characterized auxin efflux carrier (AEC) family (2.A.69) is found in10 of the 11 organisms examined with between 1 and 5 members per organism. The fewcharacterized prokaryotic members of this family catalyze uptake of dicarboxylates such asmalate and malonate [90,91]. Many of these anion-transporting families are members of theIT superfamily [92].

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10.4. Amino acids and peptidesIn addition to the APC superfamily, several other families represented in Table 5 includemembers that catalyze amino acid uptake [93]. The NSS family (2.A.22) is represented onlyin Bli. Glycine and alanine permeases of the AGCS family of semipolar amino acid uptakeporters (2.A.25) are represented in 9 of the eleven organisms.

Three families of amino acid efflux porters are represented in Table 5. The first of these is theLysE basic amino acid exporters (2.A.75), present only in Bli. The LysE family is a memberof the LysE superfamily, all members of which catalyze substrate efflux [94]. The secondamino acid efflux family represented is the RhtB semipolar amino acid exporters (2.A.76),present in Sth, Lme and Bli. This family is also a member of the LysE superfamily [94]. Finally,members of the LIV-E hydrophobic amino acid exporter family (2.A.78) are present in Ppe,Lbr, Lcr, Sth, Lme, Blo and Bli. The LIV-E family is unrelated to the LysE superfamily, andall characterized members of this family export hydrophobic amino acids [95,96].

Peptide uptake permeases represented include those of the POT family (2.A.17) found in 7organisms, the OPT family (2.A.67) found in 2 organisms, and the AbgT family (2.A.68) foundin 1 organism. The Tat family of pmf-driven protein secretion permeases (2.A.64) was foundin Sth and Bli. The Oxa1 family (2.A.9), involved in the insertion of integral membrane proteinsinto the plasma membrane bilayer, is represented in all organisms examined (Table 5).

10.5. Nucleobases and nucleosidesThree families found in Table 5 are concerned primarily with the uptake of nucleobases andnucleosides. These are the NCS1 family (2.A.39) (4 organisms represented), the NCS2 family(2.A.40), present in all 11 organisms, and the CNT family (2.A.41), present in 4 of the 11bacteria. Multiple such systems are present in all eleven bacteria.

11. Primary Active TransportersPrimary active transporters couple a primary source of energy to transport, and they areconsequently often multicomponent and always multidomain systems. They couple transportto processes such as ATP hydrolysis, organic acid decarboxylation and electron transfer. Aquick look at Table 5 reveals that each of the eleven organisms has multiple (66–175) ABCsystem proteins, a single F-type ATPase, always with 8 dissimilar subunits as expected, several(4–13) P-type ATPases, and a number of ATP-driven macromolecular transport systems. Oneorganism has an organic acid decarboxylation-driven Na+ exporter, but with the exception ofBli, none has a monovalent cation (H+ or Na+)-translocating electron carrier. The individualfamilies of primary active transporters and the members identified will be discussed below. Inthe following sections we will discuss homologues of known primary active transporters.

11.1. The ABC superfamily (3.A.1)ABC transporters segregate phylogenetically into uptake and efflux systems [97,98]. Theformer but not the latter act in conjunction with extracytoplasmic solute binding receptors (R)that feed the solute into the transmembrane channel (M) [99] which is energized by thecytoplasmic ATP hydrolyzing subunit/domain (C) (see TCDB). The receptors can be lipidanchored or attached to non-cleavable, N-terminal, leader sequences. In some cases they areeven fused to the transmembrane transporter component. Sometimes several receptors can feeddifferent, but structurally related solutes into a single ABC channel [100]. Transport isenergized by the hydrolysis of ATP, catalyzed by a membrane-associated ATP-binding cassette(ABC) protein or protein domain [101].

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Both the integral membrane channel forming protein (CFP; M) and the ABC protein (C) areusually present as dimers, each consisting either of a single gene product, yielding ahomodimer, or two gene products, yielding a heterodimer [102]. Sometimes, but seldom, thesethree protein types are fused to form multidomain proteins. ABC uptake permease systemstherefore normally consist of 3–5 distinct proteins although the number can be smaller (due togene fusions) or larger (due to the presence of multiple receptors) (see TCDB). The averagenumber of proteins per ABC uptake system is roughly 4.

By contrast, ABC efflux systems consist only of the ABC (C) and CFPs (M), and these twodomains are often fused. Consequently, there are either one or two proteins per ABC exporter;thus, about 1.5 proteins are present on an average per ABC exporter. The quantitation presentedin Table 5 is always in terms of numbers of proteins; consequently, these numbers should bedivided by about 4 and 1.5 for families of uptake and efflux systems, respectively, in order toestimate the numbers of systems represented.

As summarized in Table 5, ABC transporters occur in substantial numbers in all Gram-positivebacteria studied. The numbers of ABC proteins encoded within the various genomes vary from66 (for Ppe) to 175 (for Bli). The average number of ABC system proteins per organism is 127.

Two families within the ABC superfamily are concerned exclusively with carbohydrate uptake,the carbohydrate uptake transporter-1 and -2 (CUT1 and CUT2) families. While CUT1 systemsare largely concerned with oligosaccharide uptake, CUT2 systems usually exhibit specificityfor monosaccharides, particularly aldopentoses and hexoses. CUT1 homologues are found inall organisms except Ppe, Sth and Lme. Blo has 40, probably comprising about 10 systems. Itis interesting to note that Bli, with a larger genome, has only 5. Other bacteria probably have1–4 such transporters. Lcr and Ooe probably have four such systems. CUT2 family homologuesare found in all organisms studied, but the numbers of CUT2 family proteins never reach thelarge numbers sometimes observed for CUT1 family proteins. Note that the PepT family (3.A.1.5) includes members that like CUT1 family members can transport oligosaccharides (seebelow).

