reviews
Alphaproteobacteria
A group of Gram-negative, generally flagellated bacteria.
Plasmids
Semi-autonomous DNA sequences that are dispensable for bacterial growth but often confer useful new survival and colonization strategies on their bacterial hosts.
Partitioning
The distribution (or segregation) of newly replicated daughter plasmids to each of two nascent daughter cells.
1 Departamento de Alimentos, Universidade Federal de Ouro Preto, Morro do Cruzeiro, Ouro Preto, Minas Cerais 35400-000, Brazil. 2Department of Genetics & Biotechnology, Faculty of Biology, University of Athens, Athens 15701, Greece 3Department of Microbiology, 361A Wing Hall, Cornell University, Ithaca, New York 14853, USA. Correspondence to S.C W. e-mail: scw2@cornell. edu doi:10.1058/nrmicro2882
The ABCs of plasmid replication and segregation
Uelinton M. Pinto', Katherine M. Pappas2 and Stephen C. Winans5
Abstract | To ensure faithful transmission of low-copy plasmids to daughter cells, these plasmids must replicate once per cell cycle and distribute the replicated DNA to the nascent daughter cells. RepABC family plasmids are found exclusively in alphaproteobacteria and carry a combined replication and partitioning locus, the repABC cassette, which is also found on secondary chromosomes in this group. RepC and a replication origin are essential for plasmid replication, and RepA, RepB and the partitioning sites distribute the replicons to predivisional cells. Here, we review our current understanding of the transcriptional and post-transcriptional regulation of the Rep proteins and of their functions in plasmid replication and partitioning.
The class Alphaproteobacteria contains a fascinatingly diverse group of bacteria, including the human and animal pathogens Brucella spp., Bartonella spp. and Rickettsia spp., the plant-pathogenic Agrobacterium spp., the nitrogen-fixing plant symbionts Rhizobium spp., the nitrate-consuming phototroph Rhodobacter sphaeroides, the prosthecate bacterium Caulobacter crescentus, insect endosymbionts of the genus Wolbachia and many others. In many cases, the survival strategies of these bacteria require genes found on extrachromosomal genetic elements called plasmids. For example, Agrobacterium tume-faciens requires plasmid-encoded genes to cause crown gall tumours on host plants. Similarly, Rhizobium spp. require plasmid-encoded genes to form and colonize the host legume root nodules, where they reduce atmospheric nitrogen. The majority of alphaproteobacterial plasmids, particularly the larger ones, encode at least one repABC cassette, the encoded products of which direct plasmid replication and partitioning to daughter cells in a process analogous to mitosis.
Alphaproteobacteria challenge the conventional distinctions between chromosomes and plasmids. Plasmids are generally thought of as being much smaller than chromosomes and not essential for viability. However, some alphaproteobacterial plasmids are comparable in size to the main chromosome, and in most cases dispensability has not been tested. In the current genomics era, replicons that have been sequenced are designated plasmids if they seem to lack genes which are essential for core functions such as prototrophy or protein synthesis1. Many alphaproteobacteria have replicons that are referred to as secondary chromosomes or, more recently,
as chromids2'3. These replicons have one or more genes that are essential for core physiology, as well as a GC content similar to that of the primary chromosome, but possess plasmid-like replication and partitioning systems. Secondary chromosomes also tend to share synteny only within the same genus. Although we support the concept of chromids as elements that are intermediate between chromosomes and plasmids, in this Review we follow the more standard plasmid nomenclature that has been used in the genome annotations.
To paraphrase a popular aphorism, the goal of every plasmid is to become plasmids. However, if plasmids are dispensable for survival of the host in nature, one threat to their long-term survival is the birth of cured' (that is, plasmid-free) daughter cells during cell division. The long-term survival of unit-copy (that is, single-copy) and low-copy plasmids requires faithful vertical transmission from the mother cell to daughter cells, and most such plasmids optimize vertical transmission through a variety of mechanisms (FIG. 1). First, replication must occur frequently enough to ensure that sufficient copies exist to populate daughter cells. Second, low-copy plasmids rely on partitioning systems to physically distribute newly replicated plasmids into the two nascent daughter cells (high-copy plasmids, by contrast, tend to be distributed stochastically to daughter cells)4. Third, multimer resolution systems convert plasmid multimers into monomers through site-specific DNA recombination, to facilitate distribution5'6. Fourth, post-segregational killing of host cells causes plasmid-free daughter cells to lose viability owing to the action of plasmid-encoded toxin molecules, whereas plasmid-containing cells
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Replicons
Autonomously replicating DNA molecules.
Chromids
Replicons with both plasmid-and chromosome-like features: chromids have similar GC contents to cognate primary chromosomes and carry genes that are essential for core physiology, but they use plasmid-like partitioning and replication systems. The term chromid is largely synonymous with the term secondary chromosome.
Conjugative transfer
A form of interbacterial plasmid transfer that requires contact between a donor cell and a recipient cell.
Origin of replication
The minimal DNA region that supports autonomous replication. In plasmids, this is called oriV.
