The hibernation promoting factor of Betaproteobacteria Comamonas testosteroni cannot induce 100S ribosome formation but stabilizes 70S ribosomal particles.

Bacteria use several means to survive under stress conditions such as nutrient depletion. One such response is the formation of hibernating 100S ribosomes, which are translationally inactive 70S dimers. In Gammaproteobacteria (Enterobacterales), 100S ribosome formation requires ribosome modulation factor (RMF) and short hibernation promoting factor (HPF), whereas it is mediated by only long HPF in the majority of bacteria. Here, we investigated the role of HPFs of Comamonas testosteroni, which belongs to the Betaproteobacteria with common ancestor to the Gammaproteobacteria. C. testosteroni has two genes of HPF homologs of differing length (CtHPF-125 and CtHPF-119). CtHPF-125 was induced in the stationary phase, whereas CtHPF-119 conserved in many other Betaproteobacteria was not expressed in the culture conditions used here. Unlike short HPF and RMF, and long HPF, CtHPF-125 could not form 100S ribosome. We first constructed the deletion mutant of Cthpf-125 gene. When the deletion mutant grows in the stationary phase, 70S particles were degraded faster than in the wild strain. CtHPF-125 contributes to stabilizing the 70S ribosome. CtHPF-125 and CtHPF-119 both inhibited protein synthesis by transcription-translation in vitro. Our findings suggest that CtHPF-125 binds to ribosome, and stabilizes 70S ribosomes, inhibits translation without forming 100S ribosomes and supports prolonging life.

The Discovery of Ribosomal Protein bL31 from Escherichia coli: A Long Story Revisited.

Ribosomal protein bL31 in Escherichia coli was initially detected as a short form (62 amino acids) using Kaltschmidt and Wittmann's two-dimensional polyacrylamide gel electrophoresis (2D PAGE), but the intact form (70 amino acids) was subsequently identified by means of Wada's improved radical-free and highly reducing (RFHR) 2D PAGE, which was consistent with the analysis of its encoding gene rpmE. Ribosomes routinely prepared from the K12 wild-type strain contained both forms of bL31. ΔompT cells, which lack protease 7, only contained intact bL31, suggesting that protease 7 cleaves intact bL31 and generates short bL31 during ribosome preparation from wild-type cells. Intact bL31 was required for subunit association, and its eight cleaved C-terminal amino acids contributed to this function. 70S ribosomes protected bL31 from cleavage by protease 7, but free 50S did not. In vitro translation was assayed using three systems. The translational activities of wild-type and ΔrpmE ribosomes were 20% and 40% lower than those of ΔompT ribosomes, which contained one copy of intact bL31. The deletion of bL31 reduces cell growth. A structural analysis predicted that bL31 spans the 30S and 50S subunits, consistent with its functions in 70S association and translation. It is important to re-analyze in vitro translation with ribosomes containing only intact bL31.

YkgM and YkgO maintain translation by replacing their paralogs, zinc-binding ribosomal proteins L31 and L36, with identical activities.

When a cell is zinc-deficient, ykgM and ykgO, which encode paralogs of the zinc-binding ribosomal proteins L31 and L36, are expressed from the ykgM operon, which is ordinarily held inactive by the Zur repressor. In ribosomes lacking L31, ribosomal subunit association is weakened, resulting in reduced in vitro translation and the deletion mutants of rpmE, the gene encoding L31, forming small colonies. We isolated four suppressor mutants of ∆rpmE that formed normal colonies. All four mutation sites were located in zur, and ribosomes of zur mutant cells contained one copy of YkgM and had translational activities equivalent to those of ribosomes containing L31. L36 is highly conserved among bacteria, chloroplast and mitochondria. Analysis of a deletion mutant of rpmJ, which encodes L36, suggested that L36 is involved in late assembly of the 50S particle, in vitro translation and cell growth. In zur mutant cells lacking rpmJ, the paralog YkgO was expressed and took over the functions of L36. zur mutant cells contained four types of ribosomes containing combinations of L31 or YkgM, and L36 or YkgO. Copy numbers of L31 and YkgM, and L36 and YkgO, summed to 1, indicating that each paralog pair shares a binding site.

Ribosomal protein L31 in Escherichia coli contributes to ribosome subunit association and translation, whereas short L31 cleaved by protease 7 reduces both activities.