The two principal amino acid uptake families in the ABC superfamily (PAAT and HAAT) arespecific for polar and hydrophobic amino acids, respectively. Representation in the formerfamily (11–32 proteins) far exceeds that in the latter family (0–11 proteins). The PepT family,which includes members that transport peptides and oligosaccharides, is well represented (6–40 proteins per organism). The methionine uptake transporter (MUT) family (TC #3.A.1.24)is represented in all 11 organisms.

The next few uptake families listed in Table 5 are largely concerned with inorganic iontransport. Sulfate-transporting SulT family homologues are found in 5 of the 11 bacteria, butall 11 organisms have at least one phosphate transporter of the PhoT family, and five also havea phosphonate transporter of the PhnT family.

The POPT family includes a diverse group of transporters exhibiting specificity forpolyamines, opines and phosphonates. One or two systems are present in all bacteria exceptPpe and Blo. Similarly, at least one (and sometimes 2 or 3) QAT family systems for the uptakeof quaternary amine osmolytes and amino acid derivatives are present in all but threeorganisms. TauT family systems, specific for taurine, are found in a few bacteria, and thesesystems may, in addition to allowing utilization of taurine, provide defense against osmoticstress.

The next several families are concerned with heavy metal uptake. They include the FeCT andBIT iron (complex) transporters, the MZT, Mg2+, Mn2+, Zn2+, Fe2+ transporters, the CoTcobalt uptake systems and the NiCoT nickel/cobalt uptake transporters. Surprisingly, the MZT

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systems are present in all eleven bacteria although the FeCT family is present in only 5 of them,and the Bit and CoT families are represented in only three. A single NitT nitrate/nitritetransporter is found in Lbr. The NiCoT family is represented in all eleven organisms.Homologues of the putative yersiniabactin Fe3+ uptake transporter (YbtPQ) family were foundin Lbr, Lca and Ooe. Members of all other ABC uptake families are absent from all elevenorganisms.

ABC efflux systems fall into two principal subdivisions, those found primarily in prokaryotes(the 3.A.1.100 series) and those found primarily in eukaryotes (the 3.A.1.200 series). Althoughthe capsular polysaccharide exporter (CPSE) family is lacking in the 11 organisms examined,the Lipooligosaccharide Exporter (LOSE) family, previously characterized only in Gram-negative bacteria, is represented in 7 of the 11 organisms. Members of the LipopolysaccharideExporter (LPSE) and Teichoic Acid Exporter (TAE) families are also represented. AlthoughGram-positive bacteria do not make LPS, they do incorporate teichoic acids into their cell walls[103].

Four of the six prokaryotic ABC drug efflux families (Drug E2, Drug RA1, Drug RA2 andMacB) are all nearly universally present in the eleven bacteria, and the two remaining drugexport families (Drug E1 and Drug E3) are also present in select organisms. It is interestingthat many of these organisms seem to have multiple MacB macrolide exporters. Three bacteriamay also have eukaryotic-type MDR pumps, and nine may have a member of the β-exotoxin1 Exporter (βETE) family.

Four of the five ABC peptide exporter families are represented in Table 5 (the Pep1, 2, 4 and5E families). While Pep3E pumps are lacking in all organisms, and a Pep1E family memberis present in each of four organisms, the other three families, Pep2E, Pep4E and Pep5E, arewell represented. Multiple such peptide exporters are found in many of the bacteria.Homologues of the protein exporter (Prot1E) family are found in four of the eleven organisms,while a single homologue of the Prot2E family is present in Bli.

A few other ABC exporter families are represented in Table 5. These include the poorlycharacterized DevE family of putative glycolipid exporters, the LPT family of lipoproteintranslocases, and the lipid exporter (LipidE) family. Two of these families (DevE and LPT)occur in 8 of the 11 organisms examined. Most surprisingly, the LipidE family is representedin all of the 11 organisms except Ppe with as many as 11 such proteins per organism.

11.2. P-type ATPases (3.A.3)The eleven organisms studied have between 4 and 13 P-type ATPases per organism. These canbe classified according to the substrates most likely transported, based on sequence similarityto functionally characterized systems (data not shown).

One organism (Lca) has a protein most closely resembling eukaryotic Na+, K+ ATPases of P-ATPase subfamily 1, a novel finding for a prokaryote. In eukaryotes, these enzymes catalyzeuptake of 3K+ coupled to efflux of 2Na+ in an electrogenic process [104]. All organisms exceptBli have multiple Ca2+/Mn2+ ATPases of subfamily 2. The numbers of these systems perorganism vary between 1 and 5.

In subfamily 3, which includes the characterized Mn2+/Cd2+ ATPase of Lactobacillusplantarum [105] as well as H+-translocating ATPases of fungi and protozoans, we find singlemember representation in two organisms, Lbr and Sth. Lca, Lde, Lga and Lcr each have twosuch members. These four bacteria also have one member of subfamily 4, similar to theMg2+/Ni2+ ATPase of Salmonella typhimurium.

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All organisms have at least one, and as many as 3 Cu+, Ag+-ATPases of subfamily 5. InEnterococcus hirae, two such systems are characterized (TC #3.A.3.5.1 and 3.A.3.5.2) [106].One catalyzes copper uptake, the other copper efflux, presumably allowing proper maintenanceof copper homeostatic conditions in the cell [106].

Finally, all but two organisms (Sth and Blo) have 1–3 broad specificity heavy metal (Zn2+,Co2+, Cd2+, Hg2+, Ni2+, Cu2+, Pb2+ and Ag2+) efflux pumps of subfamily 6. Surprisingly, noK+ ATPases of subfamily 7, no phospholipid flippases of subfamily 8, and no monovalentalkali cation (Na+, K+) pumps of subfamily 9 were detected.

