Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus

We have identified the parC and parE genes encoding DNA topoisomerase IV (Topo IV) in Caulobacter crescentus . We have also characterized the effect of conditional Topo IV mutations on cell division and morphology. Topo IV mutants of C. crescentus are unlike mutants of Escherichia coli and S. typhimurium , which form long filamentous cells that are defective in nucleoid segregation and divide frequently to produce anucleate cells. Topo IV mutants of C. crescentus are highly pinched at multiple sites (cell separation phenotype) and they do not divide to produce cells lacking DNA. These results suggest unique regulatory mechanisms coupling nucleoid partitioning and cell division in this aquatic bacterium. In addition, distinctive nucleoid-partitioning defects are not apparent in C. crescentus Topo IV mutants as they are in E. coli and S. typhimurium . However, abnormal nucleoid segregation in parE mutant cells could be demonstrated in a genetic background containing a conditional mutation in the C. crescentus ftsA gene, an early cell division gene that is epistatic to parE for cell division and growth. We discuss these results in connection with the possible roles of C. crescentus Topo IV in the regulation of cell division, chromosome partitioning, and late events in polar morphogenesis. Although the ParC and ParE subunits of Topo IV are very similar in sequence to the GyrA and GyrB subunits of DNA gyrase, we have used DNA sequence analysis to identify a highly conserved 'GyrA box' sequence that is unique to the GyrA proteins and may serve as a hallmark of the GyrA protein family.     

New topoisomerase essential for chromosome segregation in E. coli

The nucleotide sequence of the parC gene essential for chromosome partition in E. coli was determined. The deduced amino acid sequence was homologous to that of the A subunit of gyrase. We found another new gene coding for about 70 kd protein. The gene was sequenced, and the deduced amino acid sequence revealed that the gene product was homologous to the gyrase B subunit. Mutants of this gene were isolated and showed the typical Par phenotype at nonpermissive temperature; thus the gene was named parE. Enhanced relaxation activity of supercoiled plasmid molecules was detected in the combined crude cell lysates prepared from the ParC and ParE overproducers. A topA mutation defective in topoisomerase I could be compensated by increasing both the parC and the parE gene dosage. It is suggested that the parC and parE genes code for the subunits of a new topoisomerase, named topo IV.     

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.     

A ParE-ParC fusion protein is a functional topoisomerase.

Type II topoisomerases are responsible for DNA unlinking during DNA replication and chromosome segregation. Although eukaryotic enzymes are homodimers and prokaryotic enzymes are heterotetramers, both prokaryotic and eukaryotic type II topoisomerases belong to a single protein family. The amino- and carboxyl-terminal domains of eukaryotic enzymes are homologous to the ATP-binding and catalytic subunits of prokaryotic enzymes, respectively. Topoisomerase IV, a prokaryotic type II topoisomerase, consists of the ATP-binding subunit, ParE, and the catalytic subunit, ParC. We have joined the coding regions of parE and parC in frame and constructed a fusion protein of the two subunits of topoisomerase IV. This fusion protein, ParEC, can catalyze both decatenation and relaxation reactions. The ParEC protein is also capable of decatenating replicating daughter DNA molecules during oriC DNA replication in vitro. Furthermore, the fusion gene, parEC, complements the temperature-sensitive growth of both parC and parE strains, indicating that the ParEC protein can substitute for topoisomerase IV in vivo. These results demonstrate that a fusion protein of the two subunits of topoisomerase IV is a functional topoisomerase. Thus, a heterotetrameric type II topoisomerase can be converted into a homodimeric type II topoisomerase by gene fusion.