Why is entry exclusion an essential feature of conjugative plasmids?

Plasmid 60 (2008) 1–18

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Plasmid

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Review

Why is entry exclusion an essential feature of conjugative plasmids? q

M. Pilar Garcillán-Barcia, Fernando de la Cruz *

Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC-IDICAN,C. Herrera Oria s/n, 39011 Santander, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 November 2007Revised 10 March 2008Available online 28 April 2008

Communicated by Dr. Dhruba K. Chattoraj

Keywords:Entry exclusionSurface exclusionPlasmid conjugationConjugation inhibition

0147-619X/$ - see front matter � 2008 Elsevier Incdoi:10.1016/j.plasmid.2008.03.002

q The authors want to dedicate this work to thLederberg, died on February 2nd, who started the suand entry exclusion.

* Corresponding author. Fax: +34 942 201945.E-mail address: [email protected] (F. de la Cru

Entry exclusion is a property of plasmids by which the cells that contain them become badrecipients in additional conjugation rounds. This work reviews entry exclusion essentialfeatures and analyzes the mechanisms of action of the best studied systems. We searchedfor homologs of the proteins responsible for experimentally known exclusion systems.Results were used to classify exclusion systems in families of related elements. We arriveto the conclusion that all conjugative plasmids contain at least one entry exclusion gene.Although entry exclusion genes seem to be part of the plasmid conjugative machinery, theyare systematically absent in phylogenetically related type IV protein exporting machinesinvolved in virulence for plants and animals. We infer from this fact that entry exclusionis an essential feature of conjugative plasmid biology. Mathematical models suggest thatplasmids expressing entry exclusion selectively eliminate plasmids lacking it, reinforcingits essential character and suggesting that entry exclusion plays a direct role in plasmidsurvival. Other experimental results confirm that entry exclusion is essential for the stabil-ity of a conjugative plasmid. We suggest that entry exclusion limits the damage of lethalzygosis (bacterial death produced by excessive rounds of conjugation). Additionally, itavoids competition in a host among identical plasmid backbones. Conversely, the lack ofentry exclusion in conjugative transposons can be understood as a means of generatingrapid evolutionary change.

� 2008 Elsevier Inc. All rights reserved.

1. The early story

Early in conjugation research history, it was noticedthat exponentially growing cells harboring the F sex factorwere not suitable as conjugation recipients (Lederberget al., 1952). This barrier was partially abolished eitherby treatment with periodate (Sneath and Lederberg,1961), by starvation in a saline buffer gelatine (Curtisset al., 1969) or by growing cells into stationary phase(Lederberg et al., 1952), a behavior termed ‘‘F� phenocopy-ing” (Achtman et al., 1977). Such inefficient transfer wasnamed ‘‘superinfection immunity” (Watanabe, 1963,

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e memory of Joshuabjects of conjugation

z).

1967). In the following years, abundant experimental dataaccumulated on this phenomenon, beginning the decipher-ing of its nature. If a donor cell harbored two different sexfactors and the recipient just one of them, exclusion oper-ated only against DNA transfer of the factor present in therecipient strain, underscoring the plasmid-specificity ofexclusion (Novick, 1969). Further experimental evidenceindicated an association between exclusion and sex pilustype: all sex factors that excluded one another producedthe same sex pilus type, though not all members of one pi-lus class showed exclusion to one another (Edwards andMeynell, 1968; Meynell et al., 1968; Ohki and Ozeki,1968; Watanabe et al., 1964). Nevertheless, the fact thatdifferent plasmids with similar or identical sex pili presentin different cells did not exclude each other (Watanabe andFukasawa, 1962) suggested that shared pili were necessarybut not sufficient for exclusion. Also supporting this notionwas the fact that there was inhibition of plasmid transfer

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2 M.P. Garcillán-Barcia, F. de la Cruz / Plasmid 60 (2008) 1–18

to a recipient cell harboring the same element even whenpilus formation was stringently repressed in the recipientcell (Novick, 1969).

On the other hand, whereas conjugative transfer of aplasmid was inhibited by its presence in the recipient cell,no such inhibition was observed if the donor plasmid wastransferred by transduction (Watanabe et al., 1968). Fur-thermore, neither a second F factor could be stably main-tained in an F-containing strain even when the entrybarrier was removed (Dubnau and Maas, 1968), nor a coli-cin-factor that was not excluded by F (Margolin and Bau-erle, 1966). Those observations led to the considerationthat superinfection immunity was a combination of twodifferent and independent phenomena: ‘‘plasmid incom-patibility” (an interference between closely related sex fac-tors associated to their replication) and ‘‘entry exclusion”(a barrier to the physical transfer of DNA between cells car-rying isogenic or closely related sex factors) (Novick,1969).

Kinetics of radiolabeled DNA degradation after transferwas the same in exclusion-deficient than in exclusion-proficient recipient minicells (Sheehy et al., 1972). Alsothe size of the transferred DNA molecules, as measuredby alkaline sucrose gradient centrifugation, was the samein both recipients. Thus, the possibility that entry exclu-sion resulted from events occurring after donor DNA en-ters the recipient was ruled out. Differences did occur inthe quantity of labeled DNA transferred, which wasaround 20-fold higher when non-excluding minicellswere used as recipients. Thus, it became clear that theentry exclusion barrier operated by inhibiting physicalentry of the incoming DNA into cells that exhibit theexclusion phenotype (Sheehy et al., 1972). A quantitativeexpression of entry exclusion was coined by the term‘‘exclusion index” (EI), that is, the transfer frequency ofa given plasmid to a plasmid-free recipient divided bythe frequency of transfer to a recipient carrying the sameplasmid. For instance, plasmid F showed an EI of 100–300in matings between Escherichia coli since it transferred100–300 times better to a plasmid-free recipient thanto an F+ recipient (Achtman et al., 1977; Achtman andSkurray, 1977).

2. Exclusion activity in F-like plasmids

2.1. Discovery of two exclusion genes

The F plasmid contains two exclusion systems. As willbe shown later, all conjugative plasmids contain at leastone, most of the times related to either one of the two Fsystems. Thus, we will start by first reviewing the F exclu-sion systems, which can be considered as prototypes for allothers.

Several F mutants that became good recipients inF+ � F+ conjugation were isolated and used to measurethe number of mating pairs formed after mixing donorand recipient cells (Achtman et al., 1971). A clear correla-tion between mating-pair formation and lack of exclusionwas observed. Thus, it was inferred that exclusion inhib-ited conversion of unstable to stable mating aggregates.In contrast, when using a recipient with or without an F-

like R factor, significant differences in the concentrationof mating aggregates between the R� and R+ cultures werenot detected, although there was a highly significant de-crease in the number of transconjugants (Eckerson andReynard, 1977). This fact indicated that two different phe-nomena were occurring at the same time. The first one,acting at the level of formation of stable mating aggregateswas termed ‘‘surface exclusion”, while ‘‘entry exclusion”seemed to inhibit conjugation at a stage occurring laterthan formation of mating aggregates. This apparent para-dox was solved by Achtman et al. (1977), who found thatF factor exerts exclusion by two mechanisms based intwo different F plasmid genes, traT and traS. The productof traT, independently identified as protein S (Minkleyand Ippen-Ihler, 1977), acts on the outer membrane andresults in reduced ability of recipient cells to form stablemating aggregates (Achtman et al., 1977). Thus, the TraT-dependent phenotype was hereafter called surface exclu-sion (SFX), leaving the term entry exclusion (EEX) to namethe TraS-dependent exclusion.

When donor cells harboring IncI plasmid R64-11 wereincubated with minicell recipients derived from eitherR100-1+, Col+, F+ or F� parent strains, an increase in donorconjugative DNA synthesis, ranging from three to morethan 12-fold, was observed (Fenwick and Curtiss, 1973).On the other hand, if minicell recipients were derived fromR64-11+ cells, such increment was not observed. Similarresults were obtained when using donor and recipient cellscarrying plasmid F: no increase in DNA synthesis was pro-duced in the case of F+ recipients, against more than 10-fold stimulation when F� recipient cells were used (Kings-man and Willetts, 1978). These observations led to theconclusion that potential recipient cells or minicells whichexhibited exclusion either never form mating pairs withdonor cells or, if they do, they do not generate the signalwithin the donor cells to begin conjugative transfer repli-cation. In apparent contradiction, (Ou, 1975) observed thatF+ derived minicells when acting as recipients, were able tostimulate donor conjugative DNA synthesis to the same ex-tent than minicells prepared from an F� strain. Neverthe-less, stimulation did not occur when using wholerecipient cells. Besides (Achtman et al., 1977) detected thattraS+ traT� mutants promoted higher reduction in DNAtransfer than traS� traT+ mutants. Thus, it is TraS that actsprimarily by inhibiting DNA transfer even when stablemating aggregates have been formed.

No additional genes on F showed an exclusion pheno-type (Achtman et al., 1980). Most F exclusion activitywas attributable to TraS (EI of around 200, versus 20 forTraT). It was also found that the EI was gene dosage depen-dent since, when traS and traT were cloned in a multicopyplasmid, the EI increased to 10,000 (Achtman et al., 1977;Skurray et al., 1976).

Finally, an early hint on the physiological importance ofexclusion was the observation that F mutants carrying traSand traT mutations could not be isolated by chemicalmutagenesis (Achtman et al., 1977, 1980). Thus, transfer-proficient exclusion-deficient F mutants are unstable un-der normal laboratory growth conditions (Achtman andHelmuth, 1975). This result suggests that, somehow, exclu-sion is essential for the stability of a conjugative plasmid,

Fig. 1. Genetic organization of the exclusion gene-containing regions. (A)F-like; (B) IncP-like; (C) IncN-like; (D) IncI-like; (E) IncH-like; (F) ICEsR391 and SXT; (G) ColE1-like; (H) Pheromone-responding conjugativeplasmids. Arrows corresponding to exclusion genes are shadoweddepending on the exclusion system (light gray for exclusion genes ofpheromone-responding conjugative plasmids, intermediate gray forgenes encoding inner membrane exclusion proteins and dark gray forgenes encoding outer membrane exclusion proteins). virB6 homologs aredashed. The remaining genes are filled in white.

M.P. Garcillán-Barcia, F. de la Cruz / Plasmid 60 (2008) 1–18 3

an observation that in our opinion has not drawn theattention it deserved.

2.2. Genetic organization

traS and traT genes map in adjacent loci of plasmid F,between transfer genes traG and traD (Fig. 1A). This geneticlocation is conserved in other F-like plasmids (Fig. 1A).Nevertheless, in spite of being inserted within the tra oper-on, neither traS nor traT were essential for F pilus synthesisnor for DNA transfer (Achtman et al., 1980).

Nucleotide sequence analysis of the F region comprisingtraS and traT revealed potential Shine–Dalgarno sequencesand putative promoters, PS and PT, for the respective genes,which were active as galK transcriptional fusions (Cheahet al., 1986; Jalajakumari et al., 1987). A strong transcrip-tion termination signal lies immediately downstream traT(Ham et al., 1989). traS and traT mRNAs produced fromthese promoters are unusually stable (Ham et al., 1989).TraT protein levels remain fairly constant during thegrowth cycle, even into stationary phase (Achtman et al.,1977; Frost and Manchak, 1998). Thus, the cause of exclu-sion laxity during stationary phase still remains to be elu-cidated. For other F-like plasmids such as pED208 andR100, potential traT specific promoters were also identified(Finlay and Paranchych, 1986; Ferrazza and Levy, 1980a;Ogata et al., 1982). In the case of pED208, traT was highlyexpressed independently of a vector promoter in E. coliminicells (Finlay and Paranchych, 1986).

Due to their position within the F tra region, it was sug-gested that transcriptional regulation of traS and traTshould, at least in part, coincide with that of other tra genes(Achtman et al., 1980), that is, be dependent on the tra op-eron positive regulator TraJ (Willetts, 1977). Favoring thishypothesis, reduced amounts of TraT accumulated in theouter membrane of non-leaky traJ mutant cells (Achtmanet al., 1980). Nevertheless, other experimental data pointedto an, at least partial, TraJ-independent production ofexclusion proteins: Mu-1 insertions in traJ did not com-pletely abolish function of promoter-distal tra cistrons(Helmuth and Achtman, 1975). Besides, reduced expres-sion of traJ had much less effect on traT_F expression thanon transfer (Rashtchian et al., 1983). Finally, synthesis ofTraT_R100 was found to be constitutive (Ferrazza andLevy, 1980a). These observations suggest that traT expres-sion is largely independent of TraJ.

2.3. Protein TraT

2.3.1. Biochemical characteristicsTraT is synthesized as a 25,932 Da precursor protein

(pro-TraT) (Ferrazza and Levy, 1980b; Jalajakumari et al.,1987). A 21 amino acid hydrophobic signal is cleaved offduring its transport to the outer membrane, rendering amature protein of 23,709 Da (Minkley, 1984; Jalajakumariet al., 1987) (see Table 1). After pro-TraT is synthesized,it is modified by esterification with a diacylglyceride inthe cysteine residue present at the signal sequence cleav-age site prior to processing (see Fig. 2). A third acyl groupis added via amide linkage to that cysteine after processing(Perumal and Minkley, 1984). More than 20,000 copies of

TraT protein accumulate per F+ exponentially growing cell,making it a highly expressed protein in E. coli. They localizeto the outer membrane (Achtman et al., 1977), where theyform multimeric aggregates (Manning et al., 1980). A sub-stantial fraction of the protein resides in the external face

Table 1Prediction of transmembrane helices in exclusion proteins

Protein (AccessionNo.)

Transmembrane protein prediction

MEMSAT3 DAS TMpred TMHMM TopPred SOSUI

TraT_F (BAA97971) 7(o)–25(i) 142(o)–161(i) 11–18 144–152 7–25 139(o)–156(i) 7(i)–29(o) 7(i)–27(o) 137(o)–157(i) 1–28 (s.p.)TraS_F

(NP_061479)32(i)–49(o) 65(o)–82(i) 108(i)–127(o)130(o)–147(i)

37–51 66–81 109–144

34–53 63(o)–79(i)108(i)–131(o)

35(o)–54(i) 61(i)–83(o) 98(o)–120(i)122(i)–144(o)

34(i)–54(o) 60(o)–80(i)126(i)–146(o)

32–54 59–80 108–130138–160

TraS_R100(NP_052977)

25(o)–48(i) 68(i)–86(o) 9–20 31–47 75–92 33(i)–49(o) 67(o)–95(i) 5(i)–27(o) 31(o)–48(i) 67(i)–89(o) 5(i)–25(o) 30(o)–50(i)73(i)–93(o)

1–23 (s.p.) 29–51 72–94

TrbK_RP4(AAA26437)

1–13 (s.p.) 33(o)–50(i) 8–19 6(i)–22(o) 5(i)–22(o) 4–24 1–22 (s.p.)

TrbK_R751(AAC64452)

1–8 (s.p.) 9–20 3(i)–22(o) — 3–23 1–23 (s.p.)

Eex_pKM101(NP_511192)

1–11 (s.p.) 30(o)–47(i) 6–15 4(i)–24(o) — 1–21 1–16 (s.p.)

Eex_R388(CAA57026)

1–12 (s.p.) 33(o)–52(i) 7–14 — — 1–21 —

ExcA_R64(BAA78014)

24(i)–41(o) 55(o)–78(i) 25–39 60–71 25(i)–45(o) 57–76 20(i)–42(o) 57(o)–76(i) 24–44 56–76 —

EexA_R27(NP_058232)

1–10 (s.p.) 53(o)–71(i) 7–13 3(i)–19(o) — 1(i)–21(o) 1–18 (s.p.)

EexB_R27(NP_058230)

1–10 (s.p.) 202(i)–220(o) 8–15 4(i)–23(o) — 4(i)–24(o) 1–19 (s.p.)

EexS_SXT(ABO64671)

12(i)–29(o) 41(o)–58(i) 64(i)–81(o)84(o)–101(i)

14–25 43–56 68–7887–99

12(i)–28(o) 41–5983(o)–103(i)

12(i)–31(o) 41(o)–59(i) 66(i)–83(o)87(o)–104(i)

12–32 39–59 81–101 1–33 (s.p.) 37–59 61–8385–107

MbeD_ColE1(AAB59137)

37(o)–54(i) — — — — —

Exclusion proteins were screened for transmembrane helices using several predictors available online: MEMSAT3: http://bioinf.cs.ucl.ac.uk/psipred/ (Jones, 2007; Jones et al., 1994); DAS: http://www.sbc.su.se/�miklos/DAS/ (Cserzo et al., 1997); TMpred: http://www.ch.embnet.org/software/TMPRED_form.html (Hofmann and Stoffel, 1993); TMHMM: http://www.cbs.dtu.dk/services/TMHMM-2.0/; TopPred: http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=toppred (Claros and von Heijne, 1994; von Heijne, 1992); SOSUI: http://bp.nuap.nagoya-u.ac.jp/sosui/ (Gomi et al., 2004; Hirokawa et al., 1998). Defaultparameters were used. Initial and final sequence positions of the helices are showed. When possible, orientation of the helices (outside to inside: (o)–(i) or inside to outside: (i)–(o)) is indicated. s.p., means forpossible N-terminal signal peptide.

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http://bioinf.cs.ucl.ac.uk/psipred/

http://www.sbc.su.se/~miklos/DAS/

http://www.sbc.su.se/~miklos/DAS/

http://www.ch.embnet.org/software/TMPRED_form.html

http://www.cbs.dtu.dk/services/TMHMM-2.0/

http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=toppred

http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=toppred

http://bp.nuap.nagoya-u.ac.jp/sosui/

Fig. 2. Phylogenetic tree of TraT and amino acid sequence alignment of the N-terminal part of the proteins. A neighbor-joining analysis with bootstrapvalues (1000 replicates, indicated as percentage in each node) was carried out using Mega 3.1 (Kumar et al., 2004). Pairwise matrix of genetic distances wascalculated using Poisson correction. The clade containing the homologs with more than 50% amino acid identity is shadowed in gray. In the right part of thefigure, the corresponding sequence of the N-terminal part of the proteins aligned (GenBank Accession No. in Supplementary Table S1). The putative cysteinesite of lipid attachment is indicated by an arrow in the alignment. Color legend in the alignment: white on black, invariant amino acids; black on dark gray,strongly conserved; black on light gray, similar; gray on white, weakly similar.


of the outer membrane (Aguero et al., 1984; Manning et al.,1982). Unlike major outer membrane proteins, the amountof TraT incorporated into the membrane is not affected inlipopolysaccharide-deficient mutants (Manning et al.,1980). Nevertheless, there are several chromosomal muta-tions that negatively affect TraT-mediated SFX. Mutationsin gene cpxA, whose product is involved in envelope stressresponse (McEwen and Silverman, 1980), in transcriptionalcontrol factors sfrA and sfrB (Beutin and Achtman, 1979), infexA and fexB, related to fertility expression (Lerner andZinder, 1982), or in adenylate cyclase gene cya and cyclicAMP receptor protein gene crp (Kumar and Srivastava,1983), all were shown to decrease SFX.

