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Genes to Cells (2005) 10, 733-741. doi:10.1111/j.1365-2443.2005.00872.x
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A decreased level of FtsZ is responsible for inviability of RNase E-deficient cells

Ayako Takada1, Kazuo Nagai2 and Masaaki Wachi1,*

1 Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
2 Department of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The endoribonuclease RNase E, encoded by the essential gene rne, plays a major role in cellular RNA metabolism, i.e. maturation of functional RNAs such as rRNA and tRNA, degradation of many mRNAs and processing of the ftsZ mRNA which encodes the essential cell division protein FtsZ. RNase E function is somehow regulated by the RNA binding protein Hfq. We found that temperature-sensitive colony formation of a rne-1 mutant was partially suppressed by introduction of a hfq::cat mutation. Neither accumulation of rRNA and tRNAPhe precursors nor incomplete processing of ftsZ mRNA in the rne-1 mutant was rescued by the hfq::cat mutation. However, the amount of FtsZ protein that was decreased in the rne-1 mutant was recovered up to a level similar to that of wild-type cells by the hfq::cat mutation. Overproduction of Hfq inhibited cell division because of decreased expression of FtsZ. Artificial expression of the FtsZ protein from a plasmid-borne ftsZ gene partially suppressed the temperature-sensitivity of the rne-1 mutant. These results suggest that the decreased level of FtsZ is, at least in part, responsible for the inviability of RNase E-deficient cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
RNase E is an essential endoribonuclease in Escherichia coli. The phenotype of its temperature-sensitive mutation rne-1 (originally called ams-1) was initially described as increasing the chemical half-life of total cellular RNA (Kuwano et al. 1977; Ono & Kuwano 1980). Another independently isolated mutation, rne-3071, was implicated in the maturation process of 5S rRNA from its precursor 9S rRNA (Ghora & Apirion 1978). The rne gene encodes a 1061-amino acid protein (Casarégola et al. 1992) and its N-terminal half bears the catalytic activity, whereas the C-terminal half is involved in degradosome formation (Taraeviciene et al. 1995; McDowall & Cohen 1996; Kaberdin et al. 1998; Vanzo et al. 1998). RNase E plays an important role in all aspects of RNA metabolism in E. coli, including processing and/or decay of rRNAs, tRNAs, functional RNAs and mRNAs. Endoribonucleolytic cleavage by RNase E depends on 5'-end structures of RNAs (Bouvet & Belasco 1992) and occurs within single-stranded A and/or U-rich segments (Mackie 1991, 1992; Ehretsmann et al. 1992).

Since the rne-1 and rne-3071 temperature-sensitive mutants have shown the accumulation of 5S rRNA precursors (Ghora & Apirion 1978; Babitzke et al. 1993) and an increase in the half-life of several mRNAs (Arraiano et al. 1988; Mackie 1991; Régnier & Hajnsdorf 1991), it was formerly assumed that the inviability associated with the inactivation of RNase E resulted from a defect in either 5S rRNA maturation or mRNA decay. However, experiments using several rne truncation mutants suggested that this was not correct (López et al. 1999; Ow et al. 2000). A defect of the processing of the M1 RNA subunit of RNase P could be a reason for the inviability of RNase E-deficient mutants, but this is not likely because the unprocessed precursor still retains catalytic activity (Liu & Altman 1995). Using a series of RNase E mutants, Ow & Kushner (2002) recently suggested that the processing of tRNA is the likely reason that RNase E is required for cell viability, but the opposite findings were also reported later by Deana & Belasco (2004). Thus, the reason why RNase E is essential for E. coli cell viability is not yet clearly understood.

The Hfq protein, which is encoded by the hfq gene at 94.8 min on the E. coli genetic map (Kajitani & Ishihama 1991), was initially identified as a bacterial host factor required for replication of bacteriophage Qß RNA (Fernandez et al. 1968; Su et al. 1997). The inactivation of the hfq gene in E. coli causes a wide variety of phenotypes and alters expression of many proteins (Tsui et al. 1994; Muffler et al. 1997). Hfq is required for the translation of rpoS mRNA encoding the {sigma}s subunit of RNA polymerase (Brown & Elliott 1996; Muffler et al. 1996). In the last 10 years, an increasing amount of evidence has been accumulated showing that Hfq pleiotropically regulates the expression of many genes by binding to RNAs. Hfq interacts with several small non-coding RNAs, such as DsrA, RyhB, Spot42 RNA and OxyS, by assisting their base-pairing, and therefore is required for their functions (Sledjeski et al. 2001; Massé & Gottesman 2002; Møller et al. 2002; Zhang et al. 2002, 2003). It was also reported that Hfq stimulates decay of ompA mRNA by RNase E by interfering with ribosome binding (Vytvytska et al. 1998, 2000; Moll et al. 2003). Hfq also affects polyadenylation of mRNAs (Hajnsdorf & Régnier 2000).