ParA and ParB
Proteins that mediate the partitioning of plasmids, prophages or chromosomes to nascent daughter cells.
dnaA
A gene encoding a protein that binds to bacterial replication origins and recruits other components of the replication machinery.
Cö) Co)
Cg)(oo) o
Multimer resolution fails
_____ IVIUUIIIICI ICJUIULIUII
o
Partition fails
(öö)-(ö§CD-(5ö;
Figure 11 Replication and partitioning of low-copy plasmids. Efficient transmission of low- orsingle-copy plasmids into daughter cells requires replication once per cell cycle and accurate partitioning of the two plasmids to daughter cells. Plasmids that undergo recombination-mediated multimerization must be converted back to monomers. A failure in any of these functions can lead to plasmid-free daughter cells. Thus, many plasmids contain post-segregational killing systems to eliminate plasmid-free cells. Figure is modified, with permission, from REF. 7 © (2011) American Society for Microbiology.
are protected by a plasmid-encoded antitoxin7'8. Fifth, conjugative transfer can provide a mechanism for some plasmids to recolonize cured cells9.
As described above, most of the larger alphaproteo-bacterial plasmids, as well as all secondary chromosomes, replicate their DNA and distribute it to daughter cells via proteins encoded by repA, repB and repC, which seem to be expressed as an operon. RepC is sufficient for replication, whereas RepA and RepB direct the partitioning of daughter plasmids. The origin of replication and partitioning (par) sites are also localized within or near the rep ABC operon. RepA and RepB resemble members of a large family of partitioning system proteins found throughout bacteria, whereas RepC does not resemble proteins from any other family. The fact that all three proteins are encoded in one operon is highly unusual among plasmid maintenance systems and might point to there being special challenges in ensuring appropriate levels of expression. Over the past decade, a great deal has been learned about these replication cassettes, although much work remains to be done. Here, we review recent insights into the expression of rep genes and the functions of the encoded proteins.
Distribution of RepABC-type systems
In 1989, a genetic locus bearing three genes, repA, repB and repC, was reported to be required for replication and partitioning of the octopine-type tumour-inducing (Ti) plasmids of A. tumefaciens10. RepC was found to be essential for plasmid replication, and RepA and RepB were shown to be dispensable for replication but required for efficient vertical plasmid transmission. Since that report,
homologous genes have been identified in hundreds of alphaproteobacterial isolates, almost always found to be linked in an apparent operon and in the same gene order11-14 (see Supplementary information SI (table)). More than 600 RepC-type proteins were identified in a BLAST search of the Genbank protein database (as of November 2011), all of which are found in alpha-proteobacteria; RepA and RepB homologues are found in a much wider range of bacterial plasmids, episomal prophages and chromosomes (see below).
More than one-third of the alphaproteobacterial genome sequences deposited in Genbank fall within the order Rhizobiales, and the distribution of rep ABC oper-ons in this group has been recently described111215. Of the 56 finished Rhizobiales genomes found in Genbank in late 2011,43 are annotated as having just one chromosome, 12 have two chromosomes and one has three chromosomes. The 56 genomes also collectively contain 93 plasmids ranging in size from 4.1 Kb to 2.4 Mb (see Supplementary information SI (table)).
Almost all of these 163 Rhizobiales replicons have partitioning loci that resemble those encoding ParA and ParB (see Supplementary information SI (table)). These genes fall into two well-separated clades: those found only on primary chromosomes, and those found only on secondary chromosomes and plasmids11. Genes in the primary-chromosome clade are designated/wA and parB, whereas genes in the plasmid clade are designated repA and repB. Primary chromosomes invariably contain one dnaA gene, which encodes a replication initiator (see below). In all cases, dnaA is unlinked to the parAB locus. None of the primary chromosomes contains repC.
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Box 11 Replication of enteric plasmid Rl
Plasmid Rl provides a well-studied model for replication systems of enteric plasmids8189. In this plasmid, the replication initiator RepA binds to the origin site, oriRl, which lies downstream of repA (see the figure, part a). This oriRl site contains binding sites for RepA flanked by a DnaA box at one end and three AT-rich repeats at the other (see the figure, part b). DnaA is not essential for replication of this plasmid, but seems to have an accessory role. DN A loop formation, mediated by RepA (see the figure, part c), is thought to drive DNA melting at the AT-rich region, which allows DnaC to load the replicative DNA helicase, DnaB. Replication initiates 400 nucleotides downstream of this site.
The repA gene is expressed from two promoters and is subjected to two levels of negative regulation (see the figure, part a). First, copB is co-transcribed with repAfrom promoter Pi, and the protein encoded by copB represses initiation of repA transcription from promoter P2, which remains silent under steady-state conditions (indicated by the dashed line for the transcript). Second, a counter-transcribed RNA, designated CopA, acts at a post-transcriptional level to block RepA synthesis by preventing tap translation and, therefore, repA translation, tap encodes a small leader peptide that is translationally coupled to RepA, and thus translation of tap is required for RepA synthesis. With the exception of iteron-type plasmids, most other plasmids use a counter-transcribed RNA to limit replication. Figure is reproduced from REE 89 © (2008) Henry Stewart Talks Ltd.