TraT is likely to be anchored to the outer membrane bylipid–lipid and lipid–protein interactions (Perumal andMinkley, 1984). When bound to the outer membrane, TraToligomeric form is resistant to denaturation by high tem-perature, to solubilization by detergents and to proteolyticcleavage by proteases (Manning et al., 1980; Minkley andWilletts, 1984). Although pro-TraT appears to be success-fully translocated to the outer membrane, it is defectivein forming the oligomeric structure required for SFX func-tion (Minkley, 1984). Plasmid F mature protein TraT addedin vitro to mixtures of donor and recipient cells specificallyinhibited F transfer in a dosage-dependent manner (Mink-ley and Willetts, 1984).

2.3.2. The mechanism of TraT-mediated surface exclusionTraT specificity resides in the N-terminal 180 amino

acids of the protein, since a hybrid TraT carrying an inser-tion of a foreign antigenic determinant, the C3 epitope of

polio virus, at position 180, retained partial SFX activity(Taylor et al., 1990). Mature TraT proteins from F andR100 differ only by a single residue at position 120 (Glyfor TraT_F, Ala for TraT_R100). Despite high similarity,purified preparations of TraT_F inhibited F plasmid transfer3-fold more than those of TraT_R100 (Minkley and Wil-letts, 1984). Secondary structure predictions indicated thatthe Gly to Ala change creates an additional b-turn whichmay radically alter the local structure of this region ofthe protein (Sukupolvi and O’Connor, 1990). TraT proteinsfrom plasmids belonging to different SFX specificity groupsalso have differences in this hydrophilic region (aa 116–120 of the mature proteins) that could be exposed in theouter membrane, interacting with the donor cell (Suk-upolvi and O’Connor, 1990). This segment was swappedbetween TraT proteins of plasmids R-100 and CoIB2-K98and resulted in switching of SFX specificity (Harrisonet al., 1992).

Hfr transfer was not excluded by an F+ recipient whenthe donor also contained an F-like R factor, and thus itssex pili contained, in addition to F pilin, an R-pilin of differ-ent specificity (Meynell and Ewins, 1973). (Frost et al.,1985) found a correlation between differences in the ami-no-terminal region of pilins and SFX specificities of differ-ent F-like plasmids. Those observations focused pilin as thedonor partner in the interaction with recipient TraT. It wassuggested that TraT binds to the tip of the F pilus prevent-ing its interaction with a receptor site on the recipient cellsurface (Willetts and Maule, 1974; Minkley and Willetts,1984). Later, it was proposed that TraT blocks the pilusreceptor in the recipient cell. Evidence along this line


was the detection of a direct interaction between TraT andOmpA, likely through TraT amino acids 110–150, inhibitingOmpA-specific phage binding and probably masking thepilus binding site on OmpA (Riede and Eschbach, 1986).Nevertheless, F transfer system expressing either F, R100-1 or ColB2 pilin genes was unaffected by the presence ofTraT_R100-1 in the recipient cell (Anthony et al., 1994).Thus, differences in pilin sequences were not responsiblefor recognizing specific groups in LPS, OmpA or TraT. Afterthat, it was known that TraN, an outer membrane proteinof plasmid F, and not the F pilus, interacts with OmpA dur-ing conjugation, resulting in mating-pair stabilization (Kli-mke and Frost, 1998; Klimke et al., 2005). Nevertheless, itwas ruled out TraN as responsible for TraT-dependent sur-face exclusion (Klimke and Frost, 1998). The presence of anadhesin at the pilus tip recognized by TraT was also pro-posed but not demonstrated (Anthony et al., 1994). Up today, no further candidates have been analyzed; so, the do-nor-residing counterpart interacting with TraT remains tobe established.

2.3.3. Applications of TraT in basic research andbiotechnology

TraT localization has been exploited to transport foreignantigenic epitopes to the bacterial cell surface, where thehybrid TraT proteins led to exposure of recombinant anti-gens to the medium. This E. coli display system might beuseful for novel vaccine strain construction (Taylor et al.,1990; Harrison et al., 1990; Chang et al., 1999).

TraT has also been used to study the outer cell mem-brane of gram-negative bacteria due to its effects on mem-brane permeability to hydrophobic compounds likeantibiotics. When hydrophobic amino acids of TraT secondmembrane domain (aa 117–135) were changed by chargedand proline residues, an increase in membrane permeabil-ity to hydrophobic compounds was produced (Sukupolviand O’Connor, 1987; Sukupolvi et al., 1990). On the otherhand, the introduction of a wild type traT gene restoredthe resistant phenotype against hydrophobic compoundsof a Salmonella typhimurium strain with a plasmid-carriedpermeability mutation (Sukupolvi et al., 1986).

Besides SFX, TraT plays an additional role in bacterial vir-ulence. It was identified as responsible for plasmid-speci-fied serum resistance in E. coli (Binns et al., 1982; Mollet al., 1980; Ogata et al., 1982), S. typhimurium (Rhen andSukupolvi, 1988) and Salmonella enterica (Sukupolvi et al.,1992). traT mutational inactivation eliminated serum resis-tance mediated by F-like plasmids (Moll et al., 1980; Suk-upolvi et al., 1992) and reduced the growth rate of S.typhimurium within the liver (Rhen and Sukupolvi, 1988).TraT inhibited the lysis of sensitized erythrocytes by serumcomplement (Pramoonjago et al., 1992) and decreasedE. coli sensitivity to phagocytosis by macrophages throughantagonism with complement opsonisation (Aguero et al.,1984). Cells expressing TraT altered the complement distri-bution leading to an irregular deposition of the complementcomponents. This activity was enhanced when the proteinwas produced from a high-copy-number vector (Agueroet al., 1984). It is possible that the primary role of TraT isthe modification of cell surface to favor bacterial invasive-ness, SFX being an ‘‘unintended” additional phenotype.

2.3.4. TraT as a protein familyA PSI-BLAST search (Altschul et al., 1997; Schaffer et al.,

2001) using TraT_F as a query recovered a large number ofTraT proteins from plasmids hosted by Enterobacteria(most of them F-like), as well as chromosomally-locatedhomologs exhibiting more than 77% identity (Supplemen-tary Table S1, Fig. 2). Some traT genes, like those harboredby plasmids pSCV50 and pOU1113, are placed betweengenes homologous to traG and traD genes of plasmid Fbut, interestingly, without an accompanying traS gene.Others like pYV plasmids from Yersinia enterocolitica andpCD plasmid from Yersinia pestis code for TraT homologswithout T4SS-related genes in the neighborhood. The sur-face exclusion gene ylpA from Y. enterocolitica hybridizedwith pYV plasmid of Yersinia pseudotuberculosis, suggestingits conservation among Yersinia spp. (China et al., 1990).This gene is co-regulated with the yop genes that codefor Yop virulence effectors translocated by T3SS, suggest-ing that YlpA could be also implicated in virulence,although mutations in ylpA affected neither resistance tohuman serum nor virulence in intravenously inoculatedmice (China et al., 1990). The TraT homolog present inenterohemorrhagic E. coli strain O157:H7 is coded by agene out of a tra context, placed close to a truncated ver-sion of IS600-1, in a CP4-like prophage not present in thereference E. coli strain K12 (Hayashi et al., 2001; Pernaet al., 2001). Besides, its closely related homolog from plas-mid pO113 is located out of the tra region of that plasmid.This tra region contains an exclusion protein related to nei-ther TraT nor TraS, but homologous to ExcA_R64 (see Sec-tion 3.3 and Supplementary Table S5). It can be concludedthat, in many cases, the presence of a TraT protein is inde-pendent of a conjugation-related exclusion activity. De-spite the different genetic background of the homologscommented above, all lay together in a monophyleticgroup (gray-shadowed in Fig. 2), pointing to a commonancestor.

Besides the highly related shadowed homologs, thereare other TraT-like proteins with lower but still significanthomology (28–44% identity) that are present in plasmidsand chromosomes of several bacteria, ranging from b, d, eand c-Proteobacteria or even from Fusobacteria. Many ofthem are in a context devoid of other transfer-relatedgenes. To our knowledge, no experimental evidence ofthe functionality of these proteins has been provided inthe literature and they are generally annotated like serumcomplement resistance proteins.

