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Genes to Cells (2005) 10, 1103-1112. doi:10.1111/j.1365-2443.2005.00903.x
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Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli

Masami Ueta1, Hideji Yoshida1, Chieko Wada2, Tomoya Baba3, Hirotada Mori3,4 and Akira Wada1,a,*

1 Department of Physics, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
2 Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan
3 Institute of Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0035, Japan
4 Research and Education Center of Informatics, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
When Escherichia coli cells transit from the exponential growth phase into the stationary phase, morphological and physiological changes occur. These include the appearance of small spherical cells that have condensed nucleoids and high resistance to many stresses (Hengge-Aronis 1996a). It was reported that these changes are associated with changes in the expression of more than 100 genes (Hengge-Aronis 1996b; Schellhorn et al. 1998). We have analyzed E. coli proteins using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis (RFHR 2-D PAGE) (Wada 1986a), which allowed us to detect 65 proteins expressed specifically in the stationary phase (our unpublished data). Although the functions of many of these proteins remain undetermined, it is known that several correspond to ribosome binding proteins (Wada 1986b, 1998), including RMF (ribosome modulation factor) (Wada et al. 1990), SRA (stationary-phase-induced ribosome-associated protein) (Izutsu et al. 2001a), and YfiA and YhbH (Maki et al. 2000).

RMF (MW = 6,476, pI = 11.30), a small basic protein that binds to 50S ribosomal subunits, is expressed upon ppGpp induction of the rmf gene in stationary-phase E. coli cells (Izutsu et al. 2001b). RMF stimulates the dimerization of 70S ribosomes to form 100S particles (Wada et al. 1990) that have no translational activity (Wada et al. 1995). The rmf deletion mutant does not form 100S ribosomes, and loses viability earlier than the wild-type in the stationary phase (Yamagishi et al. 1993; Wada et al. 2000). This suggests that 100S formation protects against ribosomal degradation, resulting in a longer lifetime. When stationary-phase cells are transferred to fresh media, RMF is immediately released from 100S ribosomes, which then dissociate back into translationally active 70S monomers. This process is very quick, reaching completion within one minute (Aiso et al. 2005). These observations suggest that interconversion between active 70S and inactive 100S ribosomes may regulate translation efficiency during transitions between the exponential phase and stationary phase. We have named this process ‘ribosomal hibernation’ (Yoshida et al. 2002).

SRA (MW = 5,096, pI = 11.55) binds tightly to 30S ribosomal subunits and cannot be dissociated by high salt or low magnesium concentrations. Because of this tight binding property, SRA (sra) was formerly called ribosomal protein S22 (rpsV). The number of SRA copies per ribosomal particle is 0.1 in the exponential phase but increases to about 0.4 in the stationary phase (Izutsu et al. 2001a). The function of SRA is currently unknown; an sra deletion affects neither 100S formation nor growth in the stationary phase.

Two small acidic proteins, YfiA (MW = 12 653, pI = 6.23) and YhbH (MW = 10 750, pI = 6.62), share approximately 40% homology. Homologs of YfiA and YhbH are found in many bacteria and, in protein databases, both proteins have been designated as sigma-54 modulation factors, which are related to nitrogen metabolism. Bacteria belonging to the proteobacteria gamma group, including E. coli, have both the YfiA and YhbH genes, whereas other bacteria have only one or the other. Both proteins bind to ribosomes in E. coli (Maki et al. 2000). YfiA is mainly located on 70S ribosomes and to a lesser extent on 100S ribosomes, whereas YhbH binds to 100S ribosomes exclusively. The two proteins cannot be released from the ribosome by washing with high salt concentrations, which suffices to dissociate 100S ribosomes into 70S ribosomes, but they can be released by low magnesium treatment, which dissociates 70S ribosomes into 30S and 50S subunits. The functions of these proteins in stationary phase ribosomes are unclear, but when stationary phase cells are transferred to fresh media and translation begins, YfiA and YhbH are found in the 70S fraction and not in the polysome fraction (Maki et al. 2000), suggesting that YfiA and YhbH are not necessary for the elongation step of translation in refreshed cells.