CopA RNA
oriRl I-
DnaA-binding site %     Initiation of Rl replication LZX^l RepA-binding sites <-       AT-rich repeat
would seem that rep ABC operons are poorly suited for use in primary chromosomes, and parAB and dnaA are equally poorly adapted to secondary chromosomes and plasmids.
Unlike other well-studied replication and partitioning systems, found in enterobacteria (BOXES 1,2), repABC cassettes are just beginning to be the focus of detailed studies. The best characterized repABC cassettes are found on the symbiosis plasmids p42d of Rhizobium etli and pSymA from Sinorhizobium meliloti, on two different Ti plasmids from A. tumefaciens and on plasmid pTAVl from Paracoccus versutus12'18'24. All of these model organisms, except for P. versutus, are members of the order Rhizobiales. Nonetheless, the genomes of these bacteria are dissimilar. _R. etli str. CFN42 has one circular chromosome and six circular plasmids ranging in size from 194 Kb to 642.5 Kb; all six plasmids have either one or two repABC cassettes (see Supplementary information SI (table))25, but p42d has received the most attention, as it carries the nod and nif genes that are required for root nodulation and nitrogen fixation. S. meliloti str. 1021 contains one circular chromosome and two symbiotic plasmids, pSymA (1.35 Mb) and pSymB (1.7 Mb)26, both of which are essential for root nodulation and replicate using repABC cassettes. A. tumefaciens str. C58 has a larger circular chromosome, a linear chromosome (2.1Mb), the Ti plasmid pTiC58 (0.2 Mb) and a second plasmid (0.5 Mb)27,28; both plasmids and the linear chromosome replicate using repABC cassettes. The genome of P. versutus has not been sequenced in its entirety, but it appears to have one circular chromosome and two plasmids, pTAVl (107 Kb) and pTAV3 (-400 Kb)29. Of these, pTAVl has two replication loci, a complete repABC cassette and a second copy of repC24,30, whereas pTAV3 encodes a different type of replication system31.
Conversely, none of the 14 secondary chromosomes or 93 plasmids carry dnaA. This pattern is also found in other orders within the class Alphaproteobacteria, although exceptions have been documented1617. All 14 secondary chromosomes and more than two-thirds of the plasmids include at least one repC (exceptions are found among the very small plasmids)11. Almost all repA, repB and repC genes are arranged in an apparent operon. Of the 93 plasmids, 68 have at least one complete repABC locus; five plasmids have two complete operons, and six plasmids have one complete locus and additional incomplete cassettes.
The repABC operons of secondary chromosomes do not form a separate clade from the plasmid-encoded operons. Instead, plasmid-encoded and chromosomal repABC operons are interspersed on evolutionary dendrograms and virtually impossible to distinguish from each other by sequence11. Apparently, there are no major barriers to adapting a plasmid cassette for use in a secondary chromosome or vice versa. In other words, the replication and partitioning systems of the 14 secondary chromosomes are extremely plasmid like. By contrast, the complete absence of horizontal transfer of these genes between primary chromosomes and other replicons is striking. It
Genetic structure of repABC-type cassettes
The available data indicate that the repA, repB and repC genes of each plasmid in FIG. 2 a are expressed from promoters that lie upstream of repA32'33. In the A. tumefaciens str. R10 plasmid pTiRlO (which is almost identical to other octopine-type Ti plasmids, such as pTiA6, pTiB6, pTil5955 and pTiAch5, found in other strains of A. tumefaciens), mutations that block activity of the promoters in this region also block repC expression32'34. Likewise, in p42d, disruptions of the repA promoter block repC expression, as do insertions of foreign DNA into repA or repB, owing to transcriptional polarity35.
The plasmid pTiRlO encodes a fourth gene, repD, which is 228 nucleotides in length and fully spans the gap between repA and repB36. Plasmid pTiC58 has a reading frame that is identical in position and length to repD but completely divergent in sequence. This divergence suggests that the expression of the two repD genes has no function other than to ensure the expression of downstream genes. Within repD of pTiRlO are two RepB-binding par sites36 that are highly conserved in pTiC58 and other members of the family, and are required for plasmid partitioning (see below). By contrast, no gap or small gene is found between repA and
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Box 2 | The partitioning system of prophage Pi
Many bacterial plasmids and episomal prophages, and all chromosomes are accurately distributed to predivisional daughter cells in a process referred to as replicon segregation or partitioning. Many of these replicons partition using members of the ParA-ParB family, the best studied members of which are the ParA and ParB proteins of prophage Pi (see the figure), the SopA and SopB proteins of the F plasmid and the Soj and SpoOJ proteins encoded by the Bacillus subtilis chromosome90-97. Partitioning systems also require a site, often referred to as parS or parC, that is functionally analogous to a eukaryotic centromere and is usually found closely linked to the parAB genes9798. Most members of the ParA-ParB family are encoded by bicistronic operons in which parA is located near the promoter.