A putative role in virulence could be suggested by theirpresence in the chromosome of pathogenic microorgan-isms like genital tract pathogen Campylobacter fetus (Foutsand Nelson, 2006, direct submission to NCBI database),enteropathogen Campylobacter upsaliensis RM3195 (Foutset al., 2005) and the oral disease-related bacterium Fuso-bacterium nucleatum subsp. vincentii (in the vicinity ofgenes related to hemoglobin degradation) (Kapatral et al.,2002). Curiously, every Campylobacter strain sequencedbut C. upsaliensis RM3195 is resistant to most b-lactamantibiotics, being this lack of resistance likely due to thedisruption of a class D b-lactamase gene, just placed con-tiguously to traT (Fouts et al., 2005). Although the functionTraT could have in bacteria living in such different niches


like marine water (Pseudoalteromonas haloplanktis andThiomicrospira denitrificans), wastewater (Nitrosomonaseutropha), endocellular (a small, non-conjugative plasmidharbored by the obligate endosymbiont of tsetse flies Wig-glesworthia glossinidia) is not established, it could be sus-pected that the presence of TraT produces differences inthe cell envelope that provides a selective advantage tothe host. Thus, the presence of TraT-like proteins in thechromosome of some bacteria suggests that SFX increasesbacterial fitness.

When a PSI-BLAST search against a TraT query con-verges, no protein with alternative function is retrieved.This result points to the existence of a coherent family thatcomprises only TraT-like proteins. Almost all homologsharbor the conserved cysteine residue that is lipid-modi-fied in TraT_F. No structural data on TraT proteins is avail-able. Analysis of TraT_F sequence similarity to knownatomic structures by threading (using software Phyre)did not result in significant homologs in the PDB database,perhaps a reflection of the scarce number of membraneprotein structures in the database.

2.4. TraS

2.4.1. Biochemical characteristicsThe second gene responsible for F exclusion, traS, en-

codes a 16,861 Da protein that has been less studied thanTraT. TraS is extremely hydrophobic, except for a hydro-philic domain from amino acid residues 10–24 (Jalajaku-mari et al., 1987). It does not contain an apparent signalsequence and localizes in the inner cell membrane (Acht-man et al., 1979) (see Table 1). No lipid modification hasbeen observed (Fig. 3).

2.4.2. The mechanism of TraS-mediated entry exclusionThe mechanism by which TraS mediates entry exclusion

involves the presence of inner membrane protein TraG_F inthe donor (Anthony et al., 1999; Audette et al., 2007). TheC-terminal portion of TraG is predicted to be located in theperiplasm, interacting with TraN to stabilize mating pairs,while its N-terminal domain bears homology with thelarge periplasmic domain of integral inner membrane pro-tein VirB6_pTi, involved in pilus tip formation (Firth and

Fig. 3. Amino acid sequence alignment of TraS proteins. Color legend like in Figand S2B.

Skurray, 1992). Assays in which a donor carrying a traGmutant of plasmid F was mated with a recipient containinga clone expressing both TraS and TraT resulted in exclusionwhen the mutant was complemented with traG_F but notwith the heterologous R100-1 traG gene (Anthony et al.,1999). Besides, the high level of exclusion observed wassuspected to be due to TraS since it has a greater impacton transfer reduction than TraT (Achtman et al., 1977).Plasmid-specific recognition between TraG and TraS toproduce EEX activity was later confirmed. Using TraG_F /TraG_R100 chimeras, EEX activity was mapped to aa610–673 in TraG_F, the only region that differs signifi-cantly from TraG_R100 (Audette et al., 2007). This regionis predicted to be located in the periplasm (Firth and Skur-ray, 1992; Frost et al., 1994). TraG did not interfere withEEX function of TraS when both proteins were co-producedin the recipient cell (Audette et al., 2007). To explain theseresults, it was proposed that TraG may be translocated tothe recipient cell, where it would contact the inner mem-brane, initiating transfer, a process blocked by TraS (Aud-ette et al., 2007).

2.4.3. TraS as a protein familySequence conservation is higher among traT than

among traS genes (Jalajakumari et al., 1987; Anthonyet al., 1994), so it is not surprising that only a few homo-logs appear in a PSI-BLAST search against TraS_F orTraS_R100 as queries (Supplementary Tables S2A andS2B, respectively). In contrast to what happened with TraT,TraS_R100 was not recovered as a homolog when TraS_Fwas used as query and vice versa. Plasmid pED208, the IncFmember with the lowest homology to TraT_F (80% aminoacid identity), has an exclusive TraS protein with homologyto neither TraS_F nor TraS_R100. No additional proteinscorresponding to other exclusion systems were obtainedin each case and no significant TraS structure predictionis obtained by threading.

3. Entry exclusion in other systems

Previous reviews on entry/surface exclusion by Suk-upolvi and O’Connor (1990) and Frost et al. (1994) col-lected data obtained from TraT and TraS proteins but did

. 2. GenBank Accession No. of proteins aligned in Supplementary Table S2


not extend their analysis to other conjugative systems,probably because limited information was available at thattime. The great expansion of sequenced genomes has con-tributed to broaden the range of conjugative elements forwhich the exclusion phenomenon has been studied. Now-adays, exclusion is no longer regarded as a property of F-like plasmids, but as a general characteristic of conjugativeplasmids. In most cases in which a detailed analysis wascarried out, a single gene confers the exclusion phenotypeby a mechanism that seems to be more related to EEX(TraS) than to SFX (TraT). As stated, among F-like plasmidsthe protein responsible for EEX, TraS, is highly variable inamino acid sequence. The same applies for the EEX pro-teins contained in other plasmids. Homologs to the beststudied members of the plasmid groups from Gram nega-tive bacteria listed below form coherent and independentfamilies. Into each group, synteny of the entry exclusiongene with other genes related to transfer is highly main-tained (Fig. 1B–H). Neither structural data on EEX proteinsnor precise 3D model by threading is available. Althoughautomatically annotated EEX genes can be found in manyplasmids, here we summarize data on EEX based exclu-sively on available experimental evidence.

3.1. Entry exclusion in IncP plasmids

IncPa plasmid RP4 was shown to have an EEX system(Hedges and Jacob, 1974). Two EEX proteins, TrbJ and TrbK,were initially postulated (Lyras et al., 1994)—or EexA andEexB for the probably identical plasmid R18 (Lessl et al.,1991). In fact, IncPa plasmids carry a single EEX gene, trbK,coordinately expressed with the mating-pair formationgenes of the Tra2 region (Haase et al., 1996, 1995)(Fig. 1B). Its presence in the recipient cell, but not in thedonor, is required for exclusion (Haase et al., 1996). TrbKis needed for pilus overexpression but not for conjugativetransfer (Haase et al., 1995). Its exclusion activity is dosagedependent: a 15-fold reduction is produced when RP4 ispresent in the recipient cell, but EI increases to 2 � 107

when trbK is cloned in a multicopy plasmid (Haase et al.,1995).

The unprocessed TrbK polypeptide (69 aa long) containsa lipoprotein signature (Lessl et al., 1992) that resides inthe N-terminal cysteine residue of the 47-aa mature pro-tein (Haase et al., 1996). Modification of residue Cys-23consists in a thiol-bounded bis(palmitoyloxy)-propyl andan N-terminal palmytoil group (see Fig. 4). This modifica-

Fig. 4. Amino acid sequence alignment of TrbK-like proteins. The conserved cylegend like in Fig. 2. GenBank Accession No. of proteins aligned in Supplementa

tion seems to be crucial for the cytoplasmic membranelocation of TrbK (facing the periplasm) but not for exclu-sion activity, since the mutant protein TrbKC23G—whichcannot be modified or proteolytically processed and whoselocation is mainly cytoplasmic and periplasmic—still actedin entry exclusion with a 10-fold reduced efficiency (Haaseet al., 1996). On the other hand, an 8 aa truncation at the C-terminus of TrbK—that still allows the protein to be pro-cessed—resulted in complete loss of EEX activity.

Transfer of the mobilizable plasmid RSF1010 throughthe T4SS of a co-resident RP4 was affected by the presenceof TrbK in the recipient cell to the same extent as RP4transfer itself (Haase et al., 1996). This result ruled outthe possibility of a specific interaction between TrbK andthe transferred ssDNA or any of the relaxosome proteins,constraining the donor partners to the components oftransport machinery, pilus and coupling protein.

A PSI-BLAST analysis using TrbK_RP4 as a query did notrecover any homolog, except for a 100% hit to plasmidpTB11 isolated from uncultured bacteria. Nevertheless,using protein TrbK from the IncPb plasmid R751 as a query,various homologs were obtained (Supplementary TableS3). Despite their lack of homology, TrbK_RP4 exerts exclu-sion against plasmid R751. This EI is only 3-fold lower thanthat against RP4 (Haase et al., 1996). Eye-inspection ofTrbK_RP4 and TrbK_R751 (GenBank Accession Nos.AAA26437 and AAC64452, respectively) reveals local se-quence conservation at their C-terminal ends (7 out of 9identical residues) that could be responsible for TrbK_RP4exclusion activity (see Fig. 4). All trbK_R751 homologs arelocated upstream gene trbL (Fig. 1B), even in the cases inwhich most tra genes are absent (chromosomes of Xantho-monas campestris and Pseudomonas aeruginosa). They havea Cys residue (around position 23) that could be lipid-mod-ified as Cys-23 of TrbK_RP4.