Agafonov et al. (1999) reported that YfiA (also known as pY or RaiA) binds to 30S subunits at the subunit interface. They also showed that YfiA prevents dissociation of 70S ribosomes at low magnesium concentrations and inhibits translation at the aminoacyl-tRNA binding stage (Agafonov et al. 2001). Additionally, YfiA was reported to reduce translation errors (Agafonov et al. 2004). Finally, recent analyses of X-ray crystal structures of YfiA bound to a 30S subunit revealed that YfiA binds to the P-site and A-site on the ribosome (Vila-Sanjurjo et al. 2004).

In this paper, we studied the functions of YfiA and YhbH in 70S dimer formation. We investigated ribosome profiles in the stationary phase by using a yfiA deletion mutant, a yhbH deletion mutant, and a yfiA yhbH double deletion mutant. Our results show that YfiA and YhbH have antagonistic functions in 100S ribosome formation: YfiA functions to inhibit 100S ribosome formation, whereas YhbH functions to promote it. Furthermore, we used RFHR 2-D PAGE to analyze the proteins in crude ribosomal fractions obtained from these deletion mutants in the stationary phase. More copies of YhbH were present in the yfiA deletion mutant than in the wild-type, and more copies of YfiA were present in the yhbH deletion mutant than in the wild-type, suggesting that the binding sites of these two proteins may overlap. Because of its positive contribution to 100S formation in the ribosomal hibernation process, we suggest that YhbH be named hibernation promoting factor (protein: HPF; gene name: hpf).


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Growth and viability of W3110 ({Delta}yfiA::Km), W3110 ({Delta}yhbH::Km), and W3110 ({Delta}yfiA and {Delta}yhbH::Km)

A yfiA deletion mutant, yhbH deletion mutant, and yfiA yhbH double deletion mutant of the W3110 strain were constructed as described in the Experimental procedures. The mutants were grown in EP medium at 37 °C to compare their phenotypes with those of the wild-type strain, W3110. Cell growth was measured by sample turbidity (Klett Units). There were no differences in the exponential-phase and stationary-phase growth curves between the deletion mutants and the wild-type (data not shown). In the stationary phase, the cell viability of yfiA and yhbH deletion mutants rose to a peak after 2 days and then rapidly decreased after 4 days, suggesting that these single deletion mutants may live a little longer than the wild-type (Fig. 1). The cellular viability of the double deletion mutant was similar to that of the wild-type in the stationary phase.



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  		Figure 1  Cell viability. Cultures of the wild-type strain W3110 and the deletion mutants, W3110 ({Delta}yfiA::Km), W3110 ({Delta}yhbH::Km), and W3110 ({Delta}yfiA and {Delta}yhbH::Km) were sampled at different times (2.5, 5 and 7.5 h and 1, 2, 3, 4, 5, 6, 7 and 8 d) from the exponential phase through the stationary phase of growth. Optical density and colony forming ability were determined, and the viability values (colony numbers/Klett Units) were calculated. The relative viability values obtained were normalized to that at 0 h. The data shown are the average values of the results of four experiments.

 
Effect of YfiA and YhbH on 70S dimer formation

We examined whether the absence of YfiA and/or YhbH proteins would affect the formation of 70S dimers. The wild-type strain W3110, the yfiA deletion mutant, the yhbH deletion mutant, and the yfiA yhbH double deletion mutant were grown in EP medium at 37 °C and harvested during the exponential and stationary phases. In order to investigate the ribosome profiles of these cells, cell extracts (optical density times volume (ODV) = 3) were analyzed by sucrose density gradient centrifugation as described in the Experimental procedures (Fig. 2).



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  		Figure 2  70S dimer formation in the wild-type strain W3110 and the three deletion mutants. Cells of the wild-type strain W3110 and the deletion mutants, W3110 ({Delta}yfiA::Km), W3110 ({Delta}yhbH::Km), and W3110 ({Delta}yfiA and {Delta}yhbH::Km), were grown at 37 °C in EP medium. Cultures were sampled during the log phase (2.5 h), the transition to the stationary phase (5 h), and the stationary phase (7.5 h and 1, 2, 3, 4 and 5 d). The ribosome profiles were measured as described in the Experimental procedures. The direction of the 5%-20% gradient is from left to right. S-values of 70S dimers were determined by normalizing the distance between 50S and 70S peaks as 20S. We performed the experiments at least four times.