Ribosomes routinely prepared from Escherichia coli strain K12 contain intact (70 amino acids) and short (62 amino acids) forms of ribosomal protein L31. By contrast, ribosomes prepared from ompT mutant cells, which lack protease 7, contain only intact L31, suggesting that L31 is cleaved by protease 7 during ribosome preparation. We compared ribosomal subunit association in wild-type and ompT - strains. In sucrose density gradient centrifugation under low Mg2+ , 70S content was very high in ompT - ribosomes, but decreased in the wild-type ribosomes containing short L31. In addition, ribosomes lacking L31 failed to associate ribosomal subunits in low Mg2+ . Therefore, intact L31 is required for subunit association, and the eight C-terminal amino acids contribute to the association function. In vitro translation was assayed using three different systems. Translational activities of ribosomes lacking L31 were 40% lower than those of ompT - ribosomes with one copy of intact L31, indicating that L31 is involved in translation. Moreover, in the stationary phase, L31 was necessary for 100S formation. The strain lacking L31 grew very slowly. A structural analysis predicted that the L31 protein spans the 30S and 50S subunits, consistent with the functions of L31 in 70S association, 100S formation, and translation.

Conservation of two distinct types of 100S ribosome in bacteria.

In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the 'hibernating ribosome'. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF.

Formation of 100S ribosomes in Staphylococcus aureus by the hibernation promoting factor homolog SaHPF.

In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.

Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome.

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation. The 100S ribosome is a dimer of two 70S ribosomes bridged through the 30S subunits. In some pathogens of gammaproteobacteria, such as Escherichia coli, Yersinia pestis, and Vibrio cholerae, the key factor for ribosomal dimerization is the small protein, ribosome modulation factor (RMF). When ribosomal dimerization by RMF is impaired, long-term bacterial survival is abolished. This shows that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. According to the results of several structural analyses, RMF does not directly connect two ribosomes, but binds to them and changes the conformation of their 30S subunits, thus promoting ribosomal dimerization. In this study, conserved RMF amino acids among 50 bacteria were selectively altered by mutagenesis to identify the residues involved in ribosome binding and dimerization. The activities of mutant RMF for ribosome binding and ribosome dimerization were measured using the sucrose density gradient centrifugation (SDGC) and western blotting methods. As a result, some essential amino acids of RMF for the ribosomal binding and dimerization were elucidated. Since the induction of RMF expression inhibits bacterial growth, the data on this protein could serve as information for the development of antibiotic or bacteriostatic agents.

Structural basis for regulation of bifunctional roles in replication initiator protein.

DNA replication initiator protein RepE stringently regulates F plasmid replication by its two distinct molecular association states. A predominant dimer functions as an autogenous repressor, whereas monomers act as replication initiators, and the dimer requires actions of the DnaK molecular chaperone system for monomerization. The structure of the monomeric form is known, whereas the dimeric structure and structural details of the dimer-to-monomer conversion have been unclear. Here we present the crystal structure of the RepE dimer in complex with the repE operator DNA. The dimerization interface is mainly formed by intermolecular beta-sheets with several key interactions of charged residues. The conformations of the internal N- and C-terminal domains are conserved between the dimer and monomer, whereas the relative domain orientations are strikingly different, allowing for an efficient oligomeric transition of dual-functional RepE. This domain relocation accompanies secondary structural changes in the linker connecting the two domains, and the linker is included in plausible DnaK/DnaJ-binding regions. These findings suggest an activation mechanism for F plasmid replication by RepE monomerization, which is induced and mediated by the DnaK system.

Role of HPF (hibernation promoting factor) in translational activity in Escherichia coli.

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed 'ribosomal hibernation'. Here, the contributions of HPF to 100S formation and translation were analysed in vitro. HPF bound to, but did not dimerize the 70S ribosome. RMF dimerized and formed immature 90S ribosomes. Binding of both HPF and RMF converted 90S ribosomes to mature 100S ribosomes, which is consistent with the in vivo data. The role of HPF in in vitro translation also was investigated. In an artificial mRNA poly (U)-dependent phenylalanine incorporation assay, HPF bound to ribosomal particles and inhibited translation. In contrast, in a natural MS2 mRNA-dependent leucine incorporation assay, bound HPF was removed and hardly inhibited normal translation. Multiple alignment and phylogenetic analyses indicates that the hibernation system mediated by the HPF homologue, RMF and 100S ribosome formation may be specific to the proteobacteria gamma group. In contrast, most bacteria have at least one HPF homologue, and these homologues can be classified into three types, long HPF, short HPF and YfiA.

Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA.

The replication initiator factor RepE of the F plasmid in Escherichia coli is an essential protein that stringently regulates the F-plasmid copy number. The RepE protein has a dual function: its monomer functions as a replication initiator, while its dimer acts as a transcriptional repressor of the repE gene. The wild-type dimeric RepE protein was expressed as an N-terminal histidine-tagged protein, purified under native conditions with a high salt concentration and crystallized in complex with the repE operator DNA using the sitting-drop vapour-diffusion technique. The crystals diffracted to a resolution of 3.14 A after the application of dehydration and crystal annealing and belong to space group P2(1), with unit-cell parameters a = 60.73, b = 99.32, c = 95.00 A, beta = 108.55 degrees.

