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Bacterial conjugation disseminates genes among bacteria via a process requiring direct cell contact. The cell envelope spanning secretion apparatus involved belongs to the type IV family of bacterial secretion systems, which transport protein as well as nucleoprotein substrates. This study aims to understand mechanisms leading to the initiation of type IV secretion using conjugative plasmid paradigm...
A cytometric method for the estimation of end-point conjugation rates is developed and adapted to surface conjugation. This method improves the through-put of conjugation assays based on replica-plating and results in less noisy experimental data. Although conjugation on solid surfaces deviates from ideal conditions in which cells are continuously mixed, results show that, within the limits of high...
Promiscuous, rolling-circle replication plasmid pMV158 determines tetracycline resistance to its host and can be mobilized by conjugation. Plasmid pLS1 is a deletion derivative of pMV158 that has lost its conjugative mobilization ability. Both plasmids replicate efficiently and are stably inherited in Streptococcus pneumoniae. We have analyzed the effect of pMV158 and pLS1 carriage on the bacterial...
In the absence of active partitioning, strict control of plasmid copy number is required to minimise the possibility of plasmid loss at bacterial cell division. An important cause of multicopy plasmid instability is the formation of plasmid dimers by recombination and their subsequent proliferation by over-replication in a process known as the dimer catastrophe. This leads to the formation of dimer-only...
Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region...
The region responsible for replication of Vibrio cholerae chromosome II (chrII) resembles those of plasmids that have repeated initiator binding sites (iterons) and an autorepressed initiator gene. ChrII has additional features: Its iterons require full methylation for initiator (RctB) binding, which makes them inactive for a part of the cell cycle when they are hemi-methylated. RctB also binds to...
The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have...
The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication...
The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory...
Circularized regions of the chromosome containing the origin of replication, oriC, can be maintained as autonomous minichromosomes, oriC plasmids. We show that oriC plasmids containing precise, pre-determined segments of the chromosome can be generated by a simple in vivo recombineering technique. We generated two such plasmids carrying fluorescent markers. These were transferred to a recipient strain...
Most natural conjugative IncF plasmids encode a fertility inhibition system that represses transfer gene expression in the majority of plasmid-carrying cells. The successful spread of these plasmids in clinically relevant bacteria has been suggested to be supported by a transitory derepression of transfer gene expression in newly formed transconjugants. In this study, we aimed to monitor the extent...
Peptidoglycan hydrolases that are specifically associated with bacterial conjugation systems are postulated to facilitate the assembly of the transfer apparatus by creating a temporally and spatially controlled local opening in the peptidoglycan layer. To date little is known about the role of such enzymes in conjugation systems from Gram-positive bacteria. Conjugative plasmids from the Gram-positive...
The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control...
The alpha-proteobacterium Caulobacter crescentus is characterized by its asymmetric cell division, which gives rise to a replicating stalked cell and a non-replicating swarmer cell. Thus, the initiation of chromosomal replication is tightly regulated, temporally and spatially, to ensure that it is coordinated with cell differentiation and cell cycle progression. Waves of DnaA and CtrA activities control...
The SaPIs and their relatives are phage satellites and are unique among the known bacterial pathogenicity islands in their ability to replicate autonomously. They possess a phage-like replicon, which is organized as two sets of iterons arrayed symmetrically to flank an AT-rich region that is driven to melt by the binding of a SaPI-specific initiator (Rep) to the flanking iterons. Extensive deletion...
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