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Membrane-associated DNA Transport Machines

Briana Burton1 and David Dubnau2

1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts2Public Health Research Center, New Jersey Medical School, University of Medicine and Dentistry ofNew Jersey, Newark, New Jersey

Correspondence: [email protected]

DNA pumps play important roles in bacteria during cell division and during the transfer ofgenetic material by conjugation and transformation. The FtsK/SpoIIIE proteins carry outthe translocation of double-stranded DNA to ensure complete chromosome segregationduring cell division. In contrast, the complex molecular machines that mediate conjugationand genetic transformation drive the transport of single stranded DNA. The transformationmachine also processes this internalized DNA and mediates its recombination with the res-ident chromosome during and after uptake, whereas the conjugation apparatus processesDNA before transfer. This article reviews these three types of DNA pumps, with attentionto what is understood of their molecular mechanisms, their energetics and their cellularlocalizations.

The transport of DNA across membranes bybacteria occurs during sporulation, during

cytokinesis, directly from other cells and fromthe environment. This review addresses thequestion “how is the DNA polyanion transfer-red processively across the hydrophobic mem-brane barrier”?

DNA transport must occur through water-filled channels, at least conceptually address-ing the problem posed by the hydrophobicmembrane. DNA transporters presumably usemetabolic energy directly or a coupled-flow(symporter or antiporter) mechanism to driveDNA processively through the channel. It ispossible that a Brownian ratchet mechanism,in which directionality is imposed on a diffusiveprocess, also contributes to transport.

In this article, we will consider several DNAtransport systems. We will begin with the sim-plest one, namely the FtsK/SpoIIIE systemthat is involved in cell division and sporulation.We will then turn to the more complex, multi-protein DNA uptake systems that accomplishgenetic transformation (the uptake of environ-mental DNA from the environment) and theconjugation systems of Gram-negative bacte-ria that mediate the unidirectional transfer ofDNA between cells. In each case we will discussthe proteins involved, their actions and thesources of energy that drive transport. Spacelimitations prevent discussion of other relevanttopics, such as DNA transport during bacterio-phage infection and more than a brief referenceto conjugation in Gram-positive bacteria.

Editors: Lucy Shapiro and Richard Losick

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CELL DIVISION AND SPORULATION

General Features of DNA Transport inCell Division

Proper chromosome segregation is essential forsuccessful cell division, and yet under rapidgrowth conditions bacterial cells can form adivision septum before completion of DNA seg-regation. To accomplish this, the cells have dedi-cated proteins of the FtsK/SpoIIIE family thatmove the remainder of the chromosome intothe appropriate daughter-cell compartment.Given that a single gene product codes for allthe transport functionality, this is the simplestDNA transport system discussed here.

The simplicity of the single polypeptidechain belies the fact that these transportersfunction in the division septum, one of themost topologically challenging environmentsof the bacterial cell. When chromosomes havenot fully segregated into daughter compart-ments, the constricting septal membranes con-stitute an obstacle for the final resolution of thecellular genetic material. In many of the bacteriathat contain FtsK/SpoIIIE proteins, the chro-mosomes are circular molecules. This meansthat any portion of the chromosome that is onthe wrong side of the division plane will havea loop of dsDNA that must first be resolvedproperly from its sister chromosome, and sec-ond be moved in the correct direction acrossthe division plane.

Information about the FtsK/SpoIIIE fam-ily of transporters is derived largely from thework on two founding members of the proteinfamily. FtsK from Escherichia coli has yieldedmuch functional information about the roleof these proteins in terminus resolution dur-ing chromosome segregation (a more detailedaccount of its recombination function may befound in Thanbichler 2009). SpoIIIE, whichhas a specialized function during the asym-metric division of sporulating Bacillus subtiliscells, has produced complementary informa-tion about these transporters. The focus ofthis section is on how the ATPases of theFtsK/SpoIIIE protein family transport chro-mosomal DNA in the context of cellular divi-sion membranes.

The FtsK/SpoIIIE Proteins

The FtsK/SpoIIIE family of DNA transportersis conserved among the bacteria (Iyer et al.2004). Proteins of the FtsK/SpoIIIE familyhave diverse functions including the export ofvirulence factors, conjugative transfer of plas-mid DNA and chromosome partitioning. Thosemembers involved in chromosome partitioningshare a functional domain called g (discussedlater) (Wang et al. 2006; Ausmees et al. 2007;Le Bourgeois et al. 2007; Wang et al. 2007; Valet al. 2008; Dedrick et al. 2009) and have thedistinction of functioning at a unique interfacewithin cells—the constricting division plane.

Until recently, it was thought that all chro-mosomal transporters in this family harboramino-terminal transmembrane domains.New studies on a SpoIIIE paralog in B. subtilishave prompted a revision of the group toinclude soluble FtsK proteins. Interestingly, allof these FtsK/SpoIIIE proteins localize to celldivision membranes, and they do so only atthe time of division (Wu and Errington 1997;Biller and Burkholder 2009; Kaimer et al.2009). The newly characterized soluble protein,SftA, localizes to predivisional sites, presumablythrough direct protein–protein interactionswith FtsZ, rather than through membrane teth-ering (Biller and Burkholder 2009). FtsK local-izes to vegetative cell division septa, as doesSpoIIIE, although the latter does so rarely (Wuand Errington 1997; Wang and Lutkenhaus1998; Yu et al. 1998). In the case of sporulation,SpoIIIE localization requires the presence ofDNA in the septal plane (Ben-Yehuda et al.2003). The presence of FtsK at division planesis enhanced under conditions that produce anincreased number of bisected nucleoids (Yuet al. 1998). The amino-terminal transmem-brane segments of both proteins are requiredfor proper septal localization (Begg et al. 1995;Draper et al. 1998; Yu et al. 1998; Sharp andPogliano 1999) and although there is littlesequence conservation in this region, the trans-membrane segments appear to mediate inter-action with and recruitment of other divisionmachinery proteins (Di Lallo et al. 2003). Fur-thermore, the transmembrane domains are

B. Burton and D. Dubnau

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required for proper function of SpoIIIE (Sharpand Pogliano 2003), although the precise role ofthe FtsK transmembrane domains for DNAtransport activity is still unknown.

Little is understood about the precise role ofthe variable length linker domain that followsthe transmembrane segments. However studiesin E. coli using domain deletions and chimeraswith the Haemophilus influenzae FtsK showthat the linker portion of the protein does playa role in proper cell division (Bigot et al.2004). The absence of the linker domain corre-lates with increased filamentation in thesestrains, and this phenotype could be function-ally separated from a previously characterizedrole for the linker in chromosome dimer resolu-tion (Aussel et al. 2002).

