Chieko Wada

Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli.

Deoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in 5′‐GATC‐3′ sequences and is important in many cellular processes in Escherichia coli. We performed a computational analysis of the entire E. coli genome and confirmed that GATC sequences are distributed unevenly in regulatory regions, which suggests that Dam might regulate gene transcription. To test this, a high‐density DNA microarray of 4097 E. coli genes was constructed and used to assess the gene expression profiles of the wild type and the dam‐16::kam mutant strain grown under four different conditions. We also used two‐dimensional electrophoretic analysis of the proteome to assess the protein profiles. The expression of a large number of genes was affected by the dam deficiency. Genes involved in aerobic respiration, stress and SOS responses, amino acid meta‐bolism and nucleotide metabolism were expressed at higher levels in the mutant cells, especially in aerobic conditions. In contrast, transcription of genes partici‐pating in anaerobic respiration, flagella biosynthesis, chemotaxis and motility was decreased in the dam mutant strain under both aerobic and low aerobic conditions. Thus, Dam‐controlled genes are involved in adjusting the metabolic and respiratory pathways and bacterial motility to suit particular environmental conditions. The promoters of most of these Dam‐controlled genes were also found to contain GATC sequences that overlap with recognition sites for two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator protein (CRP). We propose that Dam‐mediated methylation plays an important role in the global regulation of genes, particularly those with Fnr and CRP binding sites.

The GTP binding protein Obg homolog ObgE is involved in ribosome maturation

Obg proteins belong to a subfamily of GTP binding proteins, which are highly conserved from bacteria to human. Mutations of obgE genes cause pleiotropic defects in various species but the function remained unclear. Here we examine the function of ObgE, the Obg homolog in Escherichia coli. The growth rate correlates with the amount of ObgE in cells. Co‐fractionation experiments further suggest that ObgE binds to 30S and 50S ribosomal subunits, but not to 70S ribosome. Pull‐down assays suggest that ObgE associates with several specific ribosomal proteins of 30S and 50S subunits, as well as RNA helicase CsdA. Purified ObgE cosediments with 16S and 23S ribosomal RNAs in vitro in the presence of GTP. Finally, mutation of ObgE affects pre‐16Sr‐RNA processing, ribosomal protein levels, and ribosomal protein modification, thereby significantly reducing 70S ribosome levels. This evidence implicates that ObgE functions in ribosomal biogenesis, presumably through the binding to rRNAs and/or rRNA‐ribosomal protein complexes, perhaps as an rRNA/ribosomal protein folding chaperone or scaffold protein.

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL

In order to fold non‐native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP‐dependent reaction cycle. We constructed the real‐time three‐dimensional‐observation system at high resolution using a newly developed fast‐scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s−1). We also caught ATP/ADP‐induced open–closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP‐bound GroEL can change its conformation ‘from closed to open’ without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP (∼1.0 s) was apparently lower than those of ATP and ATP‐analogs (2–3 s), meaning that ADP‐bound open‐form is structurally less stable than ATP‐bound open‐form. These results indicate that GroEL has at least two distinct open‐conformations in the presence of nucleotide; ATP‐bound prehydrolysis open‐form and ADP‐bound open‐form, and the ATP hydrolysis in open‐form destabilizes its open‐conformation and induces the ‘from open to closed’ conformational change of GroEL.

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.

Deficiency of essential GTP‐binding protein ObgE in Escherichia coli inhibits chromosome partition

GTP‐binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc. and construct the GTPase superfamily, whose structures and sequence motifs (G‐1 to G‐5) are highly conserved from prokaryote to eukaryote. Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP. We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE. Double cross‐over experiments showed that the obgE gene is essential for growth in E. coli. From characterization of the obgE temperature‐sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated. Overproduction of ObgE also resulted in aberrant chromosome segregation. These data suggested that ObgE is involved directly or indirectly in E. coli chromosome partitioning. Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B. subtilis. We purified ObgE protein from a cell extract of E. coli, and the purified ObgE had GTPase activity and DNA‐binding ability.

Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli

During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named ‘ribosomal hibernation,’ RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild‐type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P‐ and A‐sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.

Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 Å resolution

The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N‐ and C‐terminal domains related to each other by internal pseudo 2‐fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C‐terminal domain plays the leading role in this binding, while the N‐terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.

Dynamic state of DNA topology is essential for genome condensation in bacteria

In bacteria, Dps is one of the critical proteins to build up a condensed nucleoid in response to the environmental stresses. In this study, we found that the expression of Dps and the nucleoid condensation was not simply correlated in Escherichia coli, and that Fis, which is an E. coli (gamma‐Proteobacteria)‐specific nucleoid protein, interfered with the Dps‐dependent nucleoid condensation. Atomic force microscopy and Northern blot analyses indicated that the inhibitory effect of Fis was due to the repression of the expression of Topoismerase I (Topo I) and DNA gyrase. In the Δfis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nulceoid condensation in the presence of Dps. DNA‐topology assays using the cell extract showed that the extracts from the Δfis and Topo I‐/DNA gyrase‐overexpressing strains, but not the wild‐type extract, shifted the population toward relaxed forms. These results indicate that the topology of DNA is dynamically transmutable and that the topology control is important for Dps‐induced nucleoid condensation.

Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

SummaryA subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.

Expression of ribosome modulation factor (RMF) in Escherichia coli requires ppGpp

Background During the transition from the logarithmic to the stationary phase, 70S ribosomes are dimerized into the 100S form, which has no translational activity. Ribosome Modulation Factor (RMF) is induced during the stationary phase and binds to the 50S ribosomal subunit, which directs the dimerization of 70S ribosomes. Unlike many other genes induced in the stationary phase, rmf transcription is independent of the sigma S. To identify the factors that regulate the growth phase‐dependent induction of rmf, mutant strains deficient in global regulators were examined for lacZ expression directed by the rmf promoter.