Surfing on Protein Waves: Proteophoresis as a Mechanism for Bacterial Genome Partitioning.

Efficient bacterial chromosome segregation typically requires the coordinated action of a three-component machinery, fueled by adenosine triphosphate, called the partition complex. We present a phenomenological model accounting for the dynamic activity of this system that is also relevant for the physics of catalytic particles in active environments. The model is obtained by coupling simple linear reaction-diffusion equations with a proteophoresis, or "volumetric" chemophoresis, force field that arises from protein-protein interactions and provides a physically viable mechanism for complex translocation. This minimal description captures most known experimental observations: dynamic oscillations of complex components, complex separation, and subsequent symmetrical positioning. The predictions of our model are in phenomenological agreement with and provide substantial insight into recent experiments. From a nonlinear physics view point, this system explores the active separation of matter at micrometric scales with a dynamical instability between static positioning and traveling wave regimes triggered by the dynamical spontaneous breaking of rotational symmetry.

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis.

The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created 'super-repressors', in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA's repressor activity and suggest that ATP hydrolysis is essential for plasmid localization.

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.

Stoichiometry of P1 plasmid partition complexes.

The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities.

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.

Ndd, the bacteriophage T4 protein that disrupts the Escherichia coli nucleoid, has a DNA binding activity.

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.

The effects on Escherichia coli of expression of the cloned bacteriophage T4 nucleoid disruption (ndd) gene.

Immediately after T4 bacteriophage infection, the Escherichia coli nucleoid undergoes rapid delocalization. The ndd gene of T4 is responsible for this nuclear disruption phenomenon. We have cloned two alleles of this gene and studied the effects of their expression on E. coli cells. We have shown that the Ndd protein (i) is able to reproduce the disruption of the nucleoid characteristic of T4 infection, (ii) is highly toxic and results in a logarithmic decrease in cell viability, and (iii) inhibits genomic DNA replication by blocking progression of replication forks. Induction of Ndd does not result in degradation of genomic DNA and does not significantly alter the general processes of transcription and translation during the entire period of exponential cell death. These results support the notion that the target of Ndd is some aspect of the nucleoid architecture.

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.

Direct PCR sequencing of the ndd gene of bacteriophage T4: identification of a product involved in bacterial nucleoid disruption.

The rapid disruption of the Escherichia coli nucleoid after T4 infection requires the activity of the phage-encoded ndd gene. We have genetically identified the sequence encoding ndd. Determination of the sequence of a 2.5-kb segment including ndd closed the last significant gap in the sequence of the T4 genome. This analysis was performed on PCR-amplified fragments that were purified by gel-exclusion chromatography and then submitted to linear amplification cycle sequencing. This technology permitted sequence comparison of two ndd mutants (ndd44 and ndd98) with the wild-type gene. The analysis of ndd from six bacteriophages of the T-even family indicated that the protein encoded by this nonessential gene is surprisingly conserved.