Sota Hiraga

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.

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.

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.

Cloning, sequencing, and characterization of multicopy suppressors of a mukB mutation in Escherichia coli

The mukB gene codes for a 177kDa protein, which might be a candidate for a force‐generating enzyme in chromosome positioning in Escherichia coli. The mukB106 mutant produces normal‐sized, anucleate cells and shows a temperature‐sensitive colony formation. To Identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature‐sensitive colony formation of the mukB106 mutation. The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene. The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature‐sensitive colony formation. However, none of them couid suppress both phenotypes in a mukB null mutation. DNA sequencing revealed that the msmB gene was identicai to the cspC gene and that the msmC gene had not been described before. A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold‐shock domain, such as CspA of E. coliand the Y‐box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA‐binding proteins that recognize the CCAAT sequence. Hence, the msmB and msmC genes were renamed cspC and cspE, respectively. Possible mechanisms for suppression of the mukB106 mutation are discussed.

Structure and function of the ftsH gene in Escherichia coli

The ftsH mutant Y16 shows thermosensitive filamentation with reduced amounts of penicillin-binding protein 3 (PBP3) (Ferreira et al., 1987). Genetic analysis, however, showed that the lethality of the ftsH mutation was not due to a lack of PBP3 activity alone. The ftsH gene was cloned and sequenced and the FtsH protein was deduced to be a membrane protein of 70.7 kDa which has an ATP-binding domain. Highly significant homology of amino acid sequence was observed between FtsH protein and two eukaryotic proteins, yeast Saccharomyces cerevisiae Sec 18p and its mammalian homologue NSF, which are involved in protein transport pathways. This suggests that FtsH protein may act for translocation of specific proteins including PBP3 and at least one other additional protein essential for cell growth. Suppressor mutants of Y16, which were able to grow at 42 degrees C, were isolated, and the suppressor mutations (sfh) were mapped to 16 min. A wild type chromosomal fragment able to complement the sfh mutations was cloned. We also identified another gene (ftsJ) affecting cell division in the region upstream of the ftsH gene.

Mutants defective in chromosome partitioning in E. coli

Recent experimental results suggest that replicated daughter chromosomes (nucleoids) in Escherichia coli move non-progressively and abruptly at an early stage of the D (division) period from midcell toward the cell quarter positions, which will become the centres of the daughter cells. The chromosome positioning at the quarter positions was found to be controlled by the muk gene products. In muk mutants, normal size anucleate cells are spontaneously produced during cell division. The mukA gene is identical to the tolC gene encoding an outer membrane protein. The mukB gene codes for a 177-kDa protein. The amino acid sequence of the MukB protein deduced for the nucleotide sequence suggests that the MukB protein has five characteristic secondary structure domains: an amino-terminal globular domain containing a consensus sequence binding with ATP or another nucleotide. The central region of the protein consists of two alpha-helical coiled-coil domains and one globular domain. A carboxyl-terminal globular domain is rich in cysteine and positively charged residues arginine and lysine. Two MukB protein molecules might form a homodimer in the coiled-coil regions. The predicted secondary structure of the MukB protein suggests that the protein provides the force required for the positioning of nucleoids from midcell toward the cell quarters. The mukC and mukD genes are located at 88 and 41 min of the chromosome map, respectively.

Linear multimer formation of plasmid DNA in Escherichia coli hopE (recD) mutants

SummaryThe hopE mutants of Escherichia coli, which cannot stably maintain a mini-F plasmid during cell division, have mutations in the recD gene coding for subunit D of the RecBCD enzyme (exonuclease V). A large amount of linear multimer DNA of mini-F and pBR322 plasmids accumulates in these hopE mutants. The linear multimers of plasmid DNA in the hopE (recD) mutants accumulate in sbc+ genetic backgrounds and this depends on the recA+ gene function. Linear plasmid multimers also accumulated in a recBC xthA triple mutant, but not an isogenic xthA mutant or an isogenic recBC mutant. The recBC xthA mutant is defective in the conjugative type of recombination. Linear plasmid multimers were not detected in the recBC strain. We propose models to account for linear multimer formation of plasmids in various mutants.

Characterization of high molecular weights of complexes and polymers of cytoplasmic proteins in Escherichia coli

To search for filamentous polymers of cytoplasmic proteins of Escherichia coli, high molecular weights (> 670 kDa) of protein complexes of cell extracts were fractionated by gel filtration and ion-exchange column chromatography. Proteins of 100, 77 and 52 kDa were co-purified. The 100- and 52-kDa proteins were identified to be pyruvate dehydrogenase and lipoamide dehydrogenase, respectively, by determining the N-terminal amino acid sequences. Experimental results indicate that the 77-kDa protein is identical to dihydrolipoamide acyltransferase. The 100-kDa protein was found to be identical to the 100-kDa protein described by Tomioka (1991), and was related to the formation of filaments and sheets in the presence of 100 mM KCl. However, neither long filaments nor sheets were observed in our sample containing these enzymes, which was not consistent with Tomiokas conclusion. Another 100-kDa protein which forms spirosome-like particles was purified and identified to be alcohol dehydrogenase based on the N-terminal sequence.