 
In the exponential phase (at 2.5 h), ribosomes sedimented as a major 70S peak in all strains, and no significant differences were observed between the wild-type and the deletion mutants. In wild-type cultures undergoing transition to the stationary phase (5 h), ribosomes sedimented as 90S particles, which probably correspond to an immature form of the 70S dimer. In the stationary phase (7.5 h, 1 d, and 2 d), a peak corresponding to 100S ribosomes was observed, and after 3 days, the 100S particles started to break down into 70S monomers and a broad band appeared between the 70S and 100S peaks as shown in Fig. 2.

In the yfiA deletion mutant, we observed 90S particles during the transition to the stationary phase (5 h) and 100S ribosomes in the stationary phase (7.5 h, 1 d, 2 d, and 3 d), as in the wild-type. Interestingly, more 100S ribosomes were present in the yfiA deletion mutant than in the wild-type at 1 d, 2 d, and 3 d. Furthermore, 100S ribosomes persisted in the yfiA deletion mutant even after 4 days, unlike in the wild-type. In contrast, in the yhbH deletion mutant we observed neither 90S particles during the transition to the stationary phase (5 h) nor 100S ribosomes during the stationary phase, suggesting that YhbH promotes 100S ribosome formation and YfiA inhibits 100S ribosome formation. In the yfiA yhbH double deletion mutant, we observed a shoulder of 90S particles during the transition to the stationary phase (5 h) and a 90S peak in the stationary phase (7.5 h, 1 d, and 2 d).

When cloned copies of yhbH+ genes were introduced into the deletion mutants (yhbH or yfiA and yhbH), the size of the 100S ribosome peak increased and YhbH protein was bound to the 100S ribosome as judged by Western blotting (data not shown). On the other hand, when cloned copies of the yfiA+ gene were introduced into the deletion mutants (yfiA or yfiA and yhbH), the size of the 100S ribosome peak decreased and YfiA protein was mainly bound to the 70S ribosome (data not shown). These results exclude the possibility of polar effects by these deletion mutations.

Copy numbers of YhbH and YfiA in yfiA and yhbH deletion mutants

The yhbH deletion mutant, the yfiA deletion mutant, and the double deletion mutant gave different ribosome sedimentation patterns during sucrose density gradient centrifugation (Fig. 2). To examine this in detail, we measured the copy numbers of YfiA, YhbH, and RMF in the ribosomal particles of these deletion mutants; ‘copy number’ is defined as the molar ratio of the molecule to 70S, 50S, or 30S ribosomal particles. Proteins in crude ribosomal fractions prepared from cells harvested after 2 days of growth were analyzed with RFHR 2-D PAGE (Fig. 3A). The copy numbers of the three proteins, normalized to those of the wild-type, were determined as described in the Experimental procedures (Fig. 3B). In the wild-type strain, all three ribosome binding proteins, RMF (0.59 copies), YfiA (0.3 copies), and YhbH (0.26 copies), were observed (Fig. 3Aa), and 58% of the total crude ribosome preparation was 100S particles. Therefore, the wild-type 100S particle has two molecules of RMF and one molecule of YhbH, since RMF and YhbH preferentially bind to the 70S dimer. In the yfiA deletion mutant, YfiA protein was absent, but RMF (0.74 copies) and YhbH (0.45 copies) were observed. As 75% of total crude ribosomes were present as 100S particles in the yfiA deletion mutant, the 100S particle in the yfiA deletion mutant has also two molecules of RMF and one molecule of YhbH, similar to the wild-type (Fig. 3Ab). The yhbH deletion mutant had no YhbH protein, but RMF (0.51 copies) and YfiA (0.49 copies) were observed (Fig. 3Ac). This mutant has no 70S dimer. In the double deletion mutant, both YhbH and YfiA proteins disappeared, and only RMF (0.72 copies) was observed in the gel. This double mutant formed a 90S particle that contained about two molecules of RMF (Fig. 3Ad). These results suggest that 70S dimers with only RMF bound appear as 90S particles, and those having RMF and YhbH bound appear as 100S ribosome.



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  		Figure 3  (A) RFHR 2-D PAGE analysis of crude ribosomal fractions. Cells of the wild-type strain (a), {Delta}yfiA::Km (b), {Delta}yhbH::Km (c), and the double deletion mutant (d) were grown to the stationary phase (2 days) in EP medium. Proteins were prepared from harvested cells by the acetic acid method. YfiA, YhbH, RMF, SRA, S2, S21, and L27 spots are indicated. (B) Relative copy numbers of ribosome binding proteins in the stationary phase. The copy numbers of YfiA, YhbH, and RMF were determined as described in the Experimental procedures and were normalized to those of wild-type cells. The data shown are the average values of the results from at least six gels.

