Biomedical subjects

Stuart Austin

Publications and source records attributed to Stuart Austin.

5 recordsLinked to original sources

PubMed2002

E.coli cell-cycle regulation by bacteriophage lambda.

We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the p(L) promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells. Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed. This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication. These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population. The cell division block is a result of the transient expression of the lambda kil gene. The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene. The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them. Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block. The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth.

Bacterial Proteins↗

PubMed2002

The P1 plasmid in action: time-lapse photomicroscopy reveals some unexpected aspects of plasmid partition.

The prophage of bacteriophage P1 is a low copy number plasmid in Escherichia coli and is segregated to daughter cells by an active partition system. The dynamics of the partition process have now been successfully followed by time-lapse photomicroscopy. The process appears to be fundamentally different from that previously inferred from statistical analysis of fixed cells. A focus containing several plasmid copies is captured at the cell center. Immediately before cell division, the copies eject bi-directionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter cell. These copies are free to move, associate, and disassociate. Later, they are captured to the new cell center to re-start the cycle. Studies with mutants suggest that the ability to segregate accurately at a very late stage in the cell cycle is dependent on a novel ability of the plasmid to control cell division. Should segregation be delayed, cell division is also delayed until segregation is successfully completed.

Bacteriophage P1↗

PubMed2002

The P1 plasmid is segregated to daughter cells by a 'capture and ejection' mechanism coordinated with Escherichia coli cell division.

The fate of the P1 plasmid of Escherichia coli was followed by time-lapse photomicroscopy. A GFP-ParB fusion marked the plasmid during partition (segregation) to daughter cells at slow growth rate. The process differs from that previously inferred from statistical analysis of fixed cells. A focus of plasmid copies is captured at the cell centre. Immediately before cell division, the copies eject bidirectionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter. They are not directed to a prescribed position but are free to move, associate and disassociate. Later, they are captured to the new cell centre to restart the cycle. A null P1 par mutant associates to form a focus, but it is neither captured nor ejected. A dominant negative ParB protein forms a plasmid focus that attaches to the cell centre but never ejects. It remains captive at the centre and blocks host cell division. The cells elongate. Eventually the intact focus is pushed to one side and the cells divide simultaneously in several places at the same time. This suggests that the wild-type plasmid imposes a regulatory node on the host cell cycle, preventing cell division until its own segregation is completed.

Bacterial Proteins↗

PubMed2002

The segregation of the Escherichia coli origin and terminus of replication.

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion-capture model, newly replicated DNA is fed bi-directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion-capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.

Base Sequence↗

PubMed2002

Transcriptional interference by a complex formed at the centromere-like partition site of plasmid P1.

The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for beta-galactosidase, lacZ. There was little interference with lacZ expression when ParA and ParB were supplied in trans. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down. ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription. Mutations in parA and parB that relieved the parAM314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (Par(PD)) similar to that of parAM314I. Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation. This "permanent" ParA-ParB-parS complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.

Bacterial Proteins↗