ParB of bacteriophage Pi is dimeric and binds specifically to the DN A sequence parS, which is located directly downstream of the operon (see the figure, part a). A bound ParB dimer serves to nucleate the binding of additional dimers, extending outwards from parS9799 100 (see the figure, part b). The carboxy-terminal half of ParB suffices for parS binding61, whereas the amino-terminal region is required for oligomerization and for binding to ParA101. The crystal structure of ParB-parS shows that each ParB subunit has two different DNA-binding domains, and a ParB dimer is likely to contact neighbouring plasmids102'103. ParA of Pi is also dimeric and contains Walker A and Walker B ATPase motifs. ParA is a weak ATPase, the activity of which is enhanced by binding to ParB. ParA bound to ADP can autorepress the parAB promoter (see the figure, part a), and purified ParA protects an extensive region surrounding the promoter. ParB increases repression of this promoter through stimulation of the ParA ATPase activity, leading to accumulation of the repressor form, ParA-ADP. The N-terminal domain of ParA is responsible for binding to promoter DNA through a helix-turn-helix motif in a process that requires ADP96104. ParA-ATP dimerizes and associates cooperatively to bind DNA nonspecifically in a dynamic fashion, oscillating over the nucleoid105, and this is believed to play an important part in partitioning105106. In fact, ParA-ATP bound to the nucleoid attracts the partitioning complex composed of ParB-parS, which in turn stimulates ATP hydrolysis and, eventually, the disassembly of the complex. It is proposed that this interaction drives partitioning of the daughter plasmids to the respective daughter cells107 (see the figure, part b). Figure is reproduced from REF. 90 © (2008) Henry Stewart Talks Ltd.
pcjrS
parA I  I parB
i
^ParA-ADP
Autorepression co Partitioning
Counter-transcribed RNA
A term used in plasmid biology to describe a type of antisense RNA that is synthesized from the DNA strand which is complementary to its target RNA. Like other antisense RNAs, counter-transcribed RNAs form a duplex with their targets, usually leading to degradation of both strands.
repB of p42d, pSymA or pTAVl. The par sites in these plasmids lie directly upstream or downstream of the corresponding repABC operons (FIG. 2a).
All repABC cassettes contain a gene between repB and repC that encodes a short, non-translated counter-transcribed RNA that downregulates expression of repC18'20'22, thereby controlling plasmid copy number and incompatibility (see below).
There are two other notable features of these operons (FIG. 2b). First, each has a conspicuous AT-rich region
within repC that, as described below, seems to contain the origin of plasmid replication. Second, the DNA sequence GANTC is over-represented in the putative replication origin and in the promoter of the counter-transcribed RNA. These sequences are substrates for a DNA methyl-ase that modifies the A residues, and the motifs might play a part in the timing of various events in the cell cycle37.
RepC and the origin of replication
RepC-type proteins have been found only in alpha-proteobacteria1113'23'38'39, and they bear no apparent homology to any other replication initiator proteins. Several repC genes have been shown to be essential and sufficient for plasmid replication in the hosts from which they originate18'23'40"43. The ability of a repC coding sequence to confer the ability to replicate on a plasmid indicates that the origin of replication must lie within this gene12'24,42,43. Such a location for the origin, although unusual, has precedent in several other types of plasmid and at least one bacteriophage12'44'45. The origins of many types of plasmids contain directly repeated DNA sequences called iterons, which serve as binding sites for replication initiators46-51. Origins that require RepC for activity are almost unique in their lack of iterons51. As mentioned above, all repC genes contain an AT-rich sequence of -150 nucleotides near the middle of the protein-coding sequence; such an AT-rich sequence is another common feature of diverse replication origins. Purified RepC from pTiRlO binds to the AT-rich segment via a highly conserved DNA sequence that includes imperfect dyad symmetry43. A second dyad located immediately downstream of the RepC-binding site is even more strongly conserved but does not bind RepC in vitro43, suggesting that it binds some other replication factor.
Secondary-structure predictions and amino acid conservation suggest that RepC has two domains, an amino-terminal domain (NTD) from residues 1 to 265 and a carboxy-terminal domain (CTD) from residues 295 to 439, joined by a 30 amino acid linker that is hydro-philic, unstructured and poorly conserved. A fragment containing just residues 26-158 is sufficient for DNA binding, albeit with strongly reduced sequence specificity43. This region of RepC has a structural resemblance to members of the DnaD family of low-GC-content Gram-positive bacteria. DnaD interacts with either DnaA or primosomal protein N' (PriA) at replication origins or replication restarts, respectively. In low-GC-content Gram-positive bacteria, DnaD, DnaB and Dnal together load the replicative DNA helicase onto the DNA at the origin of replication and at replication restarts52. The same region (residues 26-158) of RepC also resembles members of the MarR family of transcription factors, which bind DNA as dimers by means of a winged helix-turn-helix motif53. The role of the RepC CTD remains to be revealed; the last 39 amino acids of p42d RepC were found to play a part in plasmid incompatibility42.
Despite extensive efforts, we have never been able to detect pTiRlO RepC acting in trans*3. The same is true of p42d RepC42. Furthermore, overexpression of pTiRlO RepC causes a large increase in the copy number of the
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pTiRl0andpTiC58     H repÄ p42d ^ repA
psymA oxiaDDC^B^:
pTAVl H    repX repC]
repD
repE
xe:
repC|
repb VT"    repC| 1
repCI      I v
incA
repB
repE homologue?