The carboxy-terminal regions of these pro-teins contain the conserved portions of theproteins including the ATPase motifs and thedirectionality determining gamma domain.TrwB, a conjugation protein from the plasmidR388, provided the first structural insightsinto the ATPase domain of this protein family(Gomis-Ruth et al. 2001). The structure of thesoluble domain of TrwB (lacking the amino-terminal transmembrane region) revealed twodomains. First, an NBD similar to RecA andring helicases, and second a domain entirelycomposed of a-helices. A solution structure ofthe soluble ATPase domain of FtsK confirmedthe presence of a RecA-like fold, but also re-vealed a unique jawlike domain connected tothe RecA fold (Massey et al. 2006). Comparisonof two crystal forms suggests a hinge-like move-ment between the two domains that could leadto a rotary-inchworm translocation mechanism.The structural studies also showed that FtsK hasa large central pore, about 25 A in diameter thatcould accommodate a dsDNA molecule (Masseyet al. 2006). In contrast, TrwB which may trans-port ssDNA (see later), has a smaller, 8 A diam-eter, central pore (Gomis-Ruth et al. 2001).

The soluble ATPase domains of bothSpoIIIE and FtsK have been shown to translo-cate along double stranded DNA in an ATPdependent manner (Bath et al. 2000). In fact,in both cases, the ATPase activity is dependenton the presence of double stranded DNA. In

single-molecule experiments, FtsK producedan average translocation rate of �5 Kb/s (Salehet al. 2004; Pease et al. 2005). EM of the FtsKmotor domain in the presence of double-stranded DNA suggest that a hexamer of the pro-tein forms rings around the DNA (Aussel et al.2002; Massey et al. 2006). Further evidence thatthese rings are hexamers comes from size exclu-sion chromatography where the soluble domainof FtsK migrates as a hexamer(Aussel et al. 2002).

Recent studies have focused on SftA, thesoluble FtsK/SpoIIIE-like DNA translocasefrom B. subtilis mentioned earlier (Biller andBurkholder 2009; Kaimer et al. 2009). In con-trast to SpoIIIE, which assembles at late stagesof cell division, SftA assembles at nascent divi-sion septa with FtsZ and before membranescission (Biller and Burkholder 2009) (Fig. 1a).SftA is required for chromosome partitioningearly in cell division, in part through increasedchromosome dimer resolution, thus making itmore similar to FtsK in terms of its role in chro-mosome segregation. SpoIIIE, on the otherhand, serves as a back-up mechanism for com-pleting chromosome segregation postseptation-ally (Biller and Burkholder 2009; Kaimer et al.2009). Interestingly, some bacterial speciessuch as clostridia carry only a single g domain-containing paralog. Those with two FtsK/SpoIIIE ATPases containing g domains, clus-ter in the low GC-Gram-positive bacteria, a-proteobacteria, andb-proteobacteria (BillerandBurkholder 2009). Many of these organisms arenaturally closely linked to soil and plants but arenot endospore formers, suggesting that thepresence of the SpoIIIE-like back-up protein isnot tightly correlated with spore formation.

A remarkable feature of these chromosometransporters is their ability to move the DNAin the appropriate direction with astoundingaccuracy. This ability depends on sequencemotifs in the chromosomal DNA (Bigot et al.2005; Levy et al. 2005; Pease et al. 2005; Ptacinet al. 2008; Biller and Burkholder 2009). Shorteight-nucleotide sequences, known as KOPSand SRS sequences for FtsK- and SpoIIIE-recognition respectively, are overrepresentedon the plus strand of the chromosome andare recognized by the g domain on the very


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carboxyl terminus of the FtsK/SpoIIIE pro-teins. This domain recognizes these sequencesand uses them as a guide to accurately movethe chromosome into the appropriate daughtercell (Ptacin et al. 2006; Sivanathan et al. 2006).The co-crystal structure of three g domainsbound to KOPS sequence DNA provides struc-tural support for biochemical experiments, sug-gesting that KOPS DNA establishes directionalDNA translocation through oriented proteinloading (Bigot et al. 2006; Lowe et al. 2008).Further cytological and functional evidencesuggests that SpoIIIE follows this same orientedloading paradigm (Sharp and Pogliano 2002a;Sharp and Pogliano 2002b; Becker and Pogliano2007; Ptacin et al. 2008).

Although these insights have helped toanswer the major conundrum in the fieldregarding how the FtsK/SpoIIIE proteins faith-fully segregate chromosomal material into thecorrect daughter cell, a lingering questionremains: How do the proteins resolve loops ofDNA at the division plane? In bacteria withcircular chromosomes, it is predicted based onthe pore size of the ATPase domain, that eachindependent hexameric transporter will en-compass one double stranded “arm” of thechromosome (Fig. 1B). For the proteins thatact predivisionally, such as SftA and presumably

FtsK, the scenario is relatively simple to imag-ine. A small loop of DNA remains on the“wrong” side of the division plane, and whenthe transporters reach the loop, they might dis-engage the DNA. This release would allow theloop of DNA to move into the appropriatedaughter cell through simple Brownian motionfacilitated by the act of chromosome organiza-tion and compaction on the daughter cell side.In contrast, the resolution of a loop in a postsep-tational system, such as that of SpoIIIE, mustinvolve one of two possible mechanisms (Beckerand Pogliano 2007; Burton et al. 2007). Onepossibility is that the chromosomal DNA is lin-earized and rejoined after transport. Another isthat a rearrangement of the proteins to form asingle, larger channel could allow the loop tomove across the septum uncleaved. Lateralopening of the channels could be driven bythe mechanical force that the loop of DNAexerts on the two hexameric complexes. Thissort of mechanism is appealing because itwould allow for resolution of any DNA loopcaught in the septal space, not just the lastloop near the chromosome terminus. Furtherstudies on the link between directional trans-port and the ultimate resolution of local DNAstructures at the division plane will be neededto address this issue.

A B

Figure 1. Localization of chromosome segregation proteins, SftA and SpoIIIE, in Bacillus subtilis. (A) SftAlocalizes to early division septa in B. subtilis, whereas SpoIIIE localizes late in division and largelysporulation septa. This image shows fluorescence overlayed from FM4-64 membrane-staining (red), a SftA-YFP fusion (green) and a SpoIIIE-CFP fusion (blue), of cells from a sporulating culture. SftA fluorescence isapparent at predivisional and vegetative divsion sites, whereas SpoIIIE fluorescence colocalizes with fullyformed sporulation division membranes. (Images courtesy of J. Liu.) (B) Schematic of the chromosomecrossing the division septum through two independent DNA-conducting channels formed by SpoIIIE in asporulating B. subtilis cell. At the end of the transport process, the final loop of DNA in the largecompartment must be resolved across the division plane, by an as yet undetermined mechanism.