11.3. Macromolecular secretion systemsConstituents of three protein export systems as well as two DNA translocases were identified.The general secretory pathway (Sec) complex is ubiquitous, being found in every organismfor which it has been sought [107]. However, it can consist of as many as 8 proteins and onesmall (4.5 S) RNA, as is true for E. coli, or as few as 6 proteins plus a small RNA, as is truefor Mycoplasma genitalium. The SecA, SecY, SecE and SecG proteins as well as the Ffh andFtsY proteins (also required for membrane protein insertion) are always present. When proteinsare inserted into the membrane, YidC homologues of the Oxa1 family (2.A.9) often participate[108]. The SecDF complex (homologous to RND transporters), which serves an auxiliaryfunction, is present only in B. linens. Surprisingly, B. linens also has two SecA proteins. Finally,only Ppe has two SecYs, one for general secretion and one for specific secretion of large cellsurface adhesions [109–111]. The latter system uses different auxiliary proteins, the Asp1–4proteins. While Ppe has Asp2–4, Lme has Asp1–3 but lacks a second SecY. What these proteinsare doing in these two bacteria has yet to be determined (Table 5).

Gram-negative bacteria have multicomponent conjugal protein/DNA export systems. TheGram-positive bacterium, Staphylococcus aureus, also has such a system, the Trs system,which consists of 15 dissimilar proteins [112]. Ten of the eleven bacteria examined here havehomologues of this system (3.A.7), but the numbers of such proteins vary between 2 (for Lbr,Lga and Bli) and 8 (for Blo). It is therefore not clear that these bacteria have complete systems.

Constituents of the Bacterial Competence-related DNA Transformation Transporter (DNA-T)family (3.A.11) are found in all of the bacteria, but the numbers of such homologues varybetween 2 and 6. All but Lme, Blo and Bli have all three of the proteins required for competence[113]. Similarly, Septal DNA Translocators of the S-DNA-T family (3.A.12) are found in oneor two copies in all eleven organisms. These proteins may be required for translocation ofchromosomal or plasmid DNA across the septal membrane [114,115].

The Fimbrilin/Protein Exporter (FPE) family is represented in all nine low G+C Gram-positivebacteria under study, but it is not found in the two high G+C Gram-positive bacteria. Thesesystems consist of two proteins, an integral membrane channel protein, and an energizingATPase. Eight of the low G+C Gram-positive bacteria have both such constituents while Lcahas just the ATPase homologue.

11.4. Na+-translocating organocarboxylate decarboxylasesFour organisms, two low G+C and the two high G+C Gram-positive bacteria, have multiple(2–7) subunits of putative Na+-translocating decarboxylases (3.B.1) [116,117]. Of these, onlyone (Lca) has the decarboxylase subunit (α), the integral membrane transporter subunit (β) andthe small γ-subunit. Consequently, only Lca can be expected to export Na+ in response to thecytoplasmic decarboxylation of substrate carboxylates.

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12. The Phosphotransferase System (PTS)PTS permeases consist of 3 or 4 domains, the IIA, IIB, IIC (present in all families), and IID(present only in the Mannose (Man) family). These domains can be fused together or detachedas separate polypeptide chains [22,118]. Variations in representation of PTS proteins in theeleven organisms were extensive. The two PTS energy-coupling proteins, Enzyme I and HPr,are present in all organisms except in the high G+C Gram-positive bacterium, Bli, which alsolacks a PTS permease. The other high G+C Gram-positive bacterium, Blo, has both EnzymeI and HPr and seems to have a single PTS permease, possibly specific for β-glucosides. Lbrhas far fewer PTS permeases than the other low G+C Gram-positive bacteria with only twosystems, a lactose-type (Lac) system and a Man system.

The representation of PTS permeases in the remaining low G+C lactic acid bacteria variestremendously. Lca has the most with 63 PTS permease proteins, and all seven known familiesof these permeases are encoded within the Lca genome. The first such family, the Glucose/Glucoside (Glc) family (4.A.1) is represented in all of the low G+C lactic acid bacteria exceptLbr, with up to 12 proteins belonging to this family per organism. Surprisingly, Sth has 8 ofits 15 PTS proteins within this family while Lca, with 4 times as many PTS proteins, has only12 in the Glc family.

Within the Glc family, putative glucoside transporting systems outnumber hexose-transportingsystems 4 to 1, emphasizing the dependency of these bacteria on plant glycosides. The Fructose/Mannitol (Fru) family (4.A.2) is found in seven organisms with 1–7 proteins per organism.The Lactose/Diacetylchitobiose/Lichenan Oligosaccharide (Lac) family (4.A.3) is wellrepresented with approximately four times as many proteins as for the Fru family. This is inagreement with the observation that many of these bacteria are dependent on plant glycosidesfor growth. It contrasts with the preponderance of Fru systems in γ-proteobacteria [22]. TheGlucitol (Gut) family (4.A.4) is represented in only two organisms (Ppe and Lca) while proteinsof the GAT family (4.A.5) are present in four (Lbr, Lca, Lga and Ooe). However, Lbr has onlythe IIC component, implying that the protein does not function by a PTS-type mechanism. Itmay be a secondary carrier as discussed previously for other organisms [119,120]. The L-ascorbate (Asc) family (4.A.7) is represented in four lactic acid bacteria (Lca, Lcr, Lme andOoe). All of the low G+C Gram-positive bacteria studied have at least one mannose/fructose/sorbose (Man)-type system (4.A.6), and at least three of them have 3–6 such systems.