ZZ><1      repC| 3^)ffl>
GANTC GANTC IGANTC
GANTC
GANTC
GANTC J_
GANTC _1
repC
GANTC
GANTC / GANTC
Figure 2 | Genetic organization of repABC systems from representative RepABC plasmids. a | The replication and partitioning [repABC) modules of the Agrobacterium tumefaciens tumour-inducing (Ti) plasmids pTiRlOand pTiC58, of the Rhizobium etlistr. CFN42 plasmid p42d, of theSinorhizobium meliloti plasmid pSymAand of the Paracoccus versutus plasmid pTAVl (see main text for details). The partitioning sites are shown in orange, and the AT-rich regions that are believed to contain the plasmid origin of replication (oriV) are shown in blue. Arrows immediately upstream of repC represent counter-transcribed RNAs; in the case of pTiRlO, this RNA is called repE. The repD gene of pTiC58 is provisional and based solely on DNA sequence analysis, b | The repE-repC region of pTiRlO, showing the abundance of CANTC sites located near the repE promoter and in the AT-rich region. The CANTC sites at these locations are a common feature of repABC operons.
Copy number
The number of copies of a plasmid per bacterial cell; this number is generally held constant by the replication machinery.
Autorepression
The ability of a protein to repress the promoter of the gene encoding that protein.
plasmid expressing the protein, but has no effect on the copy number of a second RepC-dependent plasmid in the same cell, confirming that RepC functions only in cis. Although most other replication initiators function well in trans, those of the closely related plasmids Rl, NR1 and R100, as well as that of pMU720, function only in ris54-56.
Replication initiator proteins of iteron-type plasmids generally bind to the origin of replication and recruit DnaA, causing melting of the AT-rich region at the origin and thus creating the replication bubble. DNA-DnaA complexes recruit the replicative DNA helicase bound to the loading factor (DnaB and DnaC, respectively, in Escherichia coli), and the helicase in turn recruits DNA polymerase. Replication usually proceeds bidirectionally57. There are no apparent DnaA-binding motifs matching the consensus sequence for alpha-proteobacteria anywhere within repC37. It nonetheless seems plausible that RepC recruits DnaA, which would then recruit DnaB-DnaC58. It is also possible that RepC is able to recruit DnaB-DnaC directly. The resemblance between RepC and DnaD of Gram-positive bacteria suggests a role for RepC in loading the replicative helicase.
It is striking that some bacteria can have as many as six RepABC family replicons (see Supplementary information SI (table)). One bacterium, R. etli, has eight complete cassettes distributed over six replicons. One might wonder how each RepC can function only at its cognate origin rather than at heterologous origins in the same cell. Although most RepC proteins found within a single bacterium tend to be fairly divergent11, there are several surprising examples to the contrary.
One such case is the RepC proteins of the R. legumino-sarum plasmids pRL9 and pRL12; these proteins are 97% identical. Similar examples are found in plasmids from Nitrobacter hamburgensis, Mezorhizobium loti str. MAFF303099, Agrobacterium vitis str. S4 and R. leguminosarum bv. trifolii str. WSM1325 (see Supplementary information SI (table))42. Can two almost identical RepC proteins avoid acting at heterologous origins, and if so, how? We hypothesize that all RepC proteins are strictly cis acting, as was shown for those of pTiRlO and p42d (see above). If this were true, these proteins would not need to diverge, as they could never act at heterologous origins, no matter how similar the proteins were. We have constructed strains containing two different plasmids with identical repC genes and did not notice any incompatibility43. However, the opposite was reported for p42d42, so additional studies are clearly needed.
Plasmid partitioning by RepA and RepB
The RepA and RepB proteins encoded by repABC cassettes generally resemble the larger family of ParA and ParB proteins (BOX 2) and are likely to have similar general properties. Insertions, frame-shift mutations or deletions in repA or repB substantially decrease plasmid stability10'24'35'59'60. Sequence homology between RepA and the ParA of prophage PI is largely restricted to the ATPase domain, which also resembles other ATPase domains in a large range of enzymes. At least two alpha-pro teobacterial RepA proteins mediate autorepression33'34. One of these RepA proteins is that of pTiRlO; this RepA autorepresses the P4 promoter (FIG. 3), which contains a 40-nucleotide region of dyad symmetry that lies within
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a 70-nucleotide region protected by purified RepA34. Amino acid residues 50-112 of RepA are strongly predicted to have a helix-loop-helix DNA-binding motif similar to that found in residues 43-105 of the crystallized PI ParA protein61. The binding of pTiRlO RepA to its operator is stimulated by ATP (and to a lesser extent by ADP) and by the addition of RepB34.
The par sites of pTAV320, p42d, pSymA, pTiC58 and pTiRlO consist of one or more copies of a 16-nucleotide palindromic sequence with the consensus GTTNNCNGCNGNNAAC. The number and position oipar sites present in this family of rep-licons varies widely (FIG. 2a). These sites are essential
for plasmid stability, bind RepB and are incompatible with their respective parental plasmid when provided in trans24'36'59'62. This is presumably due to competition between the two plasmids for the partitioning machinery. Point mutations in the cloned par site that reduce RepB binding also eliminate plasmid incompatibility59. Purified pTiRlO RepB binds with low affinity to DNA containing the two par sites within repD, but the affinity of this binding is increased by the addition of RepA34 (FIG. 3a).