B. subtilis Sporulation as Model System

B. subtilis SpoIIIE, is required for active transportof chromosomal DNA during B. subtilis sporu-lation (Wu and Errington 1998). During sporu-lation, B. subtilis cells divide asymmetrically,creating a larger mother cell and a smaller fore-spore. Two-thirds of the chromosome destinedfor the spore compartment remains on themother cell side of the newly formed septum, andmust be actively transported into the spore (Wuand Errington 1998). Cells lacking SpoIIIE areunable to move the chromosome into the sporecompartment, and are therefore completely defe-ctive for sporulation (Wu and Errington 1994).Similarly cells specifically defective for SpoIIIEATPase activity are unable to complete the sporu-lation program (Sharp and Pogliano 2002b).

SpoIIIE is expressed in vegetative cells and isdistributed uniformly throughout the mem-brane (Foulger and Errington 1989). Duringsporulation, SpoIIIE protein levels do notchange. However, at the onset of sporulation,SpoIIIE moves to the septum, localizing to themidpoint of the converging lipid bilayers (Wuand Errington 1997). The generation of thisSpoIIIE focus requires the presence of DNA inthe septal annulus (Ben-Yehuda et al. 2003). TheSpoIIIE focus remains at the septum until theentire chromosome has moved into the forespore(Sharp and Pogliano 2003). Interestingly, SpoIIIEhas been implicated in a second function duringsporulation, apparently unrelated to chromo-some transport. After transport is complete,the protein relocalizes to the pole of the sporu-lating cell, where it plays an additional role infusing the membranes of the mother cell engulf-ing the prespore (Sharp and Pogliano 1999).

What about the proteins that normally dec-orate a chromosome? Fluorescence microscopyexperiments provided evidence that not only arethe FtsK/SpoIIIE proteins efficient movers ofDNA, they also have the ability to remove virtu-ally every protein roadblock in their way, leavingthe transported DNA to enter the daughter cellnaked (Marquis et al. 2008). The case of sporu-lation, where over three megabases of DNA aretransported to the daughter cell, highlightsthis best as transcription factors, chromosome

compaction proteins, and other site-specificallybound proteins, are all removed during DNAtransport (Marquis et al. 2008). How exactlythese translocases accomplish such a dauntingtask remains to be determined.

Protein–Protein Interactions

The most striking feature of the FtsK/SpoIIIEproteins is that despite their complex functions,the actual DNA transport activity is seeminglyaccomplished entirely by these proteins.Whereas the next nucleic acids transporter sys-tems to be discussed, competence and transfor-mation, are composed of numerous geneproducts, FtsK and SpoIIIE appear to functionentirely on their own. Nevertheless, it remainsto be seen whether there are still other accessoryproteins to be discovered that play a role in theDNA transport activity, or whether these pro-teins really function as lone powerhouses fortheir chromosome transport function.

CONJUGATION

General Features of Gram-NegativeConjugation Systems

Conjugation refers to the transfer of DNA fromone cell to another, by a process requiring cell–cell contact. As with transformation, the trans-ferred molecule is ssDNA. Conjugation andType 4 protein secretion are mediated by similarcomplex machines, which in at least some casesmediate the transfer of both DNA and proteinsubstrates. Conjugation involves the participa-tion of pilins, but these are different from theType 4 pilins needed for transformation. Ourdiscussion will emphasize the Agrobacteriumtumefaciens system that transfers DNA intoplant cells and the E. coli R388 plasmid conjuga-tion system. For more detailed descriptions ofconjugation, the reader is referred to recentreviews (Chen et al. 2005; Christie et al. 2005;Chandran et al. 2009).

The Conjugation Proteins

The core proteins required for DNA transferduring conjugation are usually divided for





convenience into two groups; the DNA process-ing and transfer (DTR) and the mating pair for-mation (MPF) proteins. DTR proteins processthe DNA to be transferred and resemble the pro-teins needed for rolling circle replication by bac-teriophage FX174 and by certain plasmids. Ithas been proposed that the MPF proteins areneeded to form a pilus that connects the donorand recipient cells, retracting to bring the cellsurfaces into more intimate contact (Panickerand Minkley 1985). Recent evidence suggeststhat at least in the case of the F-pilus, singlestranded DNA may travel through the pilus,into the recipient cell (Babic et al. 2008; Shuet al. 2008). However, the role of pili in conjuga-tion is not clear and some Type 4 secretion sys-tems appear to lack them. MPF proteins are alsoneeded to form structures that traverse the innermembrane, the periplasm and the outer mem-brane, forming a channel for the passage ofDNA. In the conjugation systems, a Type 4coupling (T4C) protein mediates interactionbetween ssDNA produced by the DTR proteinsand the MPF apparatus.

DTR and Coupling Proteins

Among the R388 DTR proteins are the relaxase/helicase TrwC, the host IHF protein and TrwA,which also functions as a transcriptional re-pressor of the dtr genes. These proteins formthe relaxosome, which binds to the oriT regionof the super-coiled plasmid DNA (Christie1997; Gomis-Ruth and Coll 2006). TrwC nicksone strand and covalently attaches by its cata-lytic tyrosine residue to the 50 end of the stranddestined for transfer. The helicase activity ofTrwC then unwinds the DNA and DNA poly-merase III replaces the transferred strand byrolling circle replication. The IHF and TrwAcomponents of the relaxosome probably playarchitectural roles, shaping the DNA into a con-formation that favors TrwC binding and activ-ity. The covalently attached TrwC then acts asa “pilot protein” accompanying the T-DNA-protein complex into the recipient cell.

Although recognition by the DNA transfermachinery requires motifs present on the relax-ase, rather than a specific DNA sequence, this

is not a mere protein secretion system, withDNA coming along for the ride. The length ofthe transferred strand demands that activeDNA transport must take place. The Type 4 cou-pling (T4CP) protein (TrwB from R388 andVirD4 from A. tumefaciens) assembles into ahexamer and has an amino-terminal mem-brane-embedded domain on each protomer, aswell as a large cytosolic moiety. The structure ofthis moiety has been solved and consists of twodomains, a nucleotide-binding domain distalto the membrane surface and a membrane-proximal a-helical domain (Gomis-Ruth et al.2001). Running through the cytosolic moietyis a central channel that is �20 A wide inTrwB but decreases to �8 A at the cytosolicextremity. It is not known if this channel contin-ues through the membrane domain and accom-modates the ssDNA during transfer, because thestructure was derived from a truncated deriva-tive lacking the membrane-embedded moiety.The narrow entrance at the cytoplasmic endmay carry out a gating function as transfer is ini-tiated. Aside from its suggestive structure andcellular location, several other attributes sup-port the role of TrwB as a DNA pump. Forinstance, TrwB is a DNA-dependent ATPase,and shows conformational changes in the sur-face residues of the central channel dependingon its ATP-binding state (Tato et al. 2005). Inaddition, the closest structural analog to TrwBis stated to be FtsK, although the latter proteinhas a wider central channel, presumably to allowthe passage of dsDNA. It is worth noting thatmembers of the HerA/FtsK/SpoIIIE proteinfamily are also required for conjugation inGram-positive bacteria (Iyer et al. 2004; Berk-man et al. 2010) The nucleotide-bindingdomain of TrwB also resembles that of thering helicases, which are known to use theenergy of ATP hydrolysis to translocate alongDNA (Gomis-Ruth et al. 2001).