13. Transmembrane Electron Transport SystemsWhen grown in the presence of heme, at least some lactic acid bacteria gain additionalmetabolic energy in the form of a proton motive force. The mechanisms are not known, andthese observations could be a result of indirect effects rather than the direct coupling of electronflow to proton efflux. In fact, none of the currently recognized bacterial H+- or Na+-translocating electron flow systems was identified (families of oxidoreduction-driven cationtransporters; TC section 3.D).

Transmembrane electron flow systems of TC classes 5.A and 5.B do not transport cationsacross the membrane, but electrons do flow from one side of it to the other. These systemstherefore contribute to or subtract from the membrane potential. Recognized transmembraneelectron flow systems are present in only one organism, the large genome high G+C Gram-positive bacterium, Bli. Bli has 4 members of the Disulfide Bond Oxidoreductase (DsbD)family (5.A.1) and three protein members of the Prokaryotic Molybdopterin-containingOxidoreductase (PMO) family (5.A.3). However, of these three proteins, two are α-subunitsand one is a β-subunit. Therefore, only one complete PMO family member may be present.Recognizable pmf-generating or -dissipating electron flow carriers are essentially absent in allother bacteria examined.

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14. Auxiliary Transport ProteinsAuxiliary transport proteins are relegated to TC class 8. Gram-positive bacteria have“membrane fusion proteins” (MFPs) of family 8.A.1. These proteins were named followingtheir discovery in Gram-negative bacteria [121], but they have since been described in Gram-positive bacteria where they can be essential for the activity of particular ABC-type exporttransporters [122].

Only two of the bacteria studied here have such a protein (Sth and Lme). These probablyfunction in conjunction with peptide efflux pumps of the ABC superfamily [122]. In fact, bothSth and Lme have genes encoding ABC transporters of the Pep2E family (TC #3.A.1.112)adjacent to the genes coding for their MFPs. Two Gram-positive bacterial MFPs have beenshown to be essential for ABC transport function [123–125].

Seven organisms have MPA1 family members (8.A.3) that regulate complex capsularpolysaccharide export via PST-type transporters of the MOP superfamily (TC #2.A.66.2)[83]. The K+ Transport/Nucleotide Binding Regulatory Subunit (KTN) family (8.A.5), whichcontrols the activities of Trk family K+ porters, is found in 9 of the 11 organisms, and all ofthe low G+C lactic acid bacteria have homologues of the eukaryotic rBAT (1–5 copies perorganism). rBAT homologues stimulate amino acid transport via members of the APCsuperfamily, but they are homologous to various glycosidases [126–128]. There is no evidencethat these homologues regulate amino acid transport in bacteria.

15. Poorly Characterized TransportersSeveral poorly characterized transporter families of TC class 9 are sparsely represented in Table5. These include MerTP Hg2+ transporters (9.A.2) (1 organism), the PnuC family (9.A.4) ofnicotinamide mononucleotide (NMN) transporters (3 organisms) and the FeoB (9.A.8) andOFeT (9.A.10) families of iron transporters (4 and 2 organisms, respectively). A putative LeadUptake Porter (PbrT family; 9.A.17) is found in each of two organisms, Sth and Blo. MgtEMg2+ transporters (9.A.19) are found in single copy in Ppe, Lme and Bli.

Several families of unknown function (TC Class 9.B) are worthy of brief mention. The putativemurein precursor exporter (9.B.3), responsible for export of lipid-linked oligosaccharideprecursors of peptidoglycan, is found in all eleven organisms. These transporters may berequired for the assembly of the bacterial cell wall. The MgtC family of putative Mg2+

transporters (9.B.20) is represented in single copy in 7 organisms. The AI-2E family (TC #2.A.86), one member of which exports the form-containing autoinducer AI-2 signalling molecule[129–131] is found in all eleven organisms (1–5 copies per organism). As AI-2 is believed tobe required for interspecies communication, it is possible that this type of signalling isimportant for the lifestyles of LABs and other Gram-positive bacteria.

The Hly III family (9.B.30) of putative hemolysins is present in 10 of the 11 organisms. It willbe interesting to know the function of these homologues. Members of the Putative VectorialGlycosyl Polymerization (VGP) family (9.B.32) are found in 9 of the 11 organisms, and asmany as 6 paralogues may be present in a single organism. It can be presumed that these proteinsfunction in the assembly of exopolysaccharides and capsular polysaccharides and possiblyteichoic acids as well [132].

16. ConclusionsThe fully sequenced genomes of eleven Gram-positive bacteria were searched for homologuesin TCDB in order to analyze the transport capabilities of these bacteria. These eleven bacteriadevote at least 13 to 18% of their genes to transport. Of the total known transport proteins

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encoded in these genomes, on average, about 48% constitute uptake systems, 32% constituteefflux systems, and 5% of the proteins encode systems that transport substrates bidirectionally(Table 6). Fifteen percent are of unknown polarity and mechanism of action. Most of thebidirectional transporters are channels that either have low specificity for their substrates orare nonspecific. The putative uptake systems in these lactic acid bacteria transport primarilyamino acids, sugars, cations, anions and peptides. The amino acid transporters are the mostabundant while peptide transporters are the least abundant of these uptake systems.