3.2. Entry exclusion in IncN and IncW plasmids

IncN plasmid pKM101 and IncW plasmid R388 bothcontain a single gene responsible for entry exclusion, eex,located within the mating-pair formation gene cluster(Bolland et al., 1990; Winans and Walker, 1985) (seeFig. 1C). EI was 6400 for Eex_pKM101 (Winans and Walker,1985) and 200 for Eex_R388 (Bolland et al., 1990). In thecase of pKM101, Tn5 insertions on neither side of geneeex affected protein Eex expression. Besides, an eex::Tn5insertion was not polar on downstream transfer genes.

steine residue that is modified in TrbK_RP4 is marked by an arrow. Colorry Table S3.


These results suggested that eex carries its own promoterbut that expression of downstream transfer genes is notdependent on it (Winans and Walker, 1985). WhenpKM101 DNA sequence became known, it was proposedthat eex initiation codon was translationally coupled totraC while its termination codon was coupled to traD, thegenes located upstream and downstream to eex, respec-tively (Pohlman et al., 1994). The EI is greatly enhancedby protein overexpression (around 60,000-fold reductionwhen eex_pKM101 is expressed from the lac promoter)(Pohlman et al., 1994). Computer analysis of the predictedEex_pKM101 protein suggests that it may be exported andcovalently modified by the addition of lipids to residueCys-15 (Pohlman et al., 1994) (see Table 1 and Supplemen-tary Fig. S1).

When Eex_pKM101 was used as query in a PSI-BLASTsearch, many EEX proteins were retrieved; most of themshowing less than 40% sequence identity (SupplementaryTable S4 and Supplementary Fig. S1). This is the broadestfamily we analyzed. Some members are harbored by plas-mids with similar T4SSs, like R46 (IncN) and R388 (IncW),but also by less related, like R6K (IncX) and pXF51 (Xylella).There are also members surrounded by tra genes located inchromosomal genomic islands of pathogenic bacteria thatcontain integrated plasmids (i.e. the cases of ECA1618,Bcen_2659, BB0489, XCC1611 and XC2620, ORF5). It is note-worthy the presence of an EEX homolog, ORF5, in an integra-tive and conjugative element (ICE) of E. coli ECOR31(Schubert et al., 2004). For all these plasmid- and chromo-some-borne members, their coding genes conserve thesynteny of Fig. 1C. However, the existence of severalchromosomal homologs lacking a tra neighborhood(PSPTO_0532, PD1221), including pairs of two differenthomologs in the same genome (CJE0694 and CJE0695,Cj0591c and Cj0592c, XF2047 and XF2161, JJD26997_1077and JJD26997_1079, CJJ81176_0619 and CJJ81176_0620)is intriguing. As the overall conserved homology is low,the phylogenetic reconstruction of the family is not reliable.In spite of that, all these proteins share some characteristicfeatures with EEX proteins from IncP plasmids: a conservedCys residue (position 15 in Eex_pKM101 and 17 inEex_R388) and an N-terminal portion rich in basic aminoacids.

3.3. Entry exclusion in IncI plasmids

An exclusion gene, exc, was found in the conjugativetransfer region of IncI plasmid R144 (Hartskeerl et al.,1983) (Fig. 1D). Such gene codes for two proteins of differ-ent sizes (Hartskeerl et al., 1985a), the small being pro-duced by in frame reinitiation of translation and not as aprocessed form of the large one (Hartskeerl et al., 1986).Simultaneous mutations in both overlapping genes re-sulted in exclusion deficiency (Hartskeerl et al., 1985a).The large protein ExcA (19 KDa) is essential for exclusionand exists in a membrane-bound form at the periplasmicside of the inner membrane, and in a soluble form residingin the cytoplasm (Hartskeerl et al., 1985a) (Table 1). Simi-lar characteristics account for another IncI plasmid, R64(Furuya and Komano, 1994), which belongs to the sameexclusion group than R144 (Hartskeerl et al., 1985b). R64

exc gene was not essential for plasmid transfer (Furuyaand Komano, 1994). Levels and kinetics of mating aggre-gate formation in mixtures of R144+ donors and exclu-sion-proficient or exclusion-deficient recipient cells weresimilar, suggesting that the exclusion by R144 does notoperate at the level of aggregate formation, but acts atthe stage of DNA transfer (Hartskeerl and Hoekstra,1984). Reinforcing this idea, the observation that R64 excgene inhibited transfer even in surface mating (700-foldreduction versus 2000-fold in liquid) was an indicationthat Exc proteins inhibit transfer at a step after recognitionof recipient cells by the thin pilus (Furuya and Komano,1994). It should be kept in mind that IncI conjugative plas-mids form two types of sex pili: a thin flexible for liquidmating and a thick rigid pilus, essential for conjugationin general (Bradley, 1983).

ExcA_R64 homologs obtained by PSI-BLAST are re-stricted to elements that have a T4SS related to the Icm/Dot system of Legionella pneumophila (Supplementary Ta-ble S5 and Supplementary Fig. S2). They do not display adetectable Cys residue homologous to that of the previ-ously described exclusion proteins.

3.4. Entry exclusion in IncH plasmids

The exclusion activity of IncHI1 plasmid R27 has beenrecently studied (Gunton et al., 2008). A cosmid screen re-vealed that the exclusion phenotype was conferred by twogenes: eexA and eexB, which are part of the Z operon (eexA-orf17-eexB-trhO) (Fig. 1E). They are not essential for conju-gative transfer (Lawley et al., 2003). Both Eex proteins con-tain predicted transmembrane domains preceding aputative signal sequence, but lack a lipoprotein signature(Gunton et al., 2008). EexB predominantly localized tothe outer membrane, while EexA localized in the cytoplas-mic membrane, in this respect resembling TraT and TraS,respectively. Unlike TraT vs TraS, EexB exerted the highestexclusion activity (EI 17,500 vs 3000 for EexA). Curiously,the EI of the entire Z operon was lower (3000) than thatobtained when the eexA gene was deleted from the operon(13,852), indicating that EexA could play a regulatory rolein EexB expression.

EexA_R27 also excluded the IncHI1 plasmid pRG1251 (EI1,781), IncHI2 plasmids pAS251-2–3 (EI 640) and R478 (EI526) and IncHII plasmid pHH1508a (EI 207). EexB_R27 EIsto plasmids pRG1251, pAS251-2-3, R478 and pHH1508awere 461, 209, 95 and 1353, respectively (Gunton et al.,2008). The significantly lower exclusion activities ofEexB_R27 on heterologous plasmids could be a conse-quence of the lower homology exhibited between EexB pro-teins from IncHI1 and IncHI2 plasmids than between thecorresponding EexA homologs (see Supplementary TableS6 and Supplementary Table S7, and Supplementary Fig. S3).

R27 overall EI dropped to background level when donorcells containing an R27 eexA mutant were mated withrecipients expressing EexA or EexB. In the same way, anR27 eexB mutant was only minimally excluded from recip-ients expressing EexA (EI 126) (Gunton et al., 2008). Therequirement for exclusion proteins both in donor and reci-pient cells for exclusion phenotypic expression constitutesa unique characteristic of the IncH system.


3.5. Entry exclusion in integrative and conjugative elements(ICEs)

The role of the mating-pair stabilisation protein TraG asthe interacting partner for exclusion in donor cells wasconfirmed for ICEs R391 and SXT (Marrero and Waldor,2005), which harbor T4SSs similar to those of F-like plas-mids (Beaber et al., 2002). R391 and SXT do not excludeeach other, despite high sequence similarity (Hochhutet al., 2001). A single entry exclusion gene (eex), sufficientfor EEX activity in recipients (EI = 40), was identified foreach ICE (Marrero and Waldor, 2005) (see Fig. 1F). ProteinEex locates in the inner membrane (see Table 1), so itsexclusion mechanism is presumably homologous to thatof TraS_F. A direct interaction is likely to occur betweenboth inner membrane proteins, Eex and TraG. The specific-ity resides on the C-terminal 56 amino acids of Eex and onjust 3 residues (amino acids 606–608) within the centralpart of TraG. Exchange of residues 606–608 betweenTraG_SXT and TraG_R391 (P-G-E to T-G-D) led to exclusionof the heterologous ICE. Nevertheless, mutations of theseTraG residues did not affect ICE transfer indicating that,although essential for EEX, they are not involved in themating-pair formation activity of the protein. Residuescritical for exclusion in proteins EexR_R391 and EexS_SXT(both 143 amino acids long) were further delimited to ami-no acids 121–132 and 121–137, respectively (Marrero andWaldor, 2007a) (Supplementary Fig. S4). The diversity ofexclusion groups within the SXT/R391 family of 21 integra-tive conjugative elements was observed to be restricted totwo types: S (SXT) or R (R391) (Marrero and Waldor,2007b). All ICEs that belong to the S exclusion group en-code P-G-E at TraG positions 606–608, while T-G-D is har-bored in TraG of ICEs belonging to the R exclusion group.