MPF Proteins

The MPF proteins are numerous, 11 in the caseof A. tumefaciens where they are best character-ized (Christie et al. 2005; Fronzes et al. 2009a).In addition to the proteins that assemble





the pilus (VirB2 and VirB5) additional pro-teins (VirB4, VirB6, VirB7, VirB8, VirB9, andVirB10) assemble across the bacterial envelope.Biochemical investigation established that VirB8,VirB10, and the NTPase VirB4, are anchored inthe inner membrane, VirB9 is located in eitherthe periplasm or in association with the outermembrane, and VirB7 is probably associatedwith the outer membrane. These proteins pre-sumably establish a channel extending fromthe T4CP protein (VirD4) to the exterior sur-face. The VirB11 ATPase is recruited to the innerface of the inner membrane where it is neededfor the assembly of the secretion channel. Thisproposed role for VirB11, in mediating assem-bly of a trans-envelope structure perhaps con-taining a pilus subunit, is analogous to thatproposed for the secretion ATPases of the T4pilus-related systems (see later), and in factVirB11 has been classified as a secretion (or traf-fic) ATPase. In addition to VirD4 and VirB11,VirB4 is also an ATPase and together these threeproteins, which contact one another, presum-ably power the assembly of the conjugationnano-machine as well as secretion of DNA.

The role of the pilin proteins is unclear. Afilamentous structure composed of pilin sub-units, perhaps a mini- or pseudopilus, may bean obligatory part of the conjugation ma-chinery, even providing a channel for DNAtransport. Another possibility is that pilin sub-units may assemble with MPF proteins to forma transport structure whereas pili per se may bean alternative structure, requiring the MPF pro-teins for its formation.

Recently, two major advances have contrib-uted to an enhanced understanding of thesecomplex systems. The first is the so-called trans-fer DNA immunoprecipitation (TrIP) assay.This technique, based on cross-linking of DNAto proteins using immunoprecipitation andPCR, has identified proteins that are proximalto the DNA during transport (Cascales andChristie 2004). This has established a provi-sional transport pathway and has suggested theidentity of channel subunits. As expected,VirD4 is the first protein contacted by theDNA following interaction with the relaxosome.Next the DNA contacts the VirB11 ATPase and

this interaction depends on the presence ofother core components, but not on the Walker Aand B residues of VirB11. Why VirB11 interactswith DNA is unknown, but it may have a directrole during transport, aside from its morphoge-netic role in building the core. In two subsequentsteps, close contacts are established betweenthe DNA and VirB6 and VirB8, located in theinner membrane, and then with the outer mem-brane/periplasmic VirB2 pilin and with VirB9.These valuable findings constrain model build-ing, based on the second major advance, theacquisition of high-resolution structural data.

Although several individual componentstructures had been solved (Yeo et al. 2000;Yeo et al. 2003; Terradot et al. 2005; Baylisset al. 2007), a big step forward derives fromthe 15-A-resolution cryoelectron microscopystructure of a complex between the VirB7-VirB9-VirB10 orthologs from the plasmidpK101 (TraN, TraO, and TraF respectively)(Fronzes et al. 2009b). This 1.05 MDa “corecomplex” is composed of 14 copies of each pro-tein, forming a double-walled two-chamberedchannel, proposed to span both membranes.The inner and outer chambers contain a chan-nel that extends 105 A, sufficient to bridge thetwo membranes. The channel is open on thecytoplasmic side and constricted near the otherend. The outer surface of the structure is com-posed mostly of TraF/VirB10, whereas TraN/VirB7 and TraO/VirB9 form the inner wall ofthe channel. It is suggested that the orthologsof the VirB4 and VirB11 ATPases may regulatethe opening at the distal end, communicatingacross the inner membrane and periplasm bysome unknown mechanism, permitting passageof the DNA. This beautiful structure providesno explicit role for these ATPases, the T4C pro-tein, or for the orthologs of VirB2, VirB6, andVirB8. Presumably one or more of these pro-teins decorates the outside of the structure,interacts with it within the membrane or mayeven be located in one of the large chambers.

More recently, the remarkable crystal struc-ture of a 0.6 MDa fragment of this core struc-ture, derived from chymotrypsin treatment, hasbeen reported (Chandran et al. 2009). Thisstructure corresponds to the outer chamber





of the cryo-EM structure. Unexpectedly, theVirB10 protein, which crosses the inner mem-brane, also comprises the outer membranechannel. It may thus represent the first bacterialprotein that traverses both membranes. Figure 2shows the crystal structure of the outer mem-brane complex superimposed on the cryo-EMstructure. Detailed comparison of the twostructures reveals suggestive differences, whichmay reflect conformational changes underlyingDNA transport (Chandran et al. 2009). It hasbeen suggested that the large chambers may besites of assembly of the MPF proteins not repre-sented in the core structure, and that these mayform the actual channel for substrate transport(Chandran et al. 2009; Christie 2009). Naturallymany questions remain concerning the detailedmechanisms of secretion and the energetics ofthe process. One wonders if the showed require-ment for the VirB2 pilin for transport hints at adynamic role for a pilus, or for a pseudopilus,analogous to that proposed below for the com-petence pseudopilus? The mechanism by whichthe DNA is transported into the recipient cellis also obscure, although a role for a pilus has

been suggested (Backert et al. 2008). Althoughthese and other major questions are unresolved,the VirB7-9-10 structure represents a break-through that should lead to rapid advances inunderstanding.

TRANSFORMATION

General Features of Bacterial Transformation

Transformation refers to the transport of envi-ronmental DNA to the cytosolic compartment,where it can meet several fates. If the incomingDNA is complementary to the resident chro-mosome, it can recombine. If it contains theinformation needed for independent replica-tion, it can do so as a plasmid or a bacterio-phage. As with conjugation, the transportedDNA is single-stranded. The nontransformingstrand is degraded, most likely by a mechanismthat is closely connected to transport across thecell membrane (Mejean and Claverys 1993).

These shared features reflect the existenceof a common set of transformation proteins(Dubnau 1999). Gram-positive and Gram-negative bacteria share the core proteins, exceptthat the latter group has additional componentsenabling DNA to cross the outer membrane.Among these conserved transformation pro-teins are those that are needed for Type 4pili (T4P) and Type 2 secretion (T2S). Helico-bacter pylori is unusual because it uses aconjugation-like system for DNA transport,rather than the usual shared transformationproteins (Hofreuter et al. 2000).