 
The copy number of the YhbH protein was approximately 1.7 times higher in the yfiA deletion mutant than in the wild-type. Similarly, the copy number of YfiA in the yhbH deletion mutant was approximately 1.6 times higher than in the wild-type strain. This suggests that YfiA and YhbH may compete for overlapping target sites on the ribosome.

The copy numbers of RMF in the wild-type and the yhbH deletion mutant were a little lower than those in the yfiA deletion mutant, suggesting that YfiA may prevent binding of RMF to the ribosome. Additionally, we could detect no differences in the copy numbers of any ribosomal protein between the deletion mutants and the wild-type strain (Fig. 3A).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Our results strongly suggest that RMF dimerizes 70S to make 90S particles, and that YhbH matures 90S to give 100S particles. In previous experiments, we performed sucrose density gradient centrifugation experiments with mixtures of purified RMF and exponential-phase 70S ribosomes lacking YhbH and found 90S particles, which are thought to be immature 70S dimers (Wada et al. 1995). We saw 100S particles in similar mixtures containing purified RMF and stationary-phase 70S ribosomes bound to both YhbH and YfiA but not RMF. In the present experiments, the yhbH and yfiA double deletion mutant expressing RMF formed 90S particles but not 100S particles. In contrast, the yfiA single deletion mutant, which has both RMF and YhbH, formed 100S particles. RMF but not YhbH can dimerize 70S ribosomes, because an rmf deletion mutant does not form 70S dimers (Yamagishi et al. 1993; Izutsu et al. 2001b).

During the transition from exponential- to stationary-phase growth, wild-type E. coli cells express RMF and YhbH in parallel with 70S dimer formation (Yamagishi et al. 1993; Izutsu et al. 2001b; A. Wada, unpublished observation). Just after the end of the exponential growth phase, a small 90S peak is transiently observed in the sucrose density gradient. This 90S peak must be formed by the binding of RMF to the 70S particle which is vacant of YhbH, because it is completely transformed to a 100S peak within 1 h following increase of YhbH expression. On the other hand, the 90S peak disappeared in the yhbH deletion mutant, and only a broad peak of 70S particles, including a small amount of 90S particles, was observed, suggesting that YfiA prevents 70S dimerization by RMF (Figs. 2 and 3). Thus, it appears that YhbH and YfiA have antagonistic functions.

Figure 1 shows that these single deletion mutants may be able to live a little longer than the wild-type strain, probably by maintaining 70S ribosomes or 70S dimers for a longer period of time during the stationary phase. Both the number of YfiA copies and the amount of 70S ribosomes at 4 and 5 days (Fig. 2) were higher in the yhbH deletion mutant than in the wild-type. The excess YfiA protein may therefore be involved in the stabilization of ribosomal subunits, which might explain why the yhbH deletion mutants live a little longer than the wild-type. Agafonov et al. (2001) found that YfiA stabilizes 70S particles in the exponential phase, and this also seems to be the case for YfiA in the stationary phase. Similarly, we observed 100S particles on day 4 and 90S particles on day 5 (Fig. 2) in the yfiA deletion mutant but not in the wild-type. The YhbH protein copy number was also higher than in the wild-type, and the excess YhbH protein may stabilize 90S and 100S particles, thereby protecting the 70S dimers from degradation and allowing the yfiA deletion mutant to live a little longer than the wild-type.

