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M Yarmolinsky

Publications and source records attributed to M Yarmolinsky.

15 recordsLinked to original sources
PubMed2001

Pairing of P1 plasmid partition sites by ParB.

The mechanisms by which bacterial plasmids and chromosomes are partitioned are largely obscure, but it has long been assumed that the molecules to be separated are initially paired, as are sister chromatids in mitosis. We offer in vivo evidence that the partition protein ParB encoded by the bacterial plasmid P1 can pair cis-acting partition sites of P1 inserted in a small, multicopy plasmid. ParB was shown previously to be capable of extensive spreading along DNA flanking the partition sites. Experiments in which ParB spreading was constrained by physical roadblocks suggest that extensive spreading is not required for the pairing process.

Bacterial Proteins↗
PubMed2000

Transcriptional silencing in bacteria.

Transcriptional silencing and repression are modes of negative control of gene expression that differ in specificity. Repressors, when present at promoter-specific binding sites, interfere locally with RNA polymerase function. Silencing proteins act by covering a continuous region of DNA, compete with a broader spectrum of proteins and are non-specific with respect to the promoters affected. Studies of transcriptional silencing promise an entrée to relatively unexplored areas of prokaryotic biology.

Bacteria↗
PubMed2000

pTAR-encoded proteins in plasmid partitioning.

Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465-478, 1987). However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host Escherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

Adenosine Triphosphatases↗
PubMed1999

Silencing of genes flanking the P1 plasmid centromere.

Partition modules stabilize bacterial plasmids and chromosomes by actively promoting their segregation into daughter cells. The partition module of plasmid P1 is typical and consists of a centromere site, parS, and genes that encode proteins ParA and ParB. We show that ParB can silence genes flanking parS (to which ParB binds), apparently by polymerizing along the DNA from a nucleation site at parS. Wild-type ParB contacts an extensive region of P1 DNA; silencing-defective ParB proteins, which were found to be partition-defective, are less able to spread. Hence, the silenced structure appears to function in partitioning.

Bacterial Proteins↗
PubMed1996

P1 plasmid partition: a mutational analysis of ParB.

P1 plasmid partition to daughter cells requires plasmid proteins, ParA and ParB, and a centromere analog, parS, to which ParB binds. ParA and ParB decrease the loss frequencies of some low copy number plasmid vectors with parS inserts, as well as that of P1 itself, more than 100-fold. Unexpectedly, we find that Par proteins can actively destabilize other parS-plasmid constructs under similar conditions. Only ParB is required for this partition dysfunction. The destabilization can be dramatic; certain parS-plasmid constructs cannot even be maintained in the presence of ParB. We take advantage of this partition dysfunction to select parB mutants unable to destabilize a particularly ParB-sensitive parS-plasmid. The mutations obtained are widely distributed in the gene. Several of the mutations affected binding of ParB to parS; others did not. Amino acid substitutions that affected binding without influencing dimerization were mapped to two separate regions of ParB. C-Terminal amino acid substitutions in ParB blocked its dimerization and binding to parS, as did deletions of the entire C-terminal part of the protein. These results are consistent with ParB binding to parS as a dimer and suggest that the C-terminal part is a dimerization domain of the protein. Cell extracts of a frameshift mutant analyzed contained, in addition to the expected truncated ParB, a second species of ParB with the normal C-terminal part. Apparently, a site of ribosomal frameshifting is encoded within parB, at which some ribosomes change reading frame in the mutant. All obtained mutant ParB proteins were unable to complement the partition defect of a parB null mutant mini-P1. This suggests that the initial reactions between ParB and parS leading to partition dysfunction and to partition function are similar.

Amino Acid Sequence↗
PubMed1994

Partition of nonreplicating DNA by the par system of bacteriophage P1.

P1 plasmid encodes a cis-acting centromere analog, parS, and two Par proteins that together stabilize plasmids by partitioning them to daughter bacteria. We infected immune bacteria with bacteriophage lambda into which parS had been inserted. The presence of P1 Par proteins in the infected cells was found to delay the appearance of cells cured of the nonreplicating, extrachromosomal lambda-parS DNA. This stabilization of lambda-parS, approximated in a computer simulation, demonstrates that active partition by the P1 par system does not require the act of plasmid replication and can be studied in its absence.

Bacterial Proteins↗
PubMed1990

Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter.

The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication. The only P1-encoded protein required for plasmid replication is the initiator, RepA. Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin. We found that repression is incomplete in E. coli strains with mutations in the dnaJ, dnaK, or grpE genes. Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter. We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains. Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.

Bacterial Proteins↗
PubMed1989

Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication.

Low-copy-number plasmids, such as P1 prophage and the fertility factor F, require a plasmid-encoded replication protein and several host products for replication. Stable maintenance also depends on active partitioning of plasmids into daughter cells. Mini-P1 par+ and par plasmids were found to be destabilized by mutations in the dnaJ, dnaK, and grpE genes of Escherichia coli. The transformation efficiency and stability of mini-F plasmids were also reduced in the mutant strains. These results indicate that heat shock proteins DnaJ, DnaK, and GrpE play roles in the replication of plasmid P1 and probably also in of F.

DNA Replication↗
PubMed1978

Analysis of bacteriophage P1 immunity by using lambda-P1 recombinants constructed in vitro.

We describe the dissection and reconstruction of a complex control circuit, the P1 immunity system, by a method that involves inserting EcoRI-generated fragments of P1 DNA into lambda vectors that can then be sequentially inserted into a bacterial cell. Using these techniques we have isolated lambda-P1 hybrid phages that express the products of P1 genes c1, c4, ant, and ban and, in appropriately constructed lysogens, confirmed the roles played by the first three of these products in phage immunity. In addition we have localized to particular P1 fragments the sites requisite for expression and repression of these gene products. The analysis leads to the conclusion that gpant acts in trans to antagonize repression mediated by gpc1, in support of one of two proposed models for gpant action. Moreover, two features of the immunity system are revealed: (i) a hitherto unknown component that effects gpc1 repression; and (ii) an unexpected ability of gpc4 to channel a superinfecting c1+ phage into the lysogenic state, which suggests that gpc4 activity regulates the establishment phase of lysogeny.

Alleles↗