In most bacteria, complex signal transduc-tion pathways regulate the proteins that mediateDNA uptake (Macfadyen 2000; Hamoen et al.2003; Claverys et al. 2006; Leisner et al. 2008).Cells that synthesize these transformation pro-teins are referred to as “competent.” Althoughthe core transformation proteins are mostlyconserved, the regulatory mechanisms are not.This is a common theme; nature often uses acommon set of tools, but uses species-specificinstructions for their use. We will not furtherdescribe the regulation of competence geneexpression. This article reflects the bias of theauthors by emphasizing the B. subtilis system,

OM

N N

IM

Figure 2. Proposed structure of the core complex forT4S system (pKM101)-mediated conjugation. Thecryo-EM structure (yellow) is superimposed on thecrystal structure of the outer-membrane complex.The location of lipid associated with TraN/VirB7is shown with black dots. The arrows illustrateproposed conformational changes (Chandran et al.2009). OM and IM indicate outer and innermembrane, respectively. (Adapted, with permission,from Chandran et al. 2009 [# Nature PublishingGroup].)





although information from other bacteria willbe included when needed. The B. subtilis no-menclature for proteins and genes will be usedthroughout. Several reviews are available thatemphasize transformation in other bacteriaor that offer more detail (Berge et al. 2002;Friedrich et al. 2002; Hamilton and Dillard2006; Claverys et al. 2009).

The Fate of Transforming DNA

It is useful to follow a DNA molecule from itsexistence in solution outside the cell, until itbecomes integrated in the resident genome ofthe transformant (reviewed in Berge et al.2002; Chen and Dubnau 2004; Chen et al.2005). Double stranded DNA (dsDNA) firstbinds to the B. subtilis cell surface withoutsequence specificity and is then fragmented bythe action of a membrane-localized nuclease(NucA) that presumably acts at the points ofattachment (Provvedi et al. 2001). After bindingand fragmentation, one strand is degraded,releasing phosphorylated products into themedium and the transforming strand is con-verted into a form that is no longer accessibleto added DNAse. DNAse resistance is taken asan indication of transport across the cell mem-brane. Indeed no difference in the kinetics ofappearance of transformants, DNAase resis-tance and degradation products has been de-tected. In S. pneumoniae, the transformingstrand is taken up with 30 to 50 polarity andthe nontransforming strand is degraded with50 to 30 polarity, consistent with close coordina-tion of the degradation and uptake steps(Mejean and Claverys 1988; Mejean and Clav-erys 1993). (For a possible difference in B. sub-tilis, see Vagner et al. 1990.) 30 ssDNA terminiinitiate recombination in all systems and inthe case of transformation, the uptake mecha-nism itself generates this required substrate.Internalized ssDNA thus interacts with the res-ident chromosome and RecA-mediated recom-bination results in formation of a heteroduplexin which one strand is from the donor and theother from the recipient chromosome. Thisheteroduplex molecule is at first stabilizedby non-covalent bonds, after which ligation

occurs. The heteroduplex is resolved by replica-tion and in S. pneumoniae by mismatch repairas well (Claverys and Lacks 1986; Majewskiand Cohan 1998).

In Gram-negative bacteria, e.g., H. influen-zae and Neisseria gonorrhoeae, dsDNA presum-ably passes across the outer membrane throughring-shaped assemblies of proteins known assecretins (Tomb et al. 1991; Collins et al. 2004).It has been reported that the secretin complexof N. gonorrhoeae contains the initial bindingsite for DNA during transformation (Assalkhouet al. 2007). On entry to the periplasm, DNA isthought to become resistant to exogenouslyadded DNAse. Transport into the cytosol ismore difficult to detect in Gram-negative bac-teria and “uptake” is (operationally) defineddifferently than in the Gram-positives. In Hae-mophilus and Neisseria, specific sequences arerequired for efficient transformation (Danneret al. 1980; Elkins et al. 1991), but the receptorfor this sequence is unknown. More typically,DNA uptake shows no discernible sequencespecificity.

Single molecule experiments (Maier et al.2004) have shown that DNA uptake in B. subtilisis processive, with no detectable pausing at atemporal resolution down to 1 s. Uptake wasobserved to occur at a rate of about 80 bp/s.(A similar rate of (�90 bp/s) has been esti-mated for S. pneumococus (Mejean and Claverys1993)). Uptake remained processive and itsvelocity was invariant against large externalforces, up to �40 pN. These measurementsestablish both the DNA transformation motorand the FtsK/SpoIIIE motors as uniquely pow-erful. They also set the parameters that must beexplained by any proposed mechanism.

The Transformation Proteins

In B. subtilis and S. pneumoniae, several cyto-solic proteins under competence-specific con-trol are required for optimal transformation;RecA, SsbB (YwpH), DprA (Smf ) and YjbF(CoiA) (reviewed in Claverys et al. 2009).RecA is required for integration of transformingDNA and in its absence, little or no transfor-mation for chromosomal markers is detected,





although transformation by plasmid DNA pro-ceeds normally. When the competence-specificssDNA binding proteins SsbB or DprA are ab-sent, incoming DNA is degraded (Berge et al.2003). DprA appears to play a role in loadingRecA onto ssDNA to form RecA filaments, pre-paratory to recombination (Mortier-Barriereet al. 2007). The molecular role of YjbF is ob-scure. Inactivation of any one of these cytosolicproteins decreases transformation, but does notprevent the uptake of transforming DNA, meas-ured by the DNAse resistance of radiolabeledDNA. Nevertheless, it is possible that they doplay a role in transport, perhaps by binding toincoming ssDNA, thereby preventing back-ward slippage. Consequently, their absencemay affect the rate or processivity of uptake.

Two membrane proteins are needed for theappearance of DNAse resistant radiolabeledDNA but are not required for binding of DNAto the cell. One of these, ComEC, is a large poly-topic transmembrane protein, which is likely toform at least part of the pore for DNA uptake(Draskovic and Dubnau 2005). In B. subtilis,inactivation of comEC prevents not only uptakeof DNA, but also the degradation of the non-transforming strand. In contrast, degradationproceeds normally in the equivalent S. pneumo-niae mutant (Berge et al. 2002). In S. pneumo-niae, a membrane endonuclease (EndA) hasbeen shown to be required for nontransformingstrand degradation (Lacks et al. 1974). The endAgene is not under competence control and noortholog of endA exists in B. subtilis. Theseobservations suggest an important differencein the coupling of DNA uptake and degradationin the two bacteria. Another membrane-associated protein, ComFA, possesses theconserved motifs typical of Super family 2helicase/translocases and is also needed forDNA uptake but not for DNA binding. Muta-tion of the ComFA Walker A motif completelyinactivates the protein for uptake (Londono-Vallejo and Dubnau 1994) and ComFA maytherefore contribute energetically to DNA up-take by translocation or may mediate strandseparation before degradation of the nontrans-forming strand. These are attractive ideas, be-cause a membrane-anchored translocase could

drive DNA into the cell and particularly becauseSuper family 2 proteins can track along a singlestrand and do so mostly with 30 to 50 polarity(Singleton et al. 2007). A null mutant of a thirdmembrane protein, ComEA, does not internal-ize DNA and was reported to reduce binding tothe cell surface, measured by incubation withradiolabeled DNA followed by centrifugal wash-ing (Inamine and Dubnau 1995). ComEA pos-sesses two helix-loop-helix motifs near itscarboxyl terminus, and is inserted in the mem-brane with these motifs located externally. Suchmotifs are known to bind DNA nonspecifically,and indeed the corresponding part of ComEAbinds dsDNA (Provvedi and Dubnau 1999).An in-frame deletion of a central portion ofComEA, has a minor effect on DNA binding,but eliminates uptake. Apparently, althoughthe dsDNA binding activity of ComEA is essen-tial, ComEA is also needed for transport.