YhbH and YfiA share high amino acid sequence homology (Fig. 4A), and it has been reported that the ratio of the sum of the copy numbers of these two proteins to the number of ribosomal particles is approximately one in the stationary phase (Maki et al. 2000). In this study, we showed that the copy number of YhbH, which is mainly bound to 100S ribosomes, was larger in the crude ribosomal fraction of the yfiA deletion mutant than in that of the wild-type. Similarly, the copy number of YfiA, which is mainly bound to 70S ribosomes, was higher in the crude ribosomal fraction of the yhbH deletion mutant than in that of the wild-type (Fig. 3B). From these data, we suggest that the binding sites of these two proteins on ribosomal particles may significantly overlap, causing the mutual exclusion of YfiA and YhbH on the same ribosomal particle. Recently, the X-ray crystal structure of YfiA bound to the 70S ribosome of E. coli was reported, and six amino acids (Arg22, Lys25, Lys28, Lys79, Arg82, and Lys86) of YfiA were found to be involved in ribosome binding (Vila-Sanjurjo et al. 2004). These amino acids form a positively charged surface and are conserved in YfiA homologs, although there are several substitutions of Arg and Lys (Fig. 4A). A comparison of the amino acid sequences of several bacterial YhbH homologs, including that of E. coli, to the YfiA sequence shows that Arg 22 and Lys28 in YfiA are changed to Lys22 and Gln28 in YhbH, respectively (Fig. 4C). However, four amino acids (Lys25, Lys79, Arg82, and Lys86) are well conserved; therefore it is possible that this region of YhbH binds to the same site on the ribosome as YfiA.



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  		Figure 4  (A) The amino acid sequences of YfiA and YhbH. Identical amino acids are marked with stars. YfiA and YhbH have about 40% homology to each other. YfiA has a C-terminal acidic extension of 18 residues that is absent in YhbH. (B, C) Comparisons of YfiA (B) and YhbH (C) homologs. An alignment of amino acid sequences of YfiA and YhbH from E. coli W3110 (ECO), Shigella flexneri (SFL), Salmonella enterica (SEN), Yersinia pestis (YPE), Photorhabdus luminescens (PLU), Vibrio vulnificus (VVU), Vibrio parahaemolyticus (VPA), Vibrio cholerae (VCH), Shewanella oneidensis (SON), Erwinia carotovora (ECA), and Photobacterium profundum (PPR) is shown. Shaded boxes indicate sequence identity. ECO, SFL, SEN, YPE, PLU, VVU, VPA, VCH, and SON also have an RMF homolog, whereas ECA and PPR do not. The amino acids required for binding of YfiA to the A-site and P-site on the 30S subunit are outlined in red. Four of these amino acids that are conserved between YfiA and YhbH homologs are also shown in red frames. Two amino acids conserved in the YhbH homologs are outlined in green.

 
It has been shown that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) center (Yoshida et al. 2002, 2004). Furthermore, it has been reported that YfiA binds to the P-site and A-site of the 30S subunit (Vila-Sanjurjo et al. 2004). The binding site of RMF must therefore be proximal to that of YfiA in the 70S particle. Interestingly, although YfiA and YhbH are highly homologous and probably share overlapping binding sites on the ribosome, these two proteins seem to have opposite effects on 100S particle formation. YfiA has a C-terminal acidic extension of 18 residues compared to YhbH (Fig. 4A). When YfiA binds to the P-site and A-site of a 30S subunit, the C-terminal 18 residues might inhibit RMF binding to the ribosome and decrease subsequent 70S dimer formation. In contrast, since YhbH lacks the C-terminal 18 residues of YfiA, YhbH binding to the P-site and A-site of a 30S subunit might stabilize the binding of RMF to the 50S subunit, resulting in enhanced 70S dimer formation. The function(s) of this 18-residue C-terminal tail will be further investigated in future studies.

There are many homologs of YfiA and YhbH in bacteria. To investigate which organisms possess both YfiA and YhbH homologs, nucleotide and protein databases were searched with the BLAST program (Fig. 4B,C). As a result, ten bacteria, including Shigella flexneri, Salmonella enterica, Yersinia pestis, Photorhabdus luminescens, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Shewanella oneidensis, Erwinia carotovora and Photobacterium profundum were found also to have YfiA and YhbH homologs. All of these belong to the proteobacteria gamma group as well as to the Gram-negative facultative anaerobic rod group, and almost all of them are Enterobacteriaceae. All of the strains except for two (Erwinia carotovora and Photobacterium profundum) have an RMF homolog. Yersinia pestis has been reported to contain two genes that display homology in their short N-terminal and long C-terminal regions to those of YhbH; however, this apparent splitting of YhbH into two genes could be due to a sequencing error. The amino acids indicated in the red frames in Fig. 4B contribute to YfiA binding at the A-site and P-site on the 30S subunit (Vila-Sanjurjo et al. 2004). These amino acids are well conserved in all the bacterial strains mentioned above. Furthermore, four of these amino acids are also conserved in YhbH homologs, whereas Arg22 and Lys28 are changed to Lys and Gln, respectively, as indicated by the two green frames (Fig. 4C).