Recently, Marrero and Waldor (2007a) determined thesubcellular localization of the TraG and Eex residues essen-tial for exclusion specificity using GFP fusions and cysteineaccessibility analysis, out of the mating context. When GFPwas fused to the carboxyl terminus of either TraG_R391 orEexS_SXT, the resulting proteins were fluorescent, an indi-cation of their cytoplasmic localization. For the second ap-proach, a Cys residue was added at the C-terminus of EexSand a mutant TraG_R391 T604C was constructed. In thecysteine accessibility analysis, the cysteine residues of aprotein located in the cytoplasm cannot be biotinylatedby 3-(N-maleimidopropinyl)biocytin, a compound thatcannot cross the inner membrane except when it is perme-abilized. Both mutants were biotinylated only when cellswere treated with the membrane permeative agent tolu-ene, confirming that the portions of both proteins impli-cated in the exclusion activity were located in thecytoplasm. Moreover, mutant TraG_R391 (T604C) wastransfer-proficient but defective in exclusion activity, sug-gesting that more residues than aa 606–608 of TraG areimplicated in the interaction with Eex.

The authors proposed several scenarios involving com-plex rearrangements for the interaction to occur in donoror recipient cytoplasm; amongst them, the translocationof either a fragment or the intact protein Eex or TraG or afragment of either protein, or a conformational change thatflips the topology of either Eex or TraG without protein

translocation. A non-related precedent comes from the pro-tein interaction SecG–SecA. SecG is an integral membraneprotein involved in the general secretion pathway. SecGhas been proposed to completely invert its membranetopology during SecA-dependent protein translocation(Nagamori et al., 2002; Nishiyama et al., 1996). Such inver-sion increases the interaction between SecA and SecG(Nagamori et al., 2002). Although SecG topology inversiontheory, being based just on accessibility studies with prote-ases and chemical reagents, is controversial (van der Sluiset al., 2006), it provides a testable working hypothesis.

3.6. Entry exclusion in mobilizable plasmids

Inselburg (1977) observed inhibition of conjugativetransfer between mating pairs carrying homologous ColE1plasmids. By deletion analysis, the exclusion function wasmapped to ColE1 HaeII-B fragment. Two of the four orfsthat lie in such region comparable in size with traS andtraT, exc1 and exc2, were predicted as entry exclusiongenes (Chan et al., 1985). A promoter region close to exc2was identified (Hale et al., 1983). A membrane locationwas suggested for Exc2 due to the presence of a lipoproteinsignal sequence at its N-terminus, which contains a cys-teine residue at position 23 susceptible to diglyceride mod-ification (Chan et al., 1985). However, (Yamada et al., 1995)observed that exclusion ability was retained by plasmidslacking exc1 and exc2, while loss of gene mbeD, containedin the mobilization region, abolished the activity. The EIwas 10, that is, not as high as for other exclusion genes.mbeD homologs are present in many ColE1-like plasmidsin the same location (Francia et al., 2004) (Fig. 1G, see alsoSupplementary Table S8). The protein is located both incytoplasm and inner membrane (Yamada et al., 1995). Itsmembrane location might be a consequence of a predictedN-terminal amphipathic helix (see Table 1). In spite of the30% dissimilarity between MbeD and the correspondingprotein of plasmid ColA, MbaD_ColA, the presence ofMbeD_ColE1 in the recipient excludes ColA (Yamadaet al., 1995). Signatures of other EEX proteins, like an N-terminus rich in basic amino acids and a cysteine residuecandidate to be lipid-modified, do not appear in MbeD-likeproteins (Supplementary Fig. S5).

The existence of an EEX system in ColE1 is not easilyreconciled with the fact that ColE1 is not self-transmissiblebut needs to use the T4SS of different conjugative plasmids(Finnegan and Sherratt, 1982), posing the question of howMbeD communicates with such a variety of auto-transmis-sible plasmids. Mobilization experiments to test ColE1exclusion used only Hfr strains as donors (Inselburg,1977; Yamada et al., 1995). Important experiments remainto be done, such as an analysis of MbeD-mediated exclu-sion in ColE1 mobilization by other conjugative plasmids,or the search of the donor counterpart of MbeD. In theabsence of these results, it is speculative to name theobserved effects as entry exclusion. Furthermore, noadditional data of exclusion mediated by mobilizable plas-mids have been reported. To our knowledge, RSF1010 isthe only other mobilizable plasmid that has been testedfor exclusion by the conjugative plasmid that brings aboutits transfer (see discussion on RSF1010 in Section 3.1).


In the absence of additional data it seems that exclusion ofmobilizable plasmids is an unspecific and secondary con-sequence of exclusion of the conjugative plasmid.

3.7. Entry exclusion in conjugative elements from low-GCgram-positive bacteria

Exclusion also occurs in pheromone-responding conju-gative plasmids of gram-positive bacteria. (Clewell andBrown, 1980), employing ‘‘male–male” matings betweenstrains carrying plasmid pAD1, suggested that there couldbe an exclusion function that reduced the recipient abilityof the cells already carrying a particular pheromone-induc-ible plasmid. This function was demonstrated in plasmidpCF-10 (Dunny et al., 1985). Pheromone induction of ‘‘malerecipients” decreased transfer by a factor of 10–300 incomparison to non induced cells or plasmid-free recipi-ents. Exclusion operated only against homologous plas-mids. Gene product Sec10 (also called PrgA) wasidentified as responsible for exclusion (Dunny, 1990; Kaoet al., 1991). It contains a putative N-terminal signal se-quence and a C-terminal hydrophobic segment that couldbe a membrane anchor. The protein is very hydrophilic,and it may be extruded to the cell exterior (Dunny, 1990;Kao et al., 1991). A protein homologous to Sec10, Sea1,was identified in pheromone-responding conjugative plas-mid pAD1 as responsible for exclusion (Martinez-Buenoet al., 1998; Weidlich et al., 1992). Hybridization studieswith DNA probes derived from gene sea1 demonstratedthat, with the exception of pAM373, all known sex phero-mone plasmids carry a homologous gene in the same loca-tion, upstream of the aggregation substance gene (Hirtet al., 1996) (see Fig. 1H). In fact, plasmid pAM373 is theonly conjugative plasmid reported that lacks an entryexclusion determinant (de Boever and Clewell, 2001). Themechanism that operates for entry exclusion in sex-phero-mone-responding plasmid is unknown, but certainlydepends on pheromone induction since in its absencepAD1-containing cells act as good recipients (Clewell andBrown, 1980). A PSI_BLAST search using Sea1_pAD1 as aquery did not result in hits belonging to a single proteinfamily, but brought in significant homologs among kine-sins, myosins and peptidases. The similarity of these exclu-sion proteins with peptidases could be a clue that they actby degrading the peptide pheromones, and thus by amechanism totally unrelated to the previously describedexclusion genes.

In ICEs from low G + C gram-positive bacteria, exclusionhas not been reported. Lack of immunity to receive anadditional copy of Tn916 by a recipient already carryingan element and the capacity of this second copy to trans-pose to new locations were demonstrated (Norgren andScott, 1991). It seems that the presence of more than onecopy of the element does not pose a problem for redundanttransfer since they normally integrate in different sites inthe host chromosome, and can also transpose intracellu-larly (Burrus et al., 2002). Those processes sometimes leadto the formation of larger multidrug resistance compositetransposons (Ayoubi et al., 1991; McDougal et al., 1998;Rice and Carias, 1998) and to resistance exchange by chro-mosome recombination between donors and recipients

through an unknown mechanism (Rice and Carias, 1998;Torres et al., 1991). This is perhaps a means of generatingrapid evolutionary changes that have contributed to theexpansion of antibiotic resistance determinants.

3.8. Entry exclusion in conjugative elements from high-GCgram-positive bacteria (Actinomycetes)

The only data concerning EEX in mycelium-formingStreptomycetes has been published for two integrativeand conjugative plasmids: SLP1 (Hagège et al., 1999) andpSAM2 (Possoz et al., 2003). They transfer like the autono-mous conjugative Streptomyces plasmids, both in pock for-mation and in the major protein involved (for a review see(Grohmann et al., 2003)). Transfer includes several steps:excision and replication in the donor, replication in the re-cipient, spreading within the mycelium and integrationinto the recipient chromosome (Hagège et al., 1994; Omerand Cohen, 1984; Possoz et al., 2001). In the case of plas-mid SLP1 of Streptomyces lividans, the presence of plasmidgenes impA and impC in recipient cells decreased SLP1transfer by more than three orders of magnitude. But, con-trary to exclusion proteins, Imp proteins act by repressingboth plasmid DNA replication and the expression of genetransfer (Hagège et al., 1999). The second case, plasmidpSAM2 from Streptomyces ambofaciens, seems to exertexclusion in a different way than SLP1. When present inboth donor and recipient strains, no excised forms are de-tected and transfer is reduced at least 1000-fold (Possozet al., 2001). A single pSAM2 gene, pif (pSAM2 immunityfactor), was found to be sufficient to abolish both transferand its initiation as effectively as the complete pSAM2 ele-ment (Possoz et al., 2003). Gene pif is transcribed even inthe absence of conjugation, maintaining the element inits integrated condition. Reciprocally, strains with an inac-tivated pif gene behave as good recipients. Its inactivationin the donor cell provoked the constant production of ex-cised pSAM2 forms that normally occur transiently duringconjugal mating (Possoz et al., 2003). Protein Pif is homol-ogous to the members of nudix (nucleotide diphosphatelinked to other moiety X) hydrolase family, and just the nu-dix motif is essential for Pif-mediated immunity to pSAM2conjugation (Possoz et al., 2003). There is neither DNAbinding motif nor membrane domain in the Pif protein se-quence, discarding a role of Pif as a transcripitional repres-sor or a bacterial surface localization to interfere directlywith the putative pSAM2 products of an interacting donor.(Possoz et al., 2003) suggested that Pif could be acting bymodifying a host component in the donor strain, prevent-ing recognition between two donors.