An important group of proteins essential fortransformation is related to Type 4 pilus (Craiget al. 2004) and Type 2 secretion (T2S) proteins(Johnson et al. 2006) of Gram-negative bacteria(Albano et al. 1989; Hobbs and Mattick 1993).Type 4 pili (T4P) are filamentous helical assem-blies of pilin proteins that emerge through asecretin pore in the outer membrane. Theassembly and disassembly of the T4P drivestwitching motility (Merz et al. 2000; Skerkerand Berg 2001; Burrows 2005). By analogy, ithas been proposed that cycles of assembly anddisassembly of a “pseudopilus” provide the me-chanical force for T2S through a secretin poreand for the uptake of DNA during transforma-tion (Mattick 2002). A T2S pseudopilus hasbeen characterized (Skerker and Berg 2001;Kohler et al. 2004), but no direct evidence forits cyclical assembly and disassembly has beenpresented.

Typically, the T4P, T2S and competence sys-tems encode several pilin or pseudopilin pro-teins, which contain conserved, hydrophobic,a-helical domains that traverse the membrane(Sauvonnet et al. 2000; Mattick 2002; Burrows2005; Chen et al. 2005; Proft and Baker2009). These systems also require dedicated,membrane-localized proteases, a conservedpolytopic integral membrane protein, and at





least one hexameric, P-loop “traffic ATPase”that is peripherally associated with the innerface of the cell membrane. This class of ATPasesis commonly associated with processes thatinvolve the transport of macromolecules andis a subclass of AAAþ proteins. Traffic ATPasesare required for formation of the pili and pseu-dopili. After removal of several amino-terminalamino acid residues from the pilin proteins bythe dedicated protease, the mature pilins aretranslocated from the membrane to the peri-plasm/wall and the T4P or Type 2 pseudopiliare assembled so that the hydrophobic a-helicesof the (pseudo)pilins are buried in the interfa-ces between the subunits of these structures(Forest and Tainer 1997; Craig et al. 2004).

This general picture applies in the case ofB. subtilis and almost certainly the other trans-formation systems. The B. subtilis orthologs ofT4P and T2S genes are encoded by comC (theprotease) and by the comG operon (reviewedin Chen and Dubnau 2004). This operon con-tains seven genes, encoding the secretionATPase (ComGA), the conserved polytopicmembrane protein (ComGB) and four typicalpseudopilins. Thus all the core components ofthese systems are present and a competencepseudopilus, composed of the major pseudopi-lin, ComGC, has been described (Chen et al.2006). Immediately after synthesis, ComGC islocated as an integral membrane protein withits carboxyl terminus extending outward fromthe membrane (Chung et al. 1998). Thecompetence-specific thiol oxido-reductase pro-teins, BdbD and BdbC then oxidize the twocysteine residues of ComGC to form an intra-molecular disulfide bond, and cleavage byComC takes place (Fig. 3A). The processedComGC is then translocated to a location out-side the membrane, and the competence pseu-dopilus is assembled (Chen et al. 2006). Inaddition to comC and bdbDC, each of thecomG ORFs are required for formation of thepseudopilus. This structure is asymmetric andhighly polydisperse. Remarkably, the ComGCsubunits are held together not only by noncova-lent interactions, but also by intermoleculardisulfide bonds, which presumably form byexchange with the pre-existing intramolecular

S-S bonds. The average length of the compe-tence pseudopilus is apparently sufficient tocross the cell wall of B. subtilis. (It is interestingthat in other, unrelated pili of Gram-positivebacteria, the subunits are covalently linked byisopeptide bonds (Mandlik et al. 2008)).

FRET measurements and microscopic co-localization studies on fluorescent fusion pro-teins have shown that many of the competenceproteins are in contact or in close proximity(Hahn et al. 2005; Kidane and Graumann2005; Kramer et al. 2007; Tadesse and Grau-mann 2007). Even the cytosolic (“soluble”)proteins are in intimate association with themembrane components of the transformationapparatus. Specifically, ComFA, ComEA,ComGA, SsbB, YjbF, DprA, and RecA havebeen shown to colocalize at the poles of B. sub-tilis (Fig. 4). This colocalization is dynamic, atleast for DprA and RecA (Kidane and Grau-mann 2005; Tadesse and Graumann 2007).DNA binding takes place preferentially at thepoles, close to foci of competence protein con-centrations, and less often near laterally locatedfoci, which are helically distributed. Single mol-ecule experiments indicate that DNA uptake,like binding, also occurs preferentially at ornear the cell poles (Hahn et al. 2005). Thesestudies show that the transformation proteinsfunction in concert and that a multicomponentprotein machine carries out transformation,from the initial binding step all the way tointegration.

Given the striking similarities of the pro-teins involved in transformation, T4P and T2S,the filamentous structures they form and therequirement in each system for movement (ofDNA, a cell, and protein respectively), it is at-tractive to consider that a mechanism like theone that powers twitching motility also drivesDNA uptake for transformation. In fact, T4P-like proteins are required for transformationin every system that has been studied, bothGram-negative and -positive, with the excep-tion of Helicobacter pylori (see later). The singlemolecule DNA uptake studies show that theforce-velocity characteristics of DNA uptakeare similar to those of T4P retraction; both areprocessive and operate with constant velocity





S

S S S

S

S

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S

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EA

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EA

SSComGB ComEC

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Figure 3. A model for transformation in Gram positive bacteria. ComGC is processed by the protease ComC andoxidized to form an intramolecular disulfide bond by BdbDC. (A) It is then translocated from the membrane ina step that requires the traffic ATPase ComGA and assembled into a pseudopilus in which the intramoleculardisulfide bonds are replaced by links between the subunits. ComGB is represented at the base of thepseudopilus without direct evidence. It is proposed that assembly and disassembly of the pseudopilus bringstransforming DNA to the ComEC channel and that interaction of the DNA with the C-terminal part of





against relatively high loads (Maier et al. 2002;Maier et al. 2004). No direct evidence yet existsdemonstrating a role for pseudopilus assemblyand disassembly in DNA transport, but evi-dence from analysis of comGA point mutationshas shown that the ATPase ComGA, and prob-ably the pseudopilus, are needed for transport(K. Briley and D. Dubnau, unpublished).ComGA, but not the pseudopilus, is also neededfor DNA binding to the cell. Because ComGA islocated on the inner surface of the membrane,the ComGA requirement for competence-specific DNA binding suggests that this protein

communicates across the membrane with anunidentified DNA receptor. This receptor isnot ComEA, and in fact when a filtration tech-nique rather than the more disruptive centrifu-gal washing measures binding of radiolabeledDNA, inactivation of comEA has very little effecton total DNA bound. It is likely that DNA bind-ing takes place first to the unknown receptor andto ComEA at a later step.