Based on these results, we propose a model for compositional and conformational changes in E. coli wild-type ribosomes through the binding of the three protein factors (Fig. 5). In the exponential phase (Fig. 5A), most of the 70S ribosomes lack RMF and YhbH and thus can actively participate in protein synthesis. During the transition to the stationary phase (Fig. 5B), newly synthesized RMF starts to bind to 70S ribosomes which are vacant of YhbH, resulting in the formation of 70S dimers which appear as 90S particles. Then YhbH binding converts the 90S particles into 100S particles (Fig. 5C). In the stationary phase, the amount of 100S particles containing RMF and YhbH increases significantly. Although YfiA-bound 70S monomers still exist in the stationary phase, active 70S ribosomes are thought to be rare. When stationary phase cells are transferred to fresh media, RMF, YhbH, and YfiA are quickly released from the ribosomal particles, and within one minute, active 70S ribosomes reappear. After another seven minutes, cell growth enters the early exponential phase (our unpublished observations, Fig. 5D).



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  		Figure 5  A model for the compositional and conformational changes in E. coli ribosomes during growth-phase transitions. Ribosome forms in the exponential phase (A), the transition to the stationary phase (B), the stationary phase (C), and in fresh media (D) are shown schematically. YfiA, YhbH, and RMF are indicated as yellow, red, and green circles, respectively. The small and large ribosomal subunits are shown as small and large ellipsoids, respectively. In the stationary phase, the 100S ribosome is a dimer of 70S ribosomes that make contact with the two 30S subunits face-to-face (Yoshida et al. 2002). RMF binds to each 50S subunit of the two 70S ribosomes in the 100S particle (Wada et al. 1990). YfiA binds to the 30S subunit of a 70S ribosome (Agafonov et al. 1999). YhbH may bind to either of the 30S subunits in a 100S ribosome. In the transition to the stationary phase (B), 70S dimers appear as 90S particles, which are thought to contain RMF, but not YhbH. See the text for details.

 
In conclusion, we have shown that there are two steps involved in the formation of 100S particles through the intermediary formation of a 90S particle. YhbH converts immature 90S particles to mature 100S ribosomal particles by promoting both particle formation and stabilization. In contrast, YfiA prevents 70S dimer formation. Thus, although YfiA and YhbH are highly homologous to each other, they have opposite functions in 70S dimer formation. From these observations, we suggest that YhbH be named ‘hibernation promoting factor (HPF).’


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Bacterial strains and plasmids

The yfiA and yhbH deletion mutants (BW25113 derived strains) were obtained from the KO collection (Systematic Knock Out strain of E. coli K-12; GenoBase: http://ecoli.aist-nara.ac.jp/gb5/Resources/deletion/deletion.html) by Baba et al. (unpublished observation). P1 vir phages were prepared from these deletion mutants as donors. Thereafter W3110 ({Delta}yhbH::Km) and W3110 ({Delta}yfiA::Km) were constructed by P1 general transduction using the recipient strain W3110 (wild-type). The deletion of the yfiA and yhbH genes in each strain was confirmed by PCR. W3110 ({Delta}yfiA) was constructed by the elimination of the kanamycin-resistance cassette from W3110 ({Delta}yfiA::Km) using a helper plasmid (pCP20) encoding FLP recombinase (Datsenko & Wanner 2000). The double deletion mutant W3110 ({Delta}yfiA and {Delta}yhbH::Km) was constructed by P1 general transduction using the recipient strain W3110 {Delta}yfiA, and the double deletion was confirmed by PCR.

