Antagonismo microbiano

Plasmids must replicate to allow their vertical transmission; failing this they would simply be lost from the host during cell replication and division. Therefore all plasmids contain a distinct locus (typically 500 bp-3 kb), known as the origin of replication (oriR) at which plasmid replication is initiated. The oriR not only allows initiation of plasmid replication but it also regulates the rate of initiation, allowing the plasmid to be maintained at a defined copy number per cell (Abeles et al., 1995). Plasmid copy numbers range from one to over a thousand per cell (Schroeter et al., 1988). In all plasmids studied to date the copy number is controlled by either an antisense RNA transcript (Osborn et al., 2000) or by the binding of replication (Rep) proteins to regulatory sequences near the initiation sites (iterons; Abeles et al., 1995).

In the RNA-binding mechanism, an anti-sense RNA targets an overlapping and complementary (sense) RNA, transcribed from the opposite strand. This target RNA would otherwise be used as a primer for DNA replication, or as a messenger for a Rep

secondary folding (Brady et al., 1983). This type of regulation, known as an “inhibitor-target mechanism”(Novick, 1987), is employed by many smaller plasmids which use basic replicons as well as several large conjugative plasmids of the incompatibility group F (IncF).

In the iteron-binding mechanism a trans-acting replication initiation/control protein (Rep) binds to a series of typically 18-22 bp direct repeats (iterons) situated near the

oriR (Hitoshi et al., 1999). Rep proteins use ATP to separate the double stranded DNA, then use their DNA helicase domains (or a host encoded DNA helicase during rolling circle replication; considered below) to partially unwind the plasmid

(Kornberg et al., 1978, Arai et al., 1981). The partially unwound (relaxed) state of the

double helix at the plasmid oriR allows the replication machinery to assemble and commence transcription. However, when Rep is in excess, it forms dimers. These dimers bind to inverse repeats in the rep gene promoter sequence preventing its own transcription (Diaz-Lopez et al., 2003).

The trans-acting feedback regulation of the oriR by either the inhibitor-target or iteron-binding mechanism means that two plasmids sharing common elements involved in their control and partitioning cannot coexist and be propagated stably in the same host. This feature has lead to the grouping of plasmids based upon their incompatibility (Scaife & Gross, 1962). Yet, there are exceptions to this rule; the plasmids may contain multiple replication origins. This feature is common to the IncF group of large plasmids (Bergquist et al., 1986). Plasmids regulated by the inhibitor- target mechanism may also become compatible by nucleotide changes in the overlapping transcript.

Following the initiation of replication, plasmids are replicated by either a Rolling- Circle (RC), a Strand Displacement or a Theta (θ) mechanism. RC replication was first seen in the 4 Kb plasmid pT181 from Staphylococcus aureus (Koepsel et al., 1985). Despite being most prevalent in Gram-positive bacteria, and used by the

Butyrivibrio plasmid pOM1 (Hefford et al., 1997), the mechanism appears to be

asymmetric process (del Solar et al., 1998) and is initiated by the incorporation of a nick in a site known as the double-stranded origin (dso) by a plasmid-encoded Rep protein (Marsin et al., 2000). The dso is found exclusively in the plus, or Crick, strand of the plasmid (del Solar et al., 1998). The resulting free 3‟-OH end is used as a primer for leading strand synthesis (Lechner & Richardson, 1983). This process is thought to involve chromosome-encoded replication machinery such as DNA polymerase III and a single-strand binding protein (McInerney & O'Donnell, 2004). Along with the plasmid-encoded Rep protein, this is known as the replisome. As replication proceeds, the Crick strand is displaced. Replication continues until the replisome reaches the dso (Khan et al., 1988). Finally the Rep protein catalyzes a DNA strand transfer reaction (Lechner et al., 1983). This process releases both a double-stranded DNA molecule, consisting of the parental minus, or Watson, strand and the newly synthesized positive strand, and a single stranded DNA molecule consisting of the parental Crick strand (Khan et al., 1988). The single-stranded molecule serves as a template for minus (Watson) strand synthesis. This process commences from the single-stranded origin (sso), distinct from the dso, but typically in the vicinity of the dso (Birch & Khan, 1992).

The Strand displacement mechanism, seen in some incompatibility group Q plasmids (del Solar et al., 1998), requires two symmetrical and adjacent single strand origins (sso) present on opposite strands (Honda et al., 1992). A rep protein binds to iterons in an adjacent AT-rich region and unwinds the duplex exposing the two sso as single- stranded entities. A plasmid-encoded primase primes the sso and replication proceeds continuously displacing the complimentary strand (Honda et al., 1992). This results in two dsDNA molecules, each possessing a parental strand.

Theta (θ) replication can be either uni- or bi-directional (Frere et al., 1993, Bruand et al., 1993), involving a leading and a lagging strand. The leading strand is synthesized continuously, while the lagging strand is synthesized in discontinuous (Okazaki) fragments, which are later joined by DNA ligase (Inselburg & Oka, 1975). Theta replication can begin from a single or multiple oriRs. Theta oriRs commonly contain a

replication initiator protein (Park & Chattoraj, 2001). Theta replication is seen in the

Butyrivibrio plasmids pRJf1 and pRJf2 (Hefford et al., 1997).

Analysis of the replication origins of all megaplasmids sequenced at the time of writing this literature review shows that most (>95%) contain genes encoding Rep proteins of the RepA or RepB-family (70%) and/or Partitioning (Par) proteins, such as ParA or ParB (82%) typically in close proximity to one another and, where defined, the replication origin. Rep proteins are thought to be exclusively encoded by plasmids, but are common to secondary chromosomes (NCBI). This may suggest that most secondary chromosomes have evolved from large or megaplasmids (Heidelberg

et al., 2000). Conversely genes encoding Par proteins are common to both plasmids

and the major chromosome (Gerdes et al., 2000). A comparison of Par proteins encoded by various plasmids to those encoded by major chromosomes shows that their phylogeny is distinct. Par proteins encoded by secondary chromosomes tend to show a phylogeny similar to that observed for plasmids (Gerdes et al., 2000). Despite these apparent differences in replication machinery the replication of major and secondary chromosomes has been shown, at least in the case of Vibrio cholerae, to be co-ordinated (Egan et al., 2004). Further, Egan et al. (2004) also found the initiation of the main and secondary chromosomes replication occurs simultaneously. Conversely, megaplasmids, like regular plasmids, are thought to autonomously regulate their initiation of replication as their copy number decreases (del Solar et al., 1998) such as following cell division.

The best characterised Butyrivibrio oriRs are those of the small cryptic plasmids pRJf1, pRJf2 and pOM1. Each of these plasmids has two ORFs, both of which are required for their self replication and maintenance. These ORFs encode similar proteins in all three plasmids, potentially indicating a conserved requirement at the oriR. The first of these ORFs encodes an acidic plasmid recombinase (Pre) protein. The second encodes a Rep protein. This oriR composition is also seen in other rumen microbial plasmids such as pRRI2 of Prevotella ruminicola (Mercer et al., 2001,