Fluorescence in situ hybridization (FISH) has shown that the origins of all repABC replicons in A. tumefa-ciens and S. meliloti are located at or near the cell pole63.
CtrA?
trb-tra/ i
C7/~l-[*D-E^MÜ
-1 copy per cell
~4 copies per cell
VirC trb-tral
CO-
VirAplus (g (j? phenolics
Partitioning proteins Replication initiator
RepA^^   ^.(kepB) (^RepC^)
repE |
^i*—^
RepE
trb-tra/ I I        repD I repE I
loo
~8 copies per cell
trb-tral f } I        repD I repE I
Figure 3 | Regulation of the repABC operon of octopine-type tumour-inducing (Ti) plasmids. a | Autoregulation. Transcription of the repABC operon is inhibited by autorepression mediated by RepA-RepB complexes at the operator region downstream of P4 (a prominent region of dyad symmetry is indicated by inverted arrows) and at the partitioning (par) sites located between repA and repB. Expression of repC is inhibited transcriptionally and post-transcriptionally by the counter-transcribed RNA RepE. In the absence of external signals, tumour-inducing (Ti) plasmids are maintained as single copies. Additional regulation may be provided by phosphorylated CtrA. b,c | The copy number of Ti plasmids is influenced by at least two diffusible chemical signals. Plant-released phenolic compounds are detected by the VirA-VirC two-component system, triggering VirA-mediated phosphorylation of VirC; phosphorylated VirC binds the vir box (vb) to activate transcription from promoter P4 (part b). Thus, in the presence of phenolics, which are encountered when bacteria enter the plant stem or root through a wound, the copy number of Ti plasmids increases to about four copies per cell. In addition, TraR-3-oxo-octanoylhomoserine lactone (OOHL) complexes, which are encountered in the environment of crown gall tumours, bind to tra box II (tbll) and tblll, activating the tral-trb operon through promoter Ptra/ and the repABC operon through promoters Pi, P2, P3 and P4 (part c). In the presence of TraR-OOHL complexes, the copy number of Ti plasmids therefore increases to about eight copies per cell.
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o
-120
-102
-140
-158
5'      RepE    3' -158      RepE     -102 Transcription
termination
repß-repC intergenic region
Translational occlusion
Figure 4 | Model of the mechanism of action of the counter-transcribed RNA RepE on the control of repC expression. AH numbers refer to the position in the mRNA relative to the repC translational start site (AUG), a | Predicted structure of the mRNA upstream of repC in the absence of RepE. Note that the ribosome-binding site (RBS) and the translational start site of repC are available for translation initiation, b | Predicted structure of RepE. c | Binding of RepE to the target mRNA upstream of repC could cause the formation of several hairpins, one of which resembles a Rho-independent transcription terminator, d | RNA molecules that are not terminated are predicted to have an alternative stem-loop that sequesters the RBS and the translational start site of repC.
In double-labelling experiments in A. tumefaciens, it was shown that the origins of two different replicons rarely colocalize; rather, they occupy close but clearly distinct sites.
Above, we asked how up to eight different RepC proteins can coexist in a single bacterium. The same question arises for RepA and RepB. In a survey of bacteria from the order Rhizobiales, multiple RepA and RepB proteins within the same bacterium were in all cases rather divergent111215. In this order, no two RepA proteins in the same bacterium are more than 61% identical, no two RepB proteins are more than 51% identical, and most pairs show lower similarity. This divergence could help to minimize heterologous interactions. It is also noteworthy that the evolutionary dendrograms of RepA and RepB within the order Rhizobiales are congruent, indicating that these proteins have co-evolved without horizontal exchange. A completely different picture emerges when comparing the phylogenies of RepA and RepB with that of RepC: this comparison provides clear evidence of extensive exchange of repC genes between heterologous cassettes11.
CtrA
A transcription factor of Caulobacter crescentus that is synthesized and phosphorylated during a particular portion of the cell cycle to regulate the expression of various promoters.
VirG
A transcription factor of Agrobacterium spp. that is phosphorylated by VirAin response to plant-released phenolic compounds and activates transcription of Plasmid tumour-inducing vir genes, which direct the transfer of tumorigenic DNA fragments into host cell nuclei.
Quorum sensing
A form of transcriptional regulation in bacteria. Quorum sensing systems consist of a bacterial pheromone (which accumulates at high population density), a pheromone synthase and a pheromone receptor, and they most often function to activate target genes in the presence of the pheromone.
Regulation of repC by a counter-transcribed RNA
Each repABC operon shown in FIG. 2a is thought to have a gene lying between repB and repC, but in the opposite orientation; this gene encodes a non-translated RNA of approximately 50 nucleotides in length. The RNA includes a predicted stem-loop that might serve as a Rho-independent transcription termination site and might also play a part in contacting complementary
mRNA sequences18,64 (FIG. 4). Similar genes are predicted to be conserved in most or perhaps all members of the repABC family. Plasmids that have a cloned copy of repE (and that replicate using a different replication system) are incompatible with plasmids that replicate using the cognate repABC cassette18- The same is true of at least two other homologous systems20,64. Point mutations that alter the structure of the RepE RNA or decrease its expression decrease this incompatibility10,18,20,22,35,64,65.