The deletion mutants were constructed to be non-polar by using a primer containing an idealized ribosome binding site and start codon for downstream gene expression (Datsenko & Wanner 2000). Furthermore, we confirmed that arabinose induction restored the phenotypes of the single and the double deletion mutants ({Delta}yfiA::Km, {Delta}yhbH::Km, or {Delta}yfiA and {Delta}yhbH::Km) transformed with plasmid carrying yfiA+(pKV7300-PBAD-yfiA+) or yhbH+(pKV7300-PBAD-yhbH+), respectively. The yfiA or yhbH fragment was amplified using W3110 genome DNA as template and PCR primers 5'-tcccccgggatgacaatgaacattaccagc-3' and 5'-cccaagcttctactcttcttcaacttcttcg-3' for yfiA, and 5'-tcccccgggatgcagctcaacattaccgga-3' and 5'-cccaagcttttagtgttgtttcagtttatc-3' for yhbH. The underlined portions indicate the restriction sites for SmaI and HindIII, respectively. The amplified fragments were treated with the restriction enzymes SmaI and HindIII and cloned between the SmaI and HindIII sites of pKV7300 (Uga et al. 1999) to generate the plasmids pKV7300-PBAD-yfiA+ and pKV7300-PBAD-yhbH+.

Growth conditions

Each strain was grown in medium E (Vogel & Bonner 1956) containing 2% polypeptone (EP medium) at 37°C. When necessary, kanamycin (12.5 µg/ml) was added to the medium. Cell growth was followed by measuring turbidity using a Klett Summerson photoelectric colorimeter (Bel-Art Product, USA) with a green filter (#54). Cell viability was determined by measuring colony numbers on L-broth plates. Cells for sucrose density gradient analysis were harvested in the exponential and stationary phases and stored at –80 °C until use.

Sucrose density gradient centrifugation

Cell pellets harvested in either the exponential or stationary phase were resuspended in association buffer (20 mM Tris-HCl [pH 7.6], 15 mM magnesium acetate, 100 mM ammonium acetate, and 6 mM 2-mercaptoethanol), mixed with an equal volume of glass beads (425-600 microns, Sigma), and vortexed five times for 30 s. After centrifugation at 18 500 g for 10 min, the absorbance of the supernatant was measured at 260 nm with a UV-1700 spectrometer (Shimadzu). The supernatants were layered on a 5–20% linear sucrose density gradient made with association buffer, such that the ODV (optical density at 260 nm times volume (ml)) values were equal. The samples were then analyzed by centrifugation in an SW40Ti rotor (Beckman) at 25 000 g for 20 h at 4 °C. The absorbance of each fraction was measured at 260 nm using a flow cell within a UV-1700 spectrometer. The amounts of particles were determined from the areas of the corresponding peaks in the OD patterns of the sucrose density gradient centrifugation.

Preparation of crude ribosomal fractions

Cells were harvested after growing for 2 days. Each cell pellet was ground with an approximately equal volume of quartz sand (Wako) and then extracted with association buffer. Crude ribosomal fractions were prepared from the cell extract essentially according to the method of Noll et al. (1973) with slight modifications as described (Horie et al. 1981).

Two-dimensional gel electrophoresis

Crude ribosomal fractions were resuspended in association buffer, and ribosomal proteins were prepared by the acetic acid method (Hardy et al. 1969). One-tenth volume of 1 M MgCl2 and two volumes of acetic acid were added, and the mixture was stirred for 1 h at 4 °C. After centrifugation at 5600 g for 10 min, the supernatant was dialyzed against 2% acetic acid three times. The proteins were lyophilized and stored at –80 °C until use. Two milligrams of the lyophilized protein was analyzed with RFHR 2-D PAGE (Wada 1986a). The protein spots on the gels were scanned with a GS-800 Calibrated Densitometer (Bio-Rad Laboratories Inc.). The optical density values of these proteins were calculated as a function of their molecular weights, and values of YhbH and YfiA were normalized against the value for ribosomal protein S2. S2 was used as a marker to estimate the copy numbers of YhbH and YfiA because S2 was previously defined to exist as a unit copy protein (Hardy 1975; Tal et al. 1990) and has a similar isoelectric point to YhbH and YfiA. ‘Copy number’ means the molar ratio of the molecule to the 70S ribosome, 50S subunit, or 30S subunit. In the same way, the copy number of RMF was calculated relative to S14, L27, L29, and L30.


   Acknowledgements
 
This work was supported by Grants-in-Aid for Scientific Research on Priority Area ‘Spatio-temporal Network of RNA Information Flow’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yoshikazu Nakamura

aPresent address: Department of Physics, Osaka Medical College, 2-7, Daigaku-Machi, Takatsuki, Osaka 569-8686, Japan. E-mail: phy003{at}art.osaka-med.ac.jp Back

* Correspondence: E-mail: phy003{at}art.osaka-med.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 28 May 2005
Accepted: 31 August 2005




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