Lactic acid bacteria have many drug efflux systems, but they also have substantial numbers ofpeptide and macromolecular exporters. Comparison of the relative proportions of transportproteins in lactic acid bacteria with those in Escherichia coli, Bacillus subtilis andMycobacterium tuberculosis revealed interesting features of the lactic acid bacteria (Table 7and Fig. 4). Twenty-four percent of the transport proteins in lactic acid bacteria seem to beinvolved in transport of inorganic molecules, and 23% of the transport proteins may transportamino acids and their derivatives. E. coli, B. subtilis and M. tuberculosis devote about 18%,21% and 42% to transport of inorganic molecules and 29%, 24% and 14% to transport of aminoacids and their derivatives. About 17% of the transport proteins in lactic acid bacteria may beinvolved in the uptake of carbon sources while in E. coli, B. subtilis and M. tuberculosis thesesystems constitute 29%, 20% and 6% of the total transport systems, respectively. In all of theserespects the LABs most closely resemble B. subtilis. Transport proteins that may be involvedin efflux of drugs and toxic compounds constitute 10% of the total transport proteins in theLABs unlike B. subtilis (17%) and M. tuberculosis (18%) but similar to E. coli which has 9%of its transport proteins concerned with efflux of these molecules. Eleven percent of thetransport proteins in lactic acid bacteria may be involved in transport of macromolecules unlikeE. coli, B. subtilis and M. tuberculosis which have only 2%, 2% and 4% of their transportproteins devoted to this function, respectively.

Of the 11 bacteria examined, B. linens has the largest genome, and many transporter familiesare represented that are found in none of the others (see Table 5). Families of secondary carriersthat were only found in B. linens are the UMF2, YnfM, EntS and NNP families in the MFS,KDGT, CaCA, NSS, APC(ACT), CPA3, CHR, LysE and RND (Hae1 and SecDF) families.Three ABC-type families, Prot2E, DrugE3 and CT(ABCC), were also found only in B.linens. Moreover, only in this organism were electron carriers of the DsbD, PMO, QCR andCOX families identified. Finally, certain oversized proteins with 19–25 TMS were found onlyin B. linens, and these proteins belong to the FeCT (19 TMSs), UIT10 (19 TMSs) and the CPA3(24 and 25 TMSs) families.

Analysis of the MFS transporters in these bacteria revealed that the greatest proportion of thesesystems transport drugs (Table 8). Forty to eighty-three percent of MFS transporters in thesebacteria probably exhibit specificity for drugs and other amphipathic compounds. MFStransporters that translocate anions are the second most abundant of the MFS carriers followedby those that transport sugars. However, Bli has more MFS transporters that transport anionsthan drugs or other substrates. Surprisingly, Sth has no sugar transporters of the MFS-type atall. These systems are almost ubiquitous among free-living bacteria.

Analysis of ABC transporters in the eleven bacteria revealed that of these systems, theseorganisms generally have more uptake systems than efflux systems (Table 9). However, Lca,Lde and Lga have more ABC efflux systems than ABC uptake systems. Drug efflux pumpsconstitute the largest proportion of the ABC systems, and amino acid uptake systems are thesecond most abundant. However, the opposite is true for Sth and Lme which have more aminoacid uptake systems than drug efflux pumps. Interestingly, in Lde, most ABC systems effluxlipids and hydrophobic compounds while in Lga most ABC systems efflux peptides. In Blo,

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ABC-type sugar uptake systems dominate. A large number of drug exporters is a commonfeature of soil bacteria which presumably must cope with such toxic substances frequently.

Bacterial multidrug resistance efflux pumps fall into five superfamilies, MFS (TC #2.A.1),RND (TC #2.A.6), DMT (TC #2.A.7), MOP (TC #2.A.66 and ABC (TC #3.A.1). Several ofthese superfamilies include multiple families that export drugs and other toxic molecules[80,83,133,134]. We analyzed the distribution of these various types of pumps in the elevenGram-positive bacteria as well as several other well-characterized bacteria.

Analyses of the putative drug efflux pumps (Table 10) in the eleven bacteria reveals that overall,55% of these pumps belong to the MFS followed by ABC (33%), MOP (6%), RND (4%) andDMT (2%). Some organisms provided exceptions to this trend. Sth had more ABC familymembers (54%) than MFS (23%) or MOP (23%), but this was the only organism in whichMOP and MFS drug efflux pumps are present in equal numbers. No RND or DMT familymembers were found in Sth, while no representatives of the MOP or DMT families were foundin Lde and Lga. RND and DMT systems were absent in Ooe and Blo. Although the greatestproportions of drug efflux pumps in E. coli and B. subtilis are MFS-types, the relativeproportions of these and other drug efflux pumps in these bacteria differ. E. coli has 58% MFS,16% RND, 14% ABC, 7% DMT and 5% MOP-type systems while B. subtilis has 61% MFS,14% MOP, about 11% each of ABC and DMT and only 3% (one system) of RND-type systems.

Sugar transporters can be secondary carriers, ABC transporters or PTS group translocators.Half of the sugar uptake systems in the low G+C Gram-positive lactic acid bacteria are PTS-type (50%) while 34% are secondary carriers and only 16% are ABC transporters (Table 11).However, in the high G+C Gram-positive bacteria, Bli and Blo, most of the sugar transportersare either secondary carriers (48%) or ABC-type transporters (50%). Bli does not encode anyPTS permeases while just one sugar transport system in Blo is a PTS permease.

In three representative Gram-negative bacterial pathogens, Borrelia burgdorferi (Bbu),Treponema pallidum (Tpa) and Haemophilus influenzae (Hin), 70% of all sugar transportersare ABC-type while 22% are PTS and only 8% are secondary carriers (Table 11). However,in three representative Gram-positive pathogens, Listeria monocytogenes (Lmo),Streptococcus pyogenes (Spy) and Mycoplasma genitalium (Mge), about 75% of all sugartransporters are PTS permeases and 16% are ABC-type while only 9% are secondary carriers.These considerations demonstrate the unique features of the LABs and reveal which organismaltypes they most resemble.