The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli.

During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.

Atomic force microscopy analysis of the role of major DNA-binding proteins in organization of the nucleoid in Escherichia coli.

Bacterial genomic DNA is packed within the nucleoid of the cell along with various proteins and RNAs. We previously showed that the nucleoid in log phase cells consist of fibrous structures with diameters ranging from 30 to 80 nm, and that these structures, upon RNase A treatment, are converted into homogeneous thinner fibers with diameter of 10 nm. In this study, we investigated the role of major DNA-binding proteins in nucleoid organization by analyzing the nucleoid of mutant Escherichia coli strains lacking HU, IHF, H-NS, StpA, Fis, or Hfq using atomic force microscopy. Deletion of particular DNA-binding protein genes altered the nucleoid structure in different ways, but did not release the naked DNA even after the treatment with RNase A. This suggests that major DNA-binding proteins are involved in the formation of higher order structure once 10-nm fiber structure is built up from naked DNA.

A novel bioinformatics strategy for searching industrially useful genome resources from metagenomic sequence libraries.

Although remarkable progress in metagenomic sequencing of various environmental samples has been made, large numbers of fragment sequences have been registered in the international DNA databanks, primarily without information on gene function and phylotype, and thus with limited usefulness. Industrial useful biological activity is often carried out by a set of genes, such as those constituting an operon. In this connection, metagenomic approaches have a weakness because sets of the genes are usually split up, since the sequences obtained by metagenome analyses are fragmented into 1-kb or much shorter segments. Therefore, even when a set of genes responsible for an industrially useful function is found in one metagenome library, it is usually difficult to know whether a single genome harbors the entire gene set or whether different genomes have individual genes. By modifying Self-Organizing Map (SOM), we previously developed BLSOM for oligonucleotide composition, which allowed classification (self-organization) of sequence fragments according to genomes. Because BLSOM could reassociate genomic fragments according to genomes, BLSOM may ameliorate the abovementioned weakness of metagenome analyses. Here, we have developed a strategy for clustering of metagenomic sequences according to phylotypes and genomes, by testing a gene set contributing to environment preservation.

Transcription-coupled nucleoid architecture in bacteria.

The circular bacterial genome DNA exists in cells in the form of nucleoids. In the present study, using genetic, molecular and structural biology techniques, we show that nascent single-stranded RNAs are involved in the step-wise folding of nucleoid fibers. In Escherichia coli, RNase A degraded thicker fibers (30 and 80 nm wide) into thinner fibers (10 nm wide), while RNase III and RNase H degraded 80-nm fibers into 30-nm (but not 10-nm) fibers. Similarly in Staphylococcus aureus, RNase A treatment resulted in 10-nm fibers. Treatment with the transcription inhibitor, rifampicin, in the absence of RNase A changed most nucleoid fibers to 10-nm fibers. Proteinase-K treatment of nucleoids exposed DNA. Thus, the smallest structural unit is an RNase A-resistant 10-nm fiber composed of DNA and proteins, and the hierarchical structure of the bacterial chromosome is controlled by transcription itself. In addition, the formation of 80-nm fibers from 30-nm fibers requires double-stranded RNA and RNA-DNA hetero duplex. RNA is evident in the architecture of log-phase uncondensed and stationary-phase condensed nucleoids.

Human small G proteins, ObgH1, and ObgH2, participate in the maintenance of mitochondria and nucleolar architectures.

The Obg subfamily protein is one of the P-loop small G proteins and is highly conserved in many organisms from bacteria to human. Two obg genes, obgH1 and obgH2, exist in the human genome. Both ObgH1 and ObgH2 showed similar GTPase activities (0.014 +/- 0.005 and 0.010 +/- 0.002/min for ObgH1 and ObgH2, respectively) to those of the bacterial Obg proteins and complemented the Obg function in Escherichia coli ribosome maturation, suggesting that the functions of Obg proteins are well conserved through evolution. Immunofluorescence microscopy of HeLa cells revealed that ObgH1 localizes in mitochondria, and ObgH2 in the dense fibrillar compartment region of the nucleolus. Knock-down of ObgH1 by RNAi induced mitochondria elongation, whereas knock-down of ObgH2 resulted in the disorganization of the nucleolar architecture. In conclusion, the two human Obg proteins have similar enzymatic activities that can complement bacterial Obg function, but show different cellular function(s) with different intracellular localizations.

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 Deltafis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nucleoid condensation in the presence of Dps. DNA-topology assays using the cell extract showed that the extracts from the Deltafis 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.

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 ( approximately 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.

Large-scale identification of protein-protein interaction of Escherichia coli K-12.

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology.

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.