3.9. Entry exclusion in plasmids from Archaea

Although the mechanism by which plasmids transferbetween Archaea has not been clearly elucidated yet, avail-able data point to the existence of a conjugation-like pro-cess (see Prangishvili et al., 1998; Schleper et al., 1995;Stedman et al., 2000). It was found that conjugative plas-mids of Sulfolobus present in recipient cells conferredimmunity to conjugation only if the plasmid to be trans-ferred was closely related (Prangishvili et al., 1998). Cured


cells were temporary resistant to conjugative transfer bypreviously resident plasmid (Stedman et al., 2000), sug-gesting that a stable exclusion protein could be acting bypreventing cell to cell interactions. It should be remem-bered that plasmid F is not transferred to DNA-less mini-cells derived from an F+ parent, despite of their absenceof F-DNA (Cohen et al., 1968), which suggests that TraSand/or TraT are also stable proteins.

4. Entry/surface exclusion and immunity to lethalzygosis

When F� cells are mixed with an excess of Hfr donors,many of the recipient cells die, a phenomenon known aslethal zygosis (Alfoldi et al., 1957). Lethality is dependenton cell to cell contacts mediated by pili that are establishedduring conjugation (Clowes, 1963; Gross, 1963; Skurrayand Reeves, 1973). Ou (1980) tested the role of transferredDNA in lethal zygosis by using Hfr donors and adding ornot nalidixic acid to the mating mixtures. Nalidixic acidinhibits DNA transfer without appreciable impact on mat-ing aggregate formation (Cohen et al., 1968; Hane andWood, 1969). There was lethal zygosis in both conditions,but significantly more when nalidixic acid was omitted,suggesting that DNA transfer produces some but not allkilling. In a mating between an Hfr donor and F� dap� re-cipient prelabeled with radioactive diaminopimelic acid,radioactivity in the medium steadily increased (Ou,1980). Furthermore, in the presence of the outer mem-brane disorganizing polycation polymyxin B nonapeptideduring mating, the viability in the recipient population de-creased (Viljanen, 1987). Those results suggested that cellpermeability induced by extensive damage in the recipientmembrane is the primary cause of lethal zygosis, ratherthan an effect of excessive DNA passage. An F-mediatedimmunity to lethal zygosis was proposed (Skurray and Re-eves, 1974) and afterward it was attributed to both exclu-sion genes traS and traT, being the presence of traS morecritical for survival (Ou, 1980). Kinetic experiments indi-cated that, after mixing F+ and F� bacteria, recipient cellsfully expressed surface exclusion in 80 min (or one divisiontime), but in 20 min the recipient became immune to lethalzygosis (Ou, 1980). We infer from this result that exclusioncould be an important evolutionary value for survival ofbacteria. We are tempted to speculate that pock formationin Streptomyces and Nocardia transfer (Doull et al., 1986;Akagawa et al., 1984; Shindoh et al., 1984; Pernodetet al., 1984; Moretti et al., 1985) might occur as a resultof lethal zygosis.

5. Entry/surface exclusion and recombination

As stated before, when surface exclusion is relaxed instationary phase, redundant transfer between F+ hoststakes place (Achtman et al., 1972; Peters and Benson,1995). Sequence analysis of F-like plasmids present inthe E. coli ECOR reference collection indicated that recom-bination between genes in plasmids takes place at aconsiderably higher frequency than that between chromo-somal genes (Boyd et al., 1996). A positive correlationbetween redundant F transfer and reversion of a plasmid-

encoded lac mutant has been established (Peters et al.,1996). Correspondingly, in recipient cells, precise excisionof transposons and recA-dependent recombination be-tween DNA sequences non-related to F are stimulated byconjugative transfer of large F derivatives (Hopkins et al.,1980; Syvanen et al., 1986), especially when an F traS mu-tant is used. Thus, EEX/SFX may prevent wasteful redun-dant transfer of a plasmid already residing in therecipient bacteria that would lead to a defective elementproduced by recombination, a circumstance aggravatedby the generally low copy number of most conjugativeplasmids.

6. Evolutionary implications of entry/surface exclusion

Exclusion is a common property of conjugative plas-mids which suggests that it confers some advantage tothe plasmid. This benefit may originate from the fact thatexclusion frees a plasmid from competition with relatedplasmids at segregation during bacterial division. A math-ematical model (van der Hoeven, 1985) predicted that con-jugative plasmids inducing entry/surface exclusion (Ex+)can always penetrate in a population of incompatible, con-jugative, non-excluding plasmids (Ex�) and even expelthem if exclusion has no negative effect on bacterial fit-ness. Moreover, the selective advantage of exclusion in-creases with higher transfer rate, lower fitness loss of thebacterial host due to the exclusion and lower copy numberof the Ex+ plasmid (see Fig. 5). Another mathematical for-mulation (van der Hoeven, 1984, 1986) predicted thattwo incompatible, conjugative plasmids of the same en-try/surface exclusion group can only coexist in a bacterialpopulation, both in chemostatic and discontinuous foodsupply conditions, if they develop different survival strate-gies: one with a high conjugative transfer rate and a lowerhost fitness, and the other with a low transfer rate, a higherintrinsic growth rate and a higher host fitness (Fig. 6A). Be-sides, the model forecasted that more than two plasmidswith the same exclusion specificity cannot coexist in thesame population. So, if a third plasmid penetrates a popu-lation with two plasmids already established, at least oneof them will be expelled and a new equilibrium will bereached (Fig. 6B). In that way, the coexistence of non-iden-tical elements would be privileged, opening possibilities tonew gene combinations. This appears to have been the casein novel hybrid ICEs arisen in transconjugant cells from do-nors containing SXT and R391 (Burrus and Waldor, 2004).

7. General discussion

While great efforts have been made to obtain effectiveconjugation inhibitors for avoiding antibiotic resistancespread (Fernandez-Lopez et al., 2005; Hooper et al., 1989;Michel-Briand and Laporte, 1985; Ou and Reim, 1976), nei-ther effective in vivo inhibitors have been obtained, normutations in the recipient host significantly affecting con-jugation have been isolated (Anthony et al., 1994; Manoiland Rosenbusch, 1982; Sanderson et al., 1981; Sherburneand Taylor, 1997). On the other hand, conjugative plasmidscode themselves for the most powerful inhibitors of their

Fig. 5. Competition between exclusion-deficient (Ex�) and exclusion-proficient (Ex+) plasmids. Modified from (van der Hoeven, 1984, 1985, 1986). (A)Influence of transfer rate. With lower transfer rate, the Ex+ plasmid can invade a bacterial population at equilibrium carrying Ex� plasmid and expel it, but itbecomes extinct itself (case represented by broken line). With higher transfer rate, the Ex+ plasmid expels the Ex� plasmid and also intrudes into a plasmid-free population (solid line). (B) Influence of host fitness. The minimal value of transfer rate required for an Ex+ plasmid to expel an Ex� plasmid decreases ifexclusion has less negative effect on bacterial fitness. (C) Influence of the plasmid copy number. The minimal value of transfer rate required for an Ex+

plasmid to expel an Ex� plasmid decreases if the copy number of the Ex+ plasmid is low.

Fig. 6. Coexistence of incompatible, conjugative plasmids of the sameexclusion group. Modified from (van der Hoeven, 1984, 1985, 1986). (A)Possibility of coexistence of two plasmids of the same exclusion group.The model delimited the conditions for two plasmids belonging to thesame exclusion group to coexist. Given a plasmid (black circle) in a bac-terial population, it can coexist with another plasmid if the values oftransfer rate and fitness host of the second plasmid are between thebroken and solid lines (dark gray circles). If the values of the secondplasmid are above the solid line (light gray circle), then it will expel thefirst one. If the values of the second plasmid are under the broken line(white circle), then it will be expel by the first one. (B) Possibility for athird plasmid of the same exclusion group to invade the equilibriumbetween two plasmids. The model stated that the equilibrium betweenthe two plasmids (black circles) in a population can be broken by a thirdplasmid if its values of host fitness and transfer rate are above the disc-ontinuous line (plasmids represented by gray circles). Plasmids withvalues under the line (white circles) can not expel the equilibrium of theestablished plasmids (black circles).


own transfer capacity. Exclusion of redundant transferseems to be a widely spread phenomenon among conjuga-tive plasmids.