The counter-transcribed RNA of pTiRlO, known as RepE, inhibits both the transcription and translation of repCls. Downregulation of RepE leads to increased plas-mid copy number, and a lack of RepE can lead to lethal runaway replication18. RepE is predicted to alter the balance between two alternative stem-loop structures in the mRNA upstream of the repC coding sequence. According to this model, in the absence of RepE, the ribosome-binding site of repC is available for translation initiation. In the presence of RepE, alternative mRNA folding creates a Rho-independent transcription termination site upstream of the ribosome-binding site. Any mRNA molecules that are not terminated are predicted to make an alternative stem-loop that sequesters the ribosome-binding site (FIG. 4). This regulation, combined with transcriptional repression by RepA and RepB, reduces repC expression to almost undetectable levels34. The counter-transcribed RNA of plasmid p42d is hypothesized to function in the same way64.
Transcriptional regulation of repABC
Information about transcriptional regulation is somewhat sparse for most repABC systems but is most well developed for pTiRlO. The repABC operon of this plasmid is transcribed from no fewer than four promoters, one of which (P4) provides basal expression of the operon (FIG. 3) and is autorepressed by RepA and RepB (see above). The P4 promoter region also contains a possible binding site for another two-component response regulator, CtrA, which regulates the cell cycle of Caulobacter crescentus37. A perfect copy of this motif (TTAAN?TTAA) is centred 50-nucleotides upstream of the P4 start site. The role of CtrA in Ti plasmid replication needs to be explored.
Promoter P4 of pTiRlO is also activated by the two-component response regulator VirG, which is phosphorylated by VirA in response to plant pheromones66 (FIG. 3b). Phosphorylated VirG binds to a virbox centred 71-nucleotides upstream of P4, leading to a 3-4-fold increase in Ti plasmid copy number (FIG. 3b). In addition, all four repABC promoters are activated by the quorum sensing transcription factor TraR, a Rhizobiaceae family LuxR-type transcription factor that allows cell density-dependent gene expression and which requires the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) for activity32,34,67. TraR-mediated activation of these promoters leads to an approximately eightfold increase in Ti plasmid copy number (FIG. 3c) and an increase in tumorigenesis in plants infected with bacteria containing these plasmids32,65. Transcriptional activation of repABC by TraR-OOHL has been also described for the symbiotic plasmid pRLlJI of R. leguminosarum6S. Two
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Dam methylase
ADNA methylase that is found in enterobacteria and methylates the A residues of GATC motifs. Cells can recognize newly synthesized DNA by its lack of methylation.
other rhizobial plasmids, pRL8JI of _R. leguminosarum and pNGR234a of Rhizobium sp. NGR234, have TraR-binding motifs in the same regions, although their roles remain to be tested69. Therefore, expression of the octo-pine-type Ti plasmid repABC operon and, ultimately, the collective Ti gene dosage are enhanced both by plant-released chemical signals (leading to phosphorylation of VirG) and quorum sensing pheromones (activating TraR).
DNA methylation sites in the repABC region
DNA methylases have several roles in bacterial physiology70, perhaps the best known of which is to protect DNA from cognate restriction endonucleases. Another role is thought to be enabling the cell to distinguish parental and newly synthesized daughter DNA. Distinguishing between the two is important for mismatch repair, for initiation of chromosome replication and for the activity of some promoters71. Methylation can also decrease the melting temperature of duplex DNA72"74. The E. coli origin of replication is rich in GATC sites, which are methylated by Dam methylase at the N6 position of A residues. After replication, hemimethylated GATC sites are tightly bound by SeqA, slowing the rate at which these sites are methylated by Dam and sequestering these sites from the replication initiation factor, DnaA. Eventually, Dam succeeds in methylating the sites, and this blocks SeqA binding. Hemimethylated GATC sites therefore block replication early in the cell cycle, and this block is eventually relieved when the sites become fully methylated later in the cell cycle75.
In the alphaproteobacterium C. crescentus, there is an analogous (although not homologous) methylase to Dam called cell cycle-regulated methylase (CcrM), which methylates the N6 position of A residues in the sequence GANTC76. The origin of replication in C. crescentus has five GANTC sites, and their full methylation is required for replication initiation77. GANTC sites are also found in the promoters of several C. crescentus genes that have important roles in cell cycle timing78, and methylation of these promoters influences their expression78'79. Alphaproteobacteria do not have a protein homologous to SeqA, but another protein might play an analogous role. There is at least one important difference between Dam and CcrM: Dam is thought to be active throughout the cell cycle, whereas CcrM is synthesized by and is active in predivisional cells only. Therefore, GATC sites of E. coli are hemimethylated only transiently after replication, whereas GANTC sites of C. crescentus remain hemimethylated for much of the cell cycle70-78.
