Hironori Niki

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.

Comparative Functional Genomics of the Fission Yeasts

A combined analysis of genome sequence, structure, and expression gives insights into fission yeast biology. The fission yeast clade—comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus—occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino‐terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha‐helical coiled‐coil domains and one globular domain, and a carboxyl‐terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild‐type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.

The trans‐envelope Tol–Pal complex is part of the cell division machinery and required for proper outer‐membrane invagination during cell constriction in E. coli

Fission of bacterial cells involves the co‐ordinated invagination of the envelope layers. Invagination of the cytoplasmic membrane (IM) and peptidoglycan (PG) layer is likely driven by the septal ring organelle. Invagination of the outer membrane (OM) in Gram‐negative species is thought to occur passively via its tethering to the underlying PG layer with generally distributed PG‐binding OM (lipo)proteins. The Tol–Pal system is energized by proton motive force and is well conserved in Gram‐negative bacteria. It consists of five proteins that can connect the OM to both the PG and IM layers via protein–PG and protein–protein interactions. Although the system is needed to maintain full OM integrity, and for class A colicins and filamentous phages to enter cells, its precise role has remained unclear. We show that all five components accumulate at constriction sites in Escherichia coli and that mutants lacking an intact system suffer delayed OM invagination and contain large OM blebs at constriction sites and cell poles. We propose that Tol–Pal constitutes a dynamic subcomplex of the division apparatus in Gram‐negative bacteria that consumes energy to establish transient trans‐envelope connections at/near the septal ring to draw the OM onto the invaginating PG and IM layers during constriction.

Cell Cycle–Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA–Protein Complexes in E. coli

Using immunofluorescence microscopy, we have found that SeqA protein, a regulator of replication initiation, is localized as discrete fluorescent foci in E. coli wild-type cells. Surprisingly, SeqA foci were observed also in an oriC deletion mutant. Statistical analysis revealed that a SeqA focus is localized at midcell in newborn cells. The SeqA focus is duplicated and tethered at midcell until an FtsZ ring is formed. Subsequently, these foci migrate in opposite directions toward cell quarter sites and remain tethered there until the cell divides. The cell cycle-dependent bidirectional migration of SeqA-DNA complexes is quite different from the migration pattern of oriC Dna copies. MukB protein is required for correct localization of SeqA complexes by an unknown mechanism.

Subcellular Distribution of Actively Partitioning F Plasmid during the Cell Division Cycle in E. coli

F plasmid is partitioned with fidelity to daughter cells during cell division cycle owing to two trans-acting genes, sopA and sopB, and a cis-acting site, sopC. We visualized the subcellular distribution of mini-F-plasmid molecules by fluorescence in situ hybridization. Mini-F-plasmid molecules having the sopABC segment were localized at midcell in newborn cells. Replicated plasmid molecules migrated to cell positions 1/4 and 3/4 without coupling with cell elongation and were tethered to these positions until completion of cell division. In contrast, molecules of a mini F plasmid lacking the sopABC segment were distributed randomly in spaces not occupied by nucleoids. The sopABC system caused replicated plasmid molecules to be positioned and tethered at the cell quarter sites.

E.coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA binding and ATP/GTP binding activities.

mukB mutants of Escherichia coli are defective in the correct partitioning of replicated chromosomes. This results in the appearance of normal‐sized anucleate (chromosome‐less) cells during cell proliferation. Based on the nucleotide sequence of the mukB gene, the MukB protein of 177 kDa was predicted to be a filamentous protein with globular domains at the ends, and also having DNA binding and nucleotide binding abilities. Here we present evidence that the purified MukB protein possesses these characteristics. MukB forms a homodimer with a rod‐and‐hinge structure having a pair of large, C‐terminal globular domains at one end and a pair of small, N‐terminal globular domains at the opposite end; it tends to bend at a middle hinge site of the rod section. Chromatography in a DNA‐cellulose column and the gel retardation assay revealed that MukB possesses DNA binding activity. Photoaffinity cross‐linking experiments showed that MukB binds to ATP and GTP in the presence of Zn2+. Throughout the purification steps, acyl carrier protein was co‐purified with MukB.

Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.

Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli

mukF, mukE and mukB genes are essential for the process of chromosome partitioning in Escherichia coli. We have studied protein–protein interactions among MukB, MukE and MukF proteins by co‐immunoprecipitation and sucrose gradient sedimentation experiments, using mukFEB null cells harboring plasmids carrying the wild‐type or mutant‐type mukFEB operon. MukB forms a complex with MukF and MukE. Analysis of mutant MukB proteins suggested that MukF and MukE bind the C‐terminal globular domain of MukB. MukF is indispensable for an interaction between MukB and MukE; however, MukF itself is able to associate with MukB even in the absence of MukE. We have also found that MukF has a Ca2+‐binding activity. Although purified MukF was able to make a complex either with MukE or MukB, a complex consisting of the three Muk proteins was barely detected in vitro. However, increasing the Ca2+ or Mg2+ concentration in the reaction partially restored complex formation. This suggests that Ca2+ or Mg2+ may be required for the formation of a complex consisting of the three Muk proteins, and thus may participate in a particular step during chromosome partitioning.

Bidirectional migration of SeqA‐bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli

We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions.