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.     

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 Ca(2+)-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 Ca(2+) or Mg(2+) concentration in the reaction partially restored complex formation. This suggests that Ca(2+) or Mg(2+) 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.     

Identification of two new genes,mukE andmukF,involved in chromosome partitioning inEscherichia 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.     

Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli.

The mukB gene encodes a protein involved in chromosome partitioning in Escherichia coli. To study the function of this protein, we isolated from the temperature-sensitive mukB null mutant and characterized 56 suppressor mutants which could grow at 42 degrees C. Ten of the mutants also showed cold-sensitive growth at 22 degrees C. Using one of the cold-sensitive mutants as host, the wild type of the suppressor gene was cloned. The cloned suppressor gene complemented all of the 56 suppressor mutations. DNA sequencing revealed the presence of an open reading frame of 723 bp which could encode a protein of 25,953 Da. The gene product was indeed detected. The previously undiscovered gene, named smbA (suppressor of mukB), is located at 4 min on the E. coli chromosome, between the tsf and frr genes. The smbA gene is essential for cell proliferation in the range from 22 to 42 degrees C. Cells which lacked the SmbA protein ceased macromolecular synthesis. The smbA mutants are sensitive to a detergent, sodium dodecyl sulfate, and they show a novel morphological phenotype under nonpermissive conditions, suggesting a defect in specific membrane sites.     

Two mutant alleles of mukB, a gene essential for chromosome partition in Escherichia coli

Abstract The MukB protein is essential for chromosome partitioning in Escherichia coli and consists of 1484 amino acid residues (170 kDa). We have determined the base changes at the mutated sites of the mukB106 mutant and a newly isolated mutant, mukB33 . These mutant mukB genes were each found to carry a single base-pair transition which leads to an amino acid substitution; a serine residue at position 33 was changed to phenylalanine in the case of mukB106 , and an aspartic acid residue at position 1201 was changed to asparagine in the case of mukB33 .     

Identification of two new genes,mukE andmukF, involved in chromosome partitioning inEscherichia 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.     

Mutants suppressing novobiocin hypersensitivity of a mukB null mutation

The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.     

Carboxyl terminal region of the MukB protein in Escherichia coli is essential for DNA binding activity

Abstract The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity. We have isolated a mutation, mukB1013 , causing a substitution of valine at position 1379 to leucine. This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected. A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.     

Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure

A strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas filamentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing influences on the organization of the bacterial nucleoid.     

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.     

Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.     

Crystal structure of the N-terminal domain of MukB: a protein involved in chromosome partitioning.

BACKGROUND: The 170 kDa protein MukB has been implicated in ATP-dependent chromosome partitioning during cell division in Escherichia coli. MukB shares its dimeric structure and domain architecture with the ubiquitous family of SMC (structural maintenance of chromosomes) proteins that facilitate similar functions. The N-terminal domain of MukB carries a putative Walker A nucleotide-binding region and the C-terminal domain has been shown to bind to DNA. Mutant phenotypes and a domain arrangement similar to motor proteins that move on microtubules led to the suggestion that MukB might be a motor protein acting on DNA. RESULTS: We have cloned, overexpressed and crystallized a 26 kDa protein consisting of 227 N-terminal residues of MukB from E. coli. The structure has been solved using multiple anomalous dispersion and has been refined to 2.2 A resolution. The N-terminal domain of MukB has a mixed alpha/beta fold with a central six-stranded antiparallel beta sheet. The putative nucleotide-binding loop, which is part of an unexpected helix-loop-helix motif, is exposed on the surface and no nucleotide-binding pocket could be detected. CONCLUSIONS: The N-terminal domain of MukB has no similarity to the kinesin family of motor proteins or to any other nucleotide-binding protein. Together with the finding of the exposed Walker A motif this observation supports a model in which the N- and C-terminal domains come together in the dimer of MukB to form the active site. Conserved residues on one side of the molecule delineate a region of the N-terminal domain that is likely to interact with the C-terminal domain.     

Nucleotide-dependent interaction of the N-terminal domain of MukB with microtubules

The MukB protein from Escherichia coli has a domain structure that is reminiscent of the eukaryotic motor proteins kinesin and myosin: N-terminal globular domains, a region of coiled-coil, and a specialised C-terminal domain. Sequence alignment of the N-terminal domain of MukB with the kinesin motor domain indicated an approximately 22% sequence identity. These observations raised the possibility that MukB might be a prokaryotic motor protein and, due to the sequence homology shared with kinesin, might bind to microtubules (Mts). We found that a construct encoding the first 342 residues of MukB (Muk342) binds specifically to Mts and shares a number of properties with the motor domain of kinesin. Visualisation of the Muk342 decorated Mt complexes using negative stain electron microscopy indicated that the Muk342 smoothly decorates the outside of Mts. Biochemical data demonstrate that Muk342 decorates Mts with a binding stoichiometry of one Muk342 monomer per tubulin monomer. These findings strongly suggest that MukB has a role in force generation and that it is a prokaryotic homologue of kinesin and myosin.     

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.     

The Escherichia coli histone-like protein HU affects DNA initiation, chromosome partitioning via MukB, and cell division via MinCDE.

Escherichia coli hupA hupB double mutants, lacking both subunits (HU1 and HU2) of the histone-like protein HU, accumulate secondary mutations. In some genetic backgrounds, these include mutations in the minCDE operon, inactivating this system of septation control and resulting in the formation of minicells. In the course of the characterization of hupA hupB mutants, we observed that the simultaneous absence of the HU2 subunit and the MukB protein, implicated in chromosome partitioning, is lethal for the bacteria; the integrity of either HU or MukB thus seems to be essential for bacterial growth. The HU protein has been shown to be involved in DNA replication in vitro; we show here that its inactivation in the hupA hupB double mutant disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. Our results suggest that global nucleoid structure, determined in part by the histone-like protein HU, plays a role in DNA replication initiation, in proper chromosome partitioning directed by the MukFEB proteins, and in correct septum placement directed by the MinCDE proteins.     

Partitioning of a mini-F plasmid into anucleate cells of the mukB null mutant.

The partition-proficient mini-F plasmid pXX325 was stably maintained in the mukB null mutant, which is defective in chromosome partitioning into the two daughter cells. In the null mutant, the plasmid was partitioned into both nucleate and anucleate daughter cells, independently of host chromosomes.     

Comparison of MukB homodimer versus MukBEF complex molecular architectures by electron microscopy reveals a higher-order multimerization

The complex of MukF, MukE, and MukB proteins participates in organization of sister chromosomes and partitioning into both daughter cells in Escherichia coli. We purified the MukB homodimer and the MukBEF complex and analyzed them by electron microscopy to compare both structures. A MukB homodimer shows a long rod-hinge-rod v-shape with small globular domains at both ends. The MukBEF complex shows a similar structure having larger globular domains than those of the MukB homodimer. These results suggest that MukF and MukE bind to the globular domains of a MukB homodimer. The globular domains of the MukBEF complex frequently associate with each other in an intramolecular fashion, forming a ring. In addition, MukBEF complex molecules tend to form multimers by the end-to-end joining with other MukBEF molecules in an intermolecular fashion, resulting in fibers and rosette-form structures in the absence of ATP and DNA in vitro.     

The nucleotide sequence of the uvrD gene of E. coli.

The nucleotide sequence of a cloned section of the E. coli chromosome containing the uvrD gene has been determined. The coding region for the UvrD protein consists of 2,160 nucleotides which would direct the synthesis of a polypeptide 720 amino acids long with a calculated molecular weight of 82 kd. The predicted amino acid sequence of the UvrD protein has been compared with the amino acid sequences of other known adenine nucleotide binding proteins and a common sequence has been identified, thought to contribute towards adenine nucleotide binding.     

The Symmetrical Structure of Structural Maintenance of Chromosomes (SMC) and MukB Proteins: Long,Antiparallel Coiled Coils,Folded at a Flexible Hinge

Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH₂-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180°, separating the terminal domains by 100 nm, or close to near 0°, bringing the terminal globular domains together.A surprising observation is that the ∼300–amino acid–long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35–45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH₂- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.     

The CHL 1 (CTF 1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M.

We have analyzed the CTF1 gene, identified in a screen for mutants with decreased chromosome transmission fidelity and shown to correspond to the previously identified chl1 mutation. Chl1 null mutants exhibited a 200-fold increase in the rate of chromosome III missegregation per cell division, and near wild-type rates of marker homozygosis on this chromosome by mitotic recombination. Analysis of the segregation of a marker chromosome indicated that sister chromatid loss (1:0 segregation) and sister chromatid non-disjunction (2:0 segregation) contributed equally to chromosome missegregation. A genomic clone of CHL1 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL1 revealed a 2.6 kb open reading frame with a 99 kd predicted protein sequence that contained two PEST sequences and was 23% identical to the coding region of a nucleotide excision repair gene, RAD3. Domains of homology between these two predicted protein sequences included a helix-turn-helix motif and an ATP binding site containing a helicase consensus. Mutants lacking the CHL1 gene product are viable and display two striking, and perhaps interrelated, phenotypes: extreme chromosome instability and a delay in cell cycle progression in G2/M. This delay is independent of the cell cycle checkpoint that requires the function of the RAD9 gene.