The low G+C Gram-positive lactic acid bacteria seem to encode slightly more transportersspecific for glycosides (51%) as compared with those for free sugars (49%) (Table 12). Thetwo high G+C Gram-positive bacteria, Bli and Blo, show tremendous bias in the utilization ofglycosides versus sugars, but in opposite directions. Blo has more than twice as many uptakesystems for glycosides as for free sugars while Bli has more than ten times more transportersfor free sugars than for glycosides. In comparison, E. coli and B. subtilis transport more freesugars (65% and 55%, respectively) than glycosides (35% and 45%, respectively).Representative Gram-negative pathogens (Bbu, Tpa and Hin) have five times more uptakesystems for free sugars than for glycosides while Gram-positive bacterial pathogens (Lmo, Spyand Mge) have nearly two fold more sugar uptake proteins than glycoside uptake systems.These observations illustrate the unusual sugar utilization capabilities of the Gram-positivebacteria that live primarily in association with plants.

All eleven bacteria studied here have at least one peptide uptake system (Fig. 5), and all butBli have at least one peptide efflux system. Bli has disproportionately greater numbers ofpeptide uptake systems (13 systems) while Lga has many more peptide efflux systems (11systems) than the other bacteria. Lca, Lde, Lga, Sth and Lme have more peptide efflux systems

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than peptide uptake systems, while Ppe, Ooe and Blo have more uptake systems for peptidesthan efflux systems. Lbr and Lcr have equal proportions of these two types of systems (Fig.5).

All low G+C lactic acid bacteria possess mechanosensitive channels for osmotic adaptation,suggesting that these organisms are exposed to extremes of osmotic conditions in their naturalhabitat. They also have chloride channels that may function in pH homeostasis. For the mostpart, they lack electron transfer, suggesting a predominantly fermentative mode of energygeneration. They have peptide uptake and export systems that function in nutrition, signalling,regulation and biological warfare. They have complements of transporters that distinguish themfrom other types of bacteria, and the different LABs also exhibit species-specificcharacteristics. All eleven organisms have competence-related and septal DNA translocationsystems although competence has not been demonstrated for any of these bacteria. They allpossess one or more putative murine precursor exporter(s), possibly required for extracellularpeptidoglycan assembly. Finally, they all possess the Sec/Oxa1 systems for secretion,membrane insertion and assembly of proteins that include all of the transporters discussed inthis article.

AcknowledgementsWe wish to acknowledge the enormous contribution of the Lactic Acid Bacterial Genome Sequencing Consortiumwithout the effort of which these analyses would not have been possible. We also thank Mary Beth Hiller for assistancein preparing this manuscript. This work was supported by NIH grant GM 55434.

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Figure 1.Phylogenetic tree of the organisms included in this study based on their 16S ribosomal RNAsequences. The Clustal X program [15] was used to align the sequences.

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Figure 2.Distribution of various topological types of transport proteins in eleven Gram-positive bacteria.The eleven organisms are indicated by the symbols shown to the right of the figure. The averagevalues for all eleven organisms are shown by the thick black band.

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Figure 3.Distribution of transport proteins belonging to each TC class in each of the eleven organismsanalyzed. Class 1, channels; class 2, carriers (secondary active transporters); class 3, primaryactive transporters; class 4, PTS-type group translocators.

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Figure 4.Comparison of the average percent distribution of transport proteins of various specificities inthe nine LABs with that in other bacteria, Bacillus subtilis (Bsu), Mycobacteriumtuberculosis (Mtu) and Escherichia coli (Eco).

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Figure 5.Distribution of uptake versus efflux transport systems specific for peptides in the eleven Gram-positive bacteria included in this study.

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amin

o ac

ids t

o pl

ant g

row

th h

orm

ones

.

a See

Tabl

e 1

for o

rgan

ism

al a

bbre

viat

ions

.

b The

phys

iolo

gica

l bas

is fo

r the

div

isio

n of

lact

obac

illi i

n th

ree

cate

gorie

s (G

roup

s I–I

II) i

s the

pre

senc

e or

abs

ence

of t

he k

ey e

nzym

es o

f hom

o- a

nd h

eter

ofer

men

tativ

e su

gar m

etab

olis

m, f

ruct

ose-

1,6-

bisp

hosp

hate

ald

olas

e an

d ph

osph

oket

olas

e, re

spec

tivel

y [1

35–1

37].

Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

NIH

-PA Author Manuscript

NIH

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NIH

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Lorca et al. Page 34Ta

ble

3N

umbe

rs o

f put

ativ

e tra

nspo

rt pr

otei

ns w

ith v

aryi

ng p

redi

cted

topo

logi

es

TM

SPp

eL

brL

caL

deL

gaL

crSt

hL

me

Ooe

Blo

Bli

071

8312

590

6685

8582

8161

113

122

3044

3125

4036

4734

4352

28

79

83

129

812

69

38

67

155

712

112

910

410

1323

916

2316

721

1522

519

2333

2926

1921

2730

1813

627

3645

2834

3524

3037

5159

712

921

1420

1317

1115

818

812

1212

512

158

67

1221

97

610

710

117

128

817

1015

2130

1918

1416

2016

2033

1112

1620

1311

1510

1613

625

1235

5531

1235

3114

3537

3910

213

1113

85

76

59

64

2114

1412

186

811

411

154

1815

31

11

12

116

12

11

317

118

11

192

241

251

Tota

l28

734

343

929

129

933

928

432

434

430

554

2

Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Lorca et al. Page 35Ta

ble

4To

tal n

umbe

rs (u

pper

) and

tota

l per

cent

(low

er) o

f tra

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at b

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g to

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ss

Cla

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1. C

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139

105

1014

914

88

51.

A. α

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ical

pro

tein

cha

nnel

s10

810

58

118

88

85

1.C

. Tox

in c

hann

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10

00

21

06

00

01.

E. H

olin

s2

10

00

21

00

00

2. E

lect

roch

emic

al P

oten

tial-d

riven

2.A

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ters

8312

086

5666

7443

8887

6921

03.

Prim

ary

Act

ive

Tran

spor

ters

106

154

217

173

131

176

165

155

154

171

218

3.A

. P-P

-bon

d hy

drol

ysis

-driv

ensy

stem

s10

315

121

317

113

116

916

315

314

816

520

4

3.B

. Dec

arbo

xyla

tion-

driv

en sy

stem

s3

24

20

52

23

26

3.D

. Oxi

dore

duct

ion-

driv

en sy

stem

s0

10

00

20

03

48

4. G

roup

Tra

nslo

cato

rs4.

A. P

hosp

hotra

nsfe

r-dr

iven

syst

ems

377

639

3619

1520

301

05.

Tra

nsm

embr

ane E

lect

ron

Car

riers

5.A

. Tw

o-el

ectro

n tra

nsfe

r car

riers

00

00

00

00

00

78.

Acc

esso

ry F

acto

rs In

volv

ed in

Tran

spor

t8.

A A

uxili

ary

trans

port

prot

eins

811

125

96

913

74

6

9. In

com

plet

ely

Cha

ract

eriz

edTr

ansp

ort S

yste

ms

2124

1617

1725

1918

1920

38

9.A

Rec

ogni

zed

trans

porte

rs o

fun

know

n bi

oche

mic

al m

echa

nism

22

11

03

23

02

1

9.B

Put

ativ

e un

char

acte

rized

tran

spor

tpr

otei

ns19

2215

1617

2217

1519

1837

N/A

119

1835

2630

2524

1639

3258

Tota

l28

734

343

929

129

933

928

432

434

430

554

2

Cla

ssSu

bcla

ssPp

eLb

rLc

aLd

eLg

aLc

rSt

hLm

eO

oeB

loB

li

1. C

hann

els

4.5

2.6

2.3

1.7

3.3

4.1

3.2

4.3

2.3

2.6

0.9

1.A

. α-H

elic

al p

rote

in c

hann

els

3.5

2.3

2.3

1.7

2.7

3.2

2.8

2.5

2.3

2.6

0.9

1.C

. Tox

in c

hann

els

0.3

0.0

0.0

0.0

0.7

0.3

0.0

1.9

0.0

0.0

0.0

1.E.

Hol

ins

0.7

0.3

0.0

0.0

0.0

0.6

0.4

0.0

0.0

0.0

0.0

2. E

lect

roch

emic

al P

oten

tial-d

riven

2.A

. Por

ters

28.9

35.0

19.6

19.2

22.1

21.8

15.1

27.2

25.3

22.6

38.7

3. P

rimar

y A

ctiv

e Tr

ansp

orte

rs36

.944

.949

.459

.543

.851

.958

.147

.844

.856

.140

.23.

A. P

-P-b

ond

hydr

olys

is-d

riven

syst

ems

35.9

44.0

48.5

58.8

43.8

49.9

57.4

47.2

43.0

54.1

37.6

3.B

. Dec

arbo

xyla

tion-

driv

en sy

stem

s1.

00.

60.

90.

70.

01.

50.

70.

60.

90.

71.

13.

D. O

xido

redu

ctio

n-dr

iven

syst

ems

0.0

0.3

0.0

0.0

0.0

0.6

0.0

0.0

0.9

1.3

1.5

4. G

roup

Tra

nslo

cato

rs4.

A. P

hosp

hotra

nsfe

r-dr

iven

syst

ems

12.9

2.0

14.4

3.1

12.0

5.6

5.3

6.2

8.7

0.3

0.0

5. T

rans

mem

bran

e Ele

ctro

n C

arrie

rs5.

A. T

wo-

elec

tron

trans

fer c

arrie

rs0.

00.

00.

00.

00.

00.

00.

00.

00.

00.

01.

38.

Acc

esso

ry F

acto

rs In

volv

ed in

Tran

spor

t8.

A A

uxili

ary

trans

port

prot

eins

2.8

3.2

2.7

1.7

3.0

1.8

3.2

4.0

2.0

1.3

1.1

9. In

com

plet

ely

Cha

ract

eriz

edTr

ansp

ort S

yste

ms

7.3

7.0

3.6

5.8

5.7

7.4

6.7

5.6

5.5

6.6

7.0

9.A

Rec

ogni

zed

trans

porte

rs o

fun

know

n bi

oche

mic

al m

echa

nism

0.7

0.6

0.2

0.3

0.0

0.9

0.7

0.9

0.0

0.7

0.2

9.B

Put

ativ

e un

char

acte

rized

tran

spor

tpr

otei

ns6.

66.

43.

45.

55.

76.

56.

04.

65.

55.

96.

8

N/A

a6.

65.

28.

08.

910

.07.

48.

54.

911

.310

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.7To

tal

100

100

100

100

100

100

100

100

100

100

100

a Not

ass

igne

d

Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Lorca et al. Page 36Ta

ble

5D

istri

butio

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tran

spor

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s in

each

supe

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mily

for t

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leve

n or

gani

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naly

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TC

Fam

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li

Cha

nnel

s 

1.A

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IC1

11

11

 1.

A.8

MIP

43

21

14

12

12

2 

1.A

.11

ClC

11

11

22

32

12

 1.

A.2

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11

 1.

A.2

2M

scL

11

11

11

11

11

 1.

A.2

3M

scS

11

11

11

11

11

 1.

A.2

9U

AC

1 

1.A

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Hsp

701

11

11

11

11

11

 1.