The Transformation Model

The following model for the transformationprocess is consistent with the available evidence(Fig. 3). dsDNA first binds to an unknownreceptor that communicates with ComGA.The DNA is cleaved by the membrane nucleaseNucA, providing an end for the initiation oftransport through a pore formed at least inpart by ComEC. The pseudopilus then drivesuptake by cycles of assembly and disassembly.The highly processive nature of uptake requireseither the participation of several pseudopiliacting in concert, or of another DNA bindingprotein that prevents the DNA from slippingbackwards through the pore. ComEA is anattractive candidate for this role. An unknownnuclease in B. subtilis (EndA in the case ofS. pneumoniae) degrades one strand duringtransport, and ComFA may unwind the DNAbefore this degradation step and/or may pro-vide part of the driving force for uptake afterdegradation. Following transport, ssDNA isprotected by binding to SsbB and DprA andalso interacts with RecA. Because the core com-petence proteins are conserved in (nearly) alltransformation systems, the essential featuresof this model are likely to be universal. An alter-native view, compatible with the evidence, is

Figure 4. ComGA-GFP localizes to the poles ofcompetent B. subtilis. This image shows fluorescencefrom DAPI-staining (blue), and a ComGA-GFPfusion (green), overlayed on a DIC image of tencells from a competent culture. Two competentcells are present, in both of which the ComGAfluorescence is localized near the cell poles. (Photocourtesy of J. Hahn.)

Figure 3. (Continued) ComEA is required for uptake, which is assisted by the ATPase ComFA. The transformingstrand (gray) enters the cytosol and the nontransforming strand is degraded with the products exiting the cell.The nomenclature is that of B. subtilis. In S. pneumoniae, EndA (not shown) carries out degradation of thenon-transforming strand. Also not shown is NucA, which introduces double strand breaks in the boundDNA. The large gray square represents cell wall material. (B) The pseudopilus has disassembled and theComGC subunits have returned to the membrane where they may form a pool available for further cycles ofassembly/disassembly. As the ssDNA enters the cytosol, it interacts with SsbB and DprA. These proteinsprotect the DNA and DprA also assists in loading RecA to form a filament, which is proposed to carry outthe homology search leading to recombination with the resident chromosome





that the pseudopilus functions to bring theDNA across the cell wall and periplasm, intocontact with the membrane-localized proteinsthat then mediate transport to the cytosol. Inthis view, transformation would be a two-stepprocess.

The cellular locations of DprA and RecAvary during transformation (Kidane and Grau-mann 2005; Tadesse and Graumann 2007).DprA, which shows only occasional polar local-ization before transformation, becomes dra-matically localized to the poles when DNA isadded. In contrast, RecA departs from the poleswhen DNA is added, forming filaments thatthen diffuse rapidly. A reasonable hypothesisis that ssDNA engages SsbB at the ComECpores, which serves to protect the DNA andaid assembly of ssDNA-DprA complexes.ssDNA thus acts to localize DprA near the cellpoles, although protein–protein contacts prob-ably also participate. DprA assists in the loadingof RecA on the ssDNA (Mortier-Barriere et al.2007), forming filaments, which then showdynamic behavior, conducting a diffusivesearch for sites of homology on the chromo-some. Thus, a picture is emerging of a transfor-mation machine, with moving parts thatmediate not only the binding and uptake ofDNA, but also the interaction of ssDNA withthe resident chromosome. This coordinatedaction of DNA uptake and recombination pro-teins constitutes strong evidence that transfor-mation has evolved for the acquisition ofgenetic information.

What are the sources of energy for uptake?Older experiments with inhibitors indicate arole for the pH component of proton motiveforce (PMF) (van Nieuwenhoven et al. 1982;Bremer et al. 1984). Consistent with this is thedemonstration in single DNA molecule experi-ments that uncouplers (DNP and CCCP) arrestuptake well before the ATP pool is depleted(Maier et al. 2004), suggesting that a protonsymport mechanism may drive transport. How-ever, it is fully possible that ATP also plays adirect role consistent with the essentiality ofthe Walker A motif of the helicase-like ComFAprotein for uptake (Londono-Vallejo and Dub-nau 1994).

In the T4P systems, there are two trafficATPases, one of which appears to drive assemblyand the other disassembly of the pilus (Herden-dorf et al. 2002; Burrows 2005; Chiang et al.2008; Jakovljevic et al. 2008). However, thecompetence and T2S systems each possess sin-gle known ATPases, which more closely resem-ble the assembly ATPases of T4P. Perhaps PMFdirectly disassembles the pseudopili or thesingle ATPase can operate in reverse. Recentlaser tweezer analyses of T4P retraction inN. gonorrhoeae and Myxococcus xanthus haverevealed the presence of two retraction motors;a high-force, low-velocity motor dependent onthe retraction ATPase, PilT and a high-velocity,low-force motor that is PilT-independent (atleast in M. xanthus) (Clausen et al. 2009a; Clau-sen et al. 2009b). The stalling force of the highvelocity motor (70 pN) is suggestive of theB. subtilis DNA uptake system, in which meas-urements hinted at a stalling force somewhatin excess of 40 pN (Maier et al. 2004). Thesefindings are consistent with a mode of compe-tence pseudopilus retraction that is not depend-ent on a traffic ATPase, perhaps driven insteadby PMF.