Looking at other T4SSs devoted to protein delivery, noentry exclusion gene has been found. The case of the geno-mic island trw-PAI present in Bartonella tribocorum (Seu-bert et al., 2003) is highly striking in this respect. Its T4SSis closely related to the Mpf genes of conjugative plasmidR388 (up to 80% amino acid identity for individual T4SScomponents) but it is devoted to intraerythrocytic patho-genesis. Despite its apparently recent recruitment fromconjugation to secretion (Frank et al., 2005), the Bartonellatrw system lacks the eex gene (Seubert et al., 2003). But eexabsence is not an exclusive characteristic of T4SSs that donot transport DNA. pTi-like plasmids contain two typesof T4SS that transport DNA substrates: one devoted to

T-DNA delivery into the plant cell (VirB/D4) and other in-volved in plasmid conjugal transfer between bacteria(Trb). Only the second codes for an entry exclusion protein(TrbK). Several T-DNA copies and concatemers of identicalcopies covalently linked can be found integrated in the nu-clear genome of the same plant cell, while different T-DNAs can be transferred to the same plant cell by differentbacteria (de Block and Debrouwer, 1991; Depicker et al.,1985; Tinland and Hohn, 1995), suggesting that transferof a T-DNA copy does not pose a problem for the entranceof a second one. The ultimate role of Agrobacterium-medi-ated transformation is to achieve the expression of the for-eign genes contained in the transferred T-DNA by the hostplant. To achieve its goal, T-DNA must overcome the pas-sage from the bacterium to the plant cell cytoplasm andsubsequently to the nucleus, where the integration processis frequently aborted. So it is not surprising that an entryexclusion system does not apply to T-DNA transfer. Inthe case of bacterial conjugation, entry exclusion serves(i) the conjugative element itself by preventing the possi-bility that an incompatible incoming plasmid eliminatesa preexisting one, (ii) the donor host cell by avoidinguneconomical excess of DNA transfer (iii) the recipient cellby averting death by lethal zygosis.

Since initial studies on exclusion were performed on F-like plasmids, the existence of two types of exclusion as ageneral phenomenon was suspected (Achtman et al.,1980). Nevertheless, most conjugative elements carry onlyone gene for EEX, usually an inner membrane protein that,when present in the recipient cell, blocks DNA transferwithin stable mating pairs. Entry exclusion proteins fromdiverse sources are not equal in effectiveness, but exhibitvariable EI that can be increased by over-expressing theexclusion protein. The second type of exclusion, SFXmediated by TraT, interferes with the initial attachment ofa donor bacterium to a potential recipient. It is found onlyin plasmids harboring F-like T4SSs. Besides carrying genescoded by hitchhiked elements like transposons andintegrons that are beneficial to the host, F-like plasmidssupply indeed the bacterial host with a shielding effect bymodifying their cell surface through TraT-mediated serumresistance, and thus favoring bacterial invasiveness. It is


tempting to speculate that only IncF and IncH plasmids,which produce pili that are firmly attached to the donorcell, can produce SFX besides EEX, while other plasmidtypes, whose pili detach easily from the cell, express onlyEEX.

It seems that entry exclusion genes evolve faster thanmost tra genes, from which the phylogenetic relations ofmany conjugative systems can be traced (Fernandez-Lopezet al., 2006; Frank et al., 2005; Planet et al., 2001). Se-quence conservation among homologous entry exclusionproteins of closely related plasmids is significantly lowerthan that existing between most Tra proteins of those plas-mids. Only scarce or even null sequence conservation canbe found comparing exclusion proteins of non-closely re-lated T4SSs for which significant homology in Tra proteinsstill exists.

Besides TraG in F-like plasmids and ICEs R391 and SXT,the donor interacting partner of exclusion proteins is un-known. The independence of exclusion and mating-pairactivities in TraG, the homology between the N-terminaldomain of protein TraG with VirB6 and the remarkableproximity of the eex gene to a virB6 orthologue in most sys-tems (Fig. 1A–C, F) suggest the possibility that VirB6 pro-teins could be the entry exclusion counterpart inconjugative systems not related to F. VirB6 plays a key rolein T-pilus assembly, stabilising VirB5 and influencing itsincorporation into the membrane, and mediating the for-mation of VirB7-VirB7 homodimers (Hapfelmeier et al.,2000). It is a polytopic membrane protein with severalfunctional domains: (i) a central region composed of alarge periplasmic loop that mediates the VirB6 interactionwith the exiting T-DNA, (ii) a transmembrane region car-boxy-terminal to the periplasmic loop required for T-DNAtransfer to VirB8, and (iii) the periplasmic N-terminal andcytoplasmic C-terminal regions involved in substratetransfer from VirB8 to VirB2/VirB9 (Jakubowski et al.,2004). Its C-terminal cytoplasmic domain is also involvedin the polar localization of the protein and can form anamphipathic helix that may encode a protein–proteininteraction domain (Judd et al., 2005). A virB6-deleted mu-tant retains the Vir protein-DNA interactions precedingVirB6 in the T-strand translocation pathway (DNA dockingto VirD4 and subsequent transfer to VirB11) but arrests thetransfer to outer membrane subunits (Cascales and Chris-tie, 2004).

The interaction between TraG and Eex/TraS couldinterfere with TraG’s DNA transfer function (Audetteet al., 2007; Marrero and Waldor, 2005). Entry exclusiondoes not interpose only with DNA but also with T4SS-mediated protein transport, regardless DNA transfer. Itis known that primase Sog of plasmid ColIb (Wilkinsand Thomas, 2000) and relaxase TrwC of plasmid R388(Draper et al., 2005) are transported in a DNA-indepen-dent way using conjugative pili. In both cases, no proteintransfer was observed when the entry exclusion proteinwas expressed in the recipient strain. Given this fact, weconsider that direct physical interaction between TraGand the transferred DNA is not the ultimate process af-fected by TraG–Eex interaction, but a more general pro-cess that arrests the transfer of any substrate throughthe T4SS.

The modus operandi by which exclusion works is dif-ferent from that of fertility inhibition since (i) the pres-ence of the exclusion protein in the donor cell is notrequired (with the exception of the IncH system); (ii) itdoes not act on the expression of transfer-related proteinsand (iii) it does not interfere in the conjugal transfer ofnon-related plasmids cohabiting with the excluded plas-mid. It is not related to super-infection exclusion byrestriction-modification systems either, since entry/sur-face exclusion does not promote recipient cell death toprevent propagation of a second element. The philosophyof entry/surface exclusion is more related to that ofsuper-infection immunity developed by T-even bacterio-phages. When E. coli was sequentially infected with twosuch phages, progeny resulted only from the first virionto infect the bacterial population while DNA of the sec-ondary or superinfecting phage was excluded from thecytosol of a cell already infected (Delbrück, 1945; Dul-becco, 1952). Superinfecting DNA is degraded (Andersonand Eigner, 1971). For exclusion activity, phage T4 re-quires protein Imm (Childs, 1973; Vallée and Cornett,1972). Imm, 83 residues long, is not processed (Lu et al.,1993). It contains two lipophilic stretches of 28 and 26residues presumed to be membrane anchors (Lu and Hen-ning, 1989, 1994). When the first stretch was reducedfrom 28 to 25 residues, still plenty for a membrane an-chor, the mutant protein could not effect any immunity(Lu and Henning, 1989). Fusions with alkaline phospha-tase and b-galactosidase indicated that its C-terminus(starting from residue 32) is located in the cytosolic sideof the membrane (Lu et al., 1993). The lack of require-ment of sequence specificity in the last 16 amino acidsof the protein for expression of immunity points that thisC-terminal part is not interacting with a phage compo-nent. The authors proposed that the protein Imm exertsits immunity effect by association to a component of aphage injection site, altering the conformation of thisreceptor such that the DNA transfer is blocked.

The precise way by which an interaction could takeplace between cytoplasmic (for Eex_R391/SXT, TraS_F/R100, TraG_R391/SXT) or periplasmic (for TraG_F/R100)protein regions of inner membrane proteins residing indifferent cells and how such interaction could be trans-lated into a reduction of DNA transfer are difficult toenvisage. Another intriguing question arises from the factthat such interaction is not produced when both proteinsreside in the same cell, or in case they interact, they doit in a different way that does not affect the DNA-proteintransfer. It is tempting to speculate that EEX donor coun-terpart is interacting with other T4SS components in away that protect it from interacting with Eex proteinsresiding in the same cell. When the mating signal is trig-gered, conformational changes in T4SS components mustoccur for carrying out DNA-protein transfer. The proximitybetween donor and recipient cell after pilus retraction isunknown. Maybe they are close enough for allowingT4SS components translocation between donor and recipi-ent membranes or even to partially fuse the membranes,providing a scenario where TraG and Eex/TraS could inter-act. Far from speculation, these questions remain to beelucidated.


8. Conclusions

Entry exclusion seems to be an important, or evenessential, feature of plasmid biology. Its physiologicalimportance has remained in the dark up to now, in spiteof many excellent publications. The reason might be 2-fold.On the one hand, entry exclusion proteins are membraneproteins and solid advances in membrane biochemistryare trailing behind in the conjugation field. On the other,we tend to focus our attention on genes and phenotypesthat are essential for survival under easily tested labora-tory conditions. But many more genes in the plasmid ge-netic pool are probably contributing to plasmid fitness(Fernandez-Lopez et al., 2006) and are waiting for a similardetailed analysis. Entry exclusion genes are just one exam-ple. We have attempted in this review to draw attention totheir importance. We believe a vast wealth of new infor-mation is waiting to be extracted from many hidden gemsin the plasmid genetic pool.

Acknowledgments

Work in the FdlC lab was supported by Grant BFU2005-03477/BMC from the Spanish Ministry of Education andScience and contract LSHM-CT-2005_019023 from E.U. VIFramework Programme.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.plasmid.2008.03.002.

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Why is entry exclusion an essential feature of conjugative plasmids?