David Lane

The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli.

The bacteria for which there is evidence that proteins of the ParAB family act in chromosome segregation also undergo developmental transitions that involve the ParAB homologues, raising the question of whether the partition activity is equivalent to that of plasmid partition systems. We have investigated the role in partition of the parAB locus of a free‐living bacterium, Pseudomonas putida, not known to pass through developmental phases. A parAB deletion mutant, compared with wild type, showed slightly higher frequencies of anucleate cells in exponen‐tially growing cultures but much higher frequencies in deceleration phase. This increase was growth medium dependent. Oversupply of ParA and ParB proteins also raised anucleate cell levels, specifically in the deceleration phase, in wild‐type and mutant strains and regardless of medium, as well as generating abnormal cell morphologies. Absence or oversupply of ParAB function had either slight or considerable effects on growth rate, depending on temperature and medium. The need for the Par proteins in chromosome partition thus appears to be subject to the cell’s physiological state. Three sequences similar to cis‐acting stabilization sites of Bacillus subtilis are present in the P. putida oriC–parAB region. One was inserted into an unstable mini‐F and shown to stabilize it in E. coli in a ParAB‐dependent manner.

Polymerization of SopA partition ATPase: regulation by DNA binding and SopB

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon‐specific systems which comprise a centromere, a centromere‐binding protein and an ATPase. Dynamic self‐assembly of the ATPase appears to enable active partition of replicon copies into cell‐halves, but for most ATPases (the Walker‐box type) the mechanism is unknown. Also unknown is how the host cell contributes to partition. We have examined the effects of non‐sequence‐specific DNA on in vitro self‐assembly of the SopA partition ATPase of plasmid F. SopA underwent polymerization provided ATP was present. DNA inhibited this polymerization and caused breakdown of pre‐formed polymers. Centromere‐binding protein SopB counteracted DNA‐mediated inhibition by itself binding to and masking the DNA, as well as by stimulating polymerization directly. The results suggest that in vivo, SopB smothers DNA by spreading from sopC, allowing SopA‐ATP polymerization which initiates plasmid displacement. We propose that SopB and nucleoid DNA regulate SopA polymerization and hence partition.

Kinetics of plasmid segregation in Escherichia coli.

Low copy‐number bacterial replicons occupy specific locations in their host cells. Production of a GFP‐Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid‐cell, whereas larger cells tend to have two foci, one at each quarter‐cell position. Duplication of the central focus is presumed to represent active partition of plasmid copies. We report here our investigation by time‐lapse microscopy of the subsequent movement of these copies to the quarter positions. Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter‐cell destinations, where they remained until the next cell cycle. The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation. Aberrant positioning of mini‐F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process. From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules. The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed.

F plasmid partition depends on interaction of SopA with non‐specific DNA

Bacterial ATPases belonging to the ParA family assure partition of their replicons by forming dynamic assemblies which move replicon copies into the new cell‐halves. The mechanism underlying partition is not understood for the Walker‐box ATPase class, which includes most plasmid and all chromosomal ParAs. The ATPases studied both polymerize and interact with non‐specific DNA in an ATP‐dependent manner. Previous work showed that in vitro, polymerization of one such ATPase, SopA of plasmid F, is inhibited by DNA, suggesting that interaction of SopA with the host nucleoid could regulate partition. In an attempt to identify amino acids in SopA that are needed for interaction with non‐specific DNA, we have found that mutation of codon 340 (lysine to alanine) reduces ATP‐dependent DNA binding > 100‐fold and correspondingly diminishes SopA activities that depend on it: inhibition of polymer formation and persistence, stimulation of basal‐level ATP hydrolysis and localization over the nucleoid. The K340A mutant retained all other SopA properties tested except plasmid stabilization; substitution of the mutant SopA for wild‐type nearly abolished mini‐F partition. The behaviour of this mutant indicates a causal link between interaction with the cells non‐specific DNA and promotion of the dynamic behaviour that ensures F plasmid partition.

Disruption of the F plasmid partition complex in vivo by partition protein SopA.

The SopA protein plays an essential, though so far undefined, role in partition of the mini‐F plasmid but, when overproduced, it causes loss of mini‐F from growing cells. Our investigation of this phenomenon has revealed that excess SopA protein reduces the linking number of mini‐F. It appears to do so by disturbing the partition complex, in which SopB normally introduces local positive supercoiling upon binding to the sopC centromere, as it occurs only in plasmids carrying sopC and in the presence of SopB protein. SopA‐induced reduction in linking number is not associated with altered sop promoter activity or levels of SopB protein and occurs in the absence of changes in overall supercoil density. SopA protein mutated in the ATPase nucleotide‐binding site (K120Q) or lacking the presumed SopB interaction domain does not induce the reduction in linking number, suggesting that excess SopA disrupts the partition complex by interacting with SopB to remove positive supercoils in an ATP‐dependent manner. Destabilization of mini‐F also depends on sopC and SopB, but the K120Q mutant retains some capacity for destabilizing mini‐F. SopA‐induced destabilization thus appears to be complex and may involve more than one SopA activity. The results are interpreted in terms of a regulatory role for SopA in the oligomerization of SopB dimers bound to the centromere.

Autoregulation of the ccd operon in the F plasmid

SummaryMini-F sequences, including the promoter and portions of the ccd region, were inserted upstream of lacZ in promoterless lacZ vectors, and β-galactosidase specific activities were measured. The results showed that the H (ccdA), G (ccdB) and D genes, together with a promoter, comprise an operon. Ccd operon expression was shown to be regulated at the level of transcription by the G gene product, probably in concert with the H gene product. Thus expression is autoregulated. Expression of the D gene was largely dependent on the ccd promoter, although low levels of transcription from another promoter within the ccd coding region were detected.

Mapping of Functional Domains in F Plasmid Partition Proteins Reveals a Bipartite SopB-recognition Domain in SopA

Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.

Probing plasmid partition with centromere-based incompatibility

Low‐copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini‐F, on repression of the sopAB operon and on occupancy of mini‐F DNA by the centromere‐binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini‐F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini‐P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini‐F handcuffing promotes pairing of SopB–sopC complexes that can subsequently segregate as intact aggregates.

Mapping of the drug resistance genes carried by the r-determinant of the R100.1 plasmid

SummaryWe have cloned the EcoRI fragments of pLC1, a circular DNA element found in an Escherichia coli dnaAts strain integratively suppressed by R100.1 (Chandler et al., 1977a), using the plasmid vector pCR1. All the resistance genes known to be present on the r-determinant of R100.1 were found to be present on pLC1. The isolation of pCR1 derivatives carrying various EcoRI fragments of either pLC1 or R100.1 has allowed a more precise mapping of the position of the resistance genes on the R100.1 molecule.

Analysis of the F plasmid centromere

SummaryThe nucleotide sequence of the cis-acting partition site (centromere) of the miniF plasmid has been determined. Its most notable feature is a reiterated 43 base pair unit. A series of plasmids deleted for portions of the repeat region was constructed and tested for incompatibility with R386 and for stability of inheritance. The extent of incompatibility with R386 was correlated with the number of repeat units. In contrast, the great majority of the repeats were not needed for miniF stability. An adjacent region of unique sequence was also found to be involved in centromere function.