The putative origins of replication of repABC plasmids are rich in GANTC sites, as are the promoters of the counter-transcribed RNA genes (FIG. 2b). If methylation of these sequences occurs only at a particular point in the cell cycle, this could have important consequences for origin utilization and/or expression of RepC. Methylation does not affect the binding affinity of RepC for DNA in vitro*3, but it might influence the binding of some other replication factor or might enhance origin melting72"74. Moreover, methylation of
the repE promoter might influence the production of the counter-transcribed RNA RepE, which downregulates RepC synthesis.
Prospects for future work on RepABC systems
A great deal of work remains to be done on this large family of replication and partitioning systems. Under most conditions, plasmids containing these systems are held at unit copy, and progress has been made in understanding the negative-control checkpoints that confer this property. Furthermore, in the case of A. tumefaciens, replication of the four replicons is synchronized, despite the use of different replication initiators for each replicon80. The underlying mechanisms for this synchronicity could involve CtrA abundance and phosphorylation (both of which are cell cycle regulated in another alphaproteobacterium37), methylation of GANTC sites in or near the replication origins, or both. Methylation could alter the melting temperature of the origin or the binding of replication factors. Future work on synchronized cultures could provide information about the cell cycle dependence of CtrA accumulation and DNA methylation.
As described above, changes in repABC transcription can alter plasmid copy number and thereby affect the expression of all plasmid-encoded genes. This can have major consequences for host-bacterium interactions. In the case of A. tumefaciens, increasing Ti plasmid copy number results in increased tumorigen-esis15'32. The discovery that environmental stimuli can affect plasmid copy number might be true of other plasmids that replicate using repABC cassettes as well, although this remains to be shown. If it is a common effect, the signals and regulatory systems might differ greatly in different organisms. Artificial overexpression of repC causes substantial increases in plasmid copy number43, and this effect could have profound applications in synthetic biology, including the development of new approaches for constructing transgenic plants and fungi.
Among the Par protein family members, RepA and RepB offer an unprecedented opportunity to understand how up to eight different partitioning protein pairs can function in harmony in a single cell. It will be interesting to examine the possible epistatic relationships between these multiple RepA-RepB pairs, and to determine how segregation is achieved, particularly in cases for which more than one Rep A-RepB pair is found in a single replicon. It will also be interesting to learn more about the roles of the many ParA and RepA proteins that lack cognate ParB or RepB partners.
At least some RepA and RepB proteins form hetero-multimers that can bind at par sites and can also bind repABC promoters34'36. Can a complex containing RepA and RepB contact a promoter and a. par site simultaneously, forming a DNA loop? How would loop formation affect the roles of these proteins in partitioning and in autoregulation? It is curious that the positions of the par sites can vary so widely (FIG. 2a), and it would be interesting to learn whether moving these sites would affect their function. How much or how little DNA can separate
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par sites from rep ABC promoters? One major difference between the RepA-RepB system and the homologous ParA-ParB system is that the autoregulation of Rep A and RepB also affects expression of RepC, which in turn regulates replication frequency and copy number. The Rep ABC system has evolved such that changes in transcription initiation have an impact on repC expression, but large fluctuations in RepC expression must be avoided.
RepC appears to be unique to alphaproteobacteria. The NTD of pTiRlO RepC can bind DNA, but with limited sequence specificity. The CTD of this protein is predicted to have very weak structural similarity to bacterial transcription factors, suggesting that this domain has some affinity for DNA independently of the NTD. In preliminary experiments, the CTD does not stably bind DNA in electrophoretic mobility shift assays43, but this needs to be confirmed using other RepC fragments. The prediction that RepC contains two domains separated
by a 30 amino acid linker requires experimental verification, and the two RepC domains are also attractive targets for structural studies.
We know little about the specific consequences of RepC binding to the origin of replication, although in all likelihood this binding recruits the replisome. Most replication initiators recruit DnaA to the origin, which in turn attracts DnaB-DnaC. A few plasmid replication initiators replace either DnaA alone or all three proteins. It will be interesting to determine which host replication proteins are recruited by RepC. RepC does not function at all in E. coli; perhaps a library of A. tumefaciens DNA could be screened for genes that allow RepC to function in a heterologous bacterial host, and this could help elucidate how RepC functions in its native host. As is the case for all experimental science, every new insight about these genes reveals a dozen or more new questions to be answered, and we now have enough thorny questions to last a lifetime.
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Acknowledgements
The work from the author's laboratory that is described in this article was supported by a grant from the National Institute of General Medical Sciences, US National Institutes of Health (GM042893). U.M.P. acknowledges financial support from the Brazilian government through the Coordenacao
reviews
de Aperfeicoamento de Pessoal de NTvel Superior (Capes). K.M.P. acknowledges the University of Athens (Greece) Research Committee (grant 70/4/7809).
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
Katherine M. Pappas's homepage: http://en.biol.uoa.gr/
departments/department-of-genetics-biotechnology/
pappas-katherine.html
Stephen C. Winans's homepage: http://micro.cornell.edu/
research/labs/winans-lab/index.cfm
Genbank: http://www.ncbi.nlm.nih.gov/genbank
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