Other Dynamic Aspects ofthe Transformation Machine

During the development of competence inB. subtilis, many of the relevant proteins accu-mulate at the poles of the cells and in foci thatappear to be helically distributed in associationwith the cell membrane. More or less con-comitantly with a loss of transformability, theseproteins leave the poles (Hahn et al. 2005).Time-lapse microscopy and the use of inhibi-tors suggest that movement to the poles de-pends on diffusion, with capture at the polesby an anchoring site (Hahn et al. 2009). In con-trast, departure from the poles is energy-driven,and depends on the action of the proteins McsAand B. McsA and B are known to act together asadaptors for the ClpCP and ClpEP proteases(Kirstein et al. 2007) and inactivation of thesedegradative proteins (McsA, McsB, ClpP, or ofClpCþClpE) prevents delocalization of com-petence proteins. It is postulated that the





degradative complexes degrade an anchor pro-tein, releasing the competence proteins and inagreement with this, McsB, ClpC, and ClpPmove to the poles of competent cells (Hahnet al. 2009). The localization and delocalizationof competence proteins is clearly regulated, butthere is no information as to the signaling path-ways involved. It is also not known why assem-bly of the transformation machinery and DNAuptake occur at the poles. Perhaps localizationfavors low affinity assembly interactions, andthe poles simply provide a convenient address.Alternatively, unique properties of the wall/membrane at the poles facilitate assembly andinsertion of the competence machine.

Transformation in Helicobacter pylori

The Helicobacter transformation system is aunique case in which core inner membraneand cytosolic constituents (notably the channelprotein ComEC and dprA) are conserved (Andoet al. 1999; Smeets et al. 2000; Yeh et al. 2003)whereas the T4P-like proteins are replaced byconjugation proteins, encoded by the comB2-B4and comB6-B10 operons (Karnholz et al. 2006).Helicobacter is therefore a hybrid between themore common transformation systems andthe conjugation machines.

An instructive recent paper (Stingl et al.2010) shows that the comB operons of Helico-bacter mediates high velocity, low-force uptakeof double stranded DNA into the periplasm,whereas comEC is needed for transport of singlestranded DNA into the cytosol, presumably by ahigh-force mechanism akin to that used byB. subtilis. This study has at least two importantimplications. First, it is the clearest demonstra-tion that transformation in Gram-negative bac-teria is a two-step process, with uptake into theperiplasm temporally uncoupled from trans-port into the cytosol. Second, it implies thatthe T4P-like and the conjugation-like transfor-mation proteins play analogous roles, conveyingdouble stranded DNA into the periplasm andacross the cell wall to provide accessibility ofthis DNA to the conserved inner membranetransport apparatus. This view of competenceis similar to that proposed above for the roles

of the pseudopilus and membrane proteins inB. subtilis.

COMPARISON OF THE CONJUGATION,TRANSFORMATION AND FTSK/SPOIIIESYSTEMS: UNANSWERED QUESTIONS

The conjugation and transformation systemsuse ssDNA as substrate and require the assemblyof elaborate machines composed of severalproteins, including those that span the cell enve-lope. In both systems cytosolic proteins processthe DNA substrate either before transfer or fol-lowing import and in both, these proteins inter-act with and coordinate their activities with themembrane transporters. Another striking simi-larity between these systems is the involvementof pilus-like structures. However, given ourpresent state of knowledge, the resemblancebetween these systems ends here, because thereis little obvious similarity between the conjuga-tion and transformation proteins, other thanthe prominent involvement of P-loop NTPases.It is likely that this reflects our ignorance andthat analogous roles for individual proteinswill become apparent.

Because of this ignorance, many questionsremain unanswered. Among the most impor-tant unresolved issues are the following. Whatare the immediate sources of energy drivingthe movement of DNA across the membrane(s)?What proteins does the DNA contact, particu-larly during transformation, and at what stagesduring transport? Is assembly and disassemblyof a pilus-like structures involved in transport?In the Gram-negative transformation and con-jugation systems, is there a role for pili per sein the transport process, or do the pilus proteinsassemble into alternative DNA-transport spe-cific structures? Finally, what is the meaning ofthe positioning at the cell poles of not onlytransformation, but also of the conjugationproteins of some bacteria (Judd et al. 2005;Teng et al. 2008; Berkman et al. 2010). Clearlystructural information on the component pro-teins will contribute in a major way to ourunderstanding. In the case of transformation,the interactions of transport and recombination





proteins, analogous to those of the DTR pro-teins and membrane components of the con-jugation apparatus, continue to be of majorinterest.

The FtsK system, whereas functionally quitedistinct from the conjugation and transforma-tion machinery, does share the central and de-fining role of the P-loop ATPase activity, andin fact as mentioned earlier, several conjugationproteins are members of the HerA/FtsK family.In contrast to conjugation and transformation,the FtsK/SpoIIIE proteins use dsDNA as a sub-strate, and the barrier across which FtsK/SpoIIIE translocates the DNA is variable. Thus,whereas ostensibly a more simple system thanthe multicomponent machineries of conjuga-tion and transformation, the current state ofknowledge about FtsK and SpoIIIE raise severalchallenging mechanistic questions. For exam-ple, how are loops of DNA resolved? How dothese proteins clear other DNA binding proteinsaway from the transported DNA so rapidly andeffectively? And ultimately, what is the architec-ture of these complexes in the context of thecell division machinery and septal membranes?These questions continue to fuel inquiry frommany angles.

ACKNOWLEDGMENTS

Work cited from the BB lab was supported by aMassachusetts Life Science Center Grant, andfrom the DD lab by National Institutes ofHealth grants GM057720 and GM043756.

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23, 20102010; doi: 10.1101/cshperspect.a000406 originally published online JuneCold Spring Harb Perspect Biol

Briana Burton and David Dubnau Membrane-associated DNA Transport Machines

Subject Collection Cell Biology of Bacteria

Electron CryotomographyElitza I. Tocheva, Zhuo Li and Grant J. Jensen

Cyanobacterial Heterocysts

James W. GoldenKrithika Kumar, Rodrigo A. Mella-Herrera and

Protein Subcellular Localization in BacteriaDavid Z. Rudner and Richard Losick Cell Division in Bacteria

Synchronization of Chromosome Dynamics and

Martin Thanbichler

Their Spatial RegulationPoles Apart: Prokaryotic Polar Organelles and

Clare L. Kirkpatrick and Patrick H. ViollierMicroscopyAutomated Quantitative Live Cell Fluorescence

Michael Fero and Kit Pogliano

MorphogenesisMyxobacteria, Polarity, and Multicellular

Dale Kaiser, Mark Robinson and Lee KroosHomologsThe Structure and Function of Bacterial Actin

Joshua W. Shaevitz and Zemer GitaiMembrane-associated DNA Transport Machines

Briana Burton and David DubnauBiofilms

Daniel López, Hera Vlamakis and Roberto KolterThe Bacterial Cell Envelope

WalkerThomas J. Silhavy, Daniel Kahne and Suzanne III Injectisome

Bacterial Nanomachines: The Flagellum and Type

Marc Erhardt, Keiichi Namba and Kelly T. HughesCell Biology of Prokaryotic Organelles

Dorothee Murat, Meghan Byrne and Arash Komeili Live Bacteria CellsSingle-Molecule and Superresolution Imaging in

Julie S. Biteen and W.E. Moerner

SegregationBacterial Chromosome Organization and

Esteban Toro and Lucy Shapiro

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