A.3

5M

IT2

15

11

32

12

11

 1.

C.1

Col

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2 

1.C

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Lact

ococ

cin

A1

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C.2

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6 

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11

1 

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ph1

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in1

Car

riers

 2.

A.1

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FS-S

P2

33

21

13

62

5 

2.A

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A1

611

53

49

17

85

8 

2.A

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MFS

-DH

A2

89

154

59

18

117

14 

2.A

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MFS

-OPA

11

1 

2.A

.1.5

MFS

-OH

S1

1 

2.A

.1.6

MFS

-MH

S1

12

17 

2.A

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MFS

-FH

S1

11

1 

2.A

.1.8

MFS

-NN

P2

 2.

A.1

.11

MFS

-OFA

11

11

12

 2.

A.1

.14

MFS

-AC

S1

11

2 

2.A

.1.1

5M

FS-A

AH

S1

12

12

31

15

 2.

A.1

.17

MFS

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12

11

12

 2.

A.1

.18

MFS

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2 

2.A

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9M

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1 

2.A

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1M

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31

21

11

11

42

6 

2.A

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3M

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ST1

13

 2.

A.1

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MFS

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F11

1 

2.A

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6M

FS-U

MF2

1 

2.A

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0M

FS-A

DT

11

2 

2.A

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2M

FS-A

CD

E1

13

 2.

A.1

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MFS

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M1

 2.

A.1

.37

MFS

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E1

11

22

 2.

A.1

.38

MFS

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S1

 2.

A.1

.40

MFS

-Azg

A2

21

13

11

22

11

 2.

A.1

.47

MFS

-UM

F61

 2.

A.2

GPH

510

21

12

18

53

 2.

A.3

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PC-A

AT

35

48

56

26

14

4 

2.A

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APC

-APA

22

22

 2.

A.3

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PC-C

AT

23

32

11

23

22

 2.

A.3

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PC-A

CT

2 

2.A

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APC

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T1

1 

2.A

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APC

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21

11

4 

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APC

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54

14

31

 2.

A.3

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PC-L

AT

12

11

12

 2.

A.3

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APC

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T1

21

 2.

A.3

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APC

-AG

T1

12

1

Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Lorca et al. Page 37T

C F

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F2

21

12

22

21

1 

2.A

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RN

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AE1

2 

2.A

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RN

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F2

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A.6

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E21

21

11

33

 2.

A.7

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MT-

SMR

22

11

11

 2.

A.7

.3D

MT-

DM

E3

22

22

21

13

4 

2.A

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DM

T-P-

DM

E1

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A.7

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MT-

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11

12

11

11

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MT-

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12

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A.7

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DM

T-PE

1 

2.A

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8D

MT-

LicB

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A.8

Gnt

P1

31

22

22

 2.

A.9

Oxa

12

22

22

22

22

12

 2.

A.1

0K

DG

T1

 2.

A.1

1C

itMH

S1

1 

2.A

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LctP

12

 2.

A.1

5B

CC

T1

18

 2.

A.1

7PO

T1

11

11

11

 2.

A.1

9C

aCA

1 

2.A

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PiT

13

 2.

A.2

1SS

S1

9 

2.A

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NSS

1 

2.A

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DA

AC

S1

11

11

4 

2.A

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CC

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11

1 

2.A

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AG

CS

14

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A.2

6LI

VC

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31

21

11

11

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A.2

8B

ASS

11

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A.3

3N

haA

11

 2.

A.3

5N

haC

22

11

11

1 

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CPA

12

32

11

12

11

1 

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CPA

22

42

22

12

11

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A.3

8Tr

k2

13

22

4 

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NC

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21

2 

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NC

S23

32

22

23

22

33

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A.4

1C

NT

11

11

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A.4

4FN

T1

1 

2.A

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Ars

B1

11

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A.4

7D

ASS

22

12

1 

2.A

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Am

t1

11

11

11

11

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A.5

0G

UP

1 

2.A

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CH

R2

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A.5

2N

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11

1 

2.A

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SulP

24

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A.5

5N

ram

p3

33

22

13

12

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6TR

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T1

19

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9A

CR

32

2 

2.A

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Dcu

C1

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A.6

2N

haD

1 

2.A

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CPA

310

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A.6

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t1

2 

2.A

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1M

OP-

MA

TE1

31

33

11

31

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6.2

MO

P-PS

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17

21

23

31

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A.6

6.4

MO

P-M

VF

11

3 

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OPT

11

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A.6

8A

bgT

3 

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AEC

22

21

21

15

31

Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Lorca et al. Page 38T

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11

11

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1 

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B1

12

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13

11

1 

2.A

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21

11

21

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3.A

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AB

C-C

UT1

511

39

1815

405

   

3.A

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AB

C-C

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39

63

28

58

612

11 

3.A

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AB

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1119

2213

1127

3217

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BC

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51

55

511

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BC

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T6

1917

408

1323

1012

1638

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A.1

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BC

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T1

11

14

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A.1

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BC

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T5

96

35

65

66

44

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A.1

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BC

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T4

45

33

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A.1

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AB

C-P

OPT

55

44

48

48

4 

3.A

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2A

BC

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T6

610

22

46

14

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A.1

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AB

C-F

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41

44

220

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A.1

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AB

C-M

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36

64

74

113

38

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AB

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AB

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33

31

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AB

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AB

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BC

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37

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96

64

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36

33

63

45

43

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AB

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21

31

14

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AB

C-L

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22

1 

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BC

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 39T

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 40T

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 41Ta

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 42Ta

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 43Ta

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 46Ta

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.

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Lorca et al. Page 47Ta

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Biochim Biophys Acta. Author manuscript; available in PMC 2008 December 1.