In the previous paper, we reported that the hfq mutant strain overproduced the cell division protein FtsZ (Takada et al. 1999). It was also reported that the polycistronic ftsA-ftsZ transcripts were cleaved by RNase E and that this cleavage affected the decay of ftsA and ftsZ mRNAs (Cam et al. 1996). It was suggested that this cleavage was required for the efficient expression of the ftsZ gene (Flärdh et al. 1997). In this paper, we demonstrate that temperature-sensitive colony formation of the rne-1 mutant was suppressed by the introduction of the hfq::cat mutation. Effects of the hfq::cat mutation on maturation of functional RNAs, rRNA and tRNA and the processing of ftsZ mRNA in the mutant cells were examined. The results suggest that a decreased level of FtsZ is, at least in part, responsible for the inviability of RNase E-deficient cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Suppression of temperature-sensitive colony formation of the rne-1 mutant by the hfq::cat mutation

In order to investigate the role of Hfq in intracellular RNA metabolism catalyzed by RNase E, we constructed a rne-1 hfq::cat double mutant, HAT113. We first examined the colony-forming ability of the mutant strains on Lennox (L) agar plates. The temperature-sensitive rne-1 mutant strain HAT103 showed temperature-sensitive growth above 40.5 °C on L agar plates. The rne-1 hfq::cat double mutant HAT113 grew as well as the parent strain HAT100 at 30 °C. Unexpectedly, HAT113 grew almost as well at 40.5 °C and slightly even at 42 °C, although colony size was smaller than that of the parent strain (Fig. 1). The effect of the hfq::cat mutation on growth in liquid medium was also examined. The rne-1 mutant strain HAT103 had a gradually decreased growth rate after temperature up-shift, although the turbidity continued to increase for several hours at both 40.5 and 42 °C. Introduction of the hfq::cat mutation did not eliminate the decrease in growth rates (Fig. 2). That is, the hfq::cat mutation suppressed only the inability of the rne-1 mutant to form colonies without suppressing the decreased growth rate phenotype.



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  		Figure 1  Suppression of temperature-sensitive growth of the rne-1 mutant by the hfq::cat mutation. Serial dilutions of cultures of (1) HAT100 (rne+) (2) HAT103 (rne-1) and (3) HAT113 (rne-1 hfq10::cat) were spotted onto L agar plates and plates were incubated at 30, 40.5 and 42 °C for 48 h.

 


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  		Figure 2  Growth of the rne-1 and rne-1 hfq::cat double mutant strains in a liquid medium. Precultures grown to late log phase in L medium at 30 °C were diluted 1 : 50 into prewarmed L medium at each temperature at time 0 and the subsequent increase of OD660 was monitored. (A) 30 °C, (B) 40.5 °C, (C) 42 °C. Circles, HAT100 (rne+); triangles, HAT103 (rne-1); squares, HAT113 (rne-1 hfq::cat).

 
We then examined whether the hfq::cat mutant could accept a rne deletion mutation. The rne deletion mutation, rne{Delta}1018::bla (Ow et al. 2000), could be transduced into the hfq::cat mutant strain HAT110 as well as the hfq+ strain HAT100 only when the rne gene product was supplied in trans from plasmid (data not shown). Thus, hfq::cat mutation cannot support the growth of cells that completely lack RNase E activity.

Effects of the hfq::cat mutation on maturation of functional RNA molecules

RNase E is responsible for maturation of functional RNAs. The rne-3071 temperature-sensitive allele was originally reported to accumulate unprocessed 5S rRNA intermediates (Ghora & Apirion 1978). As such, it was assumed that inviability associated with the inactivation of RNase E was due to a defect in 5S rRNA maturation. RNase E is also involved in 16S rRNA maturation, which can be processed by RNase G, although less effectively (Wachi et al. 1999a). It was also reported that the initiation of tRNA maturation by RNase E is essential for cell viability in E. coli (Ow & Kushner 2002). We therefore examined the effects of the hfq::cat mutation on the maturation of 16S rRNA, 5S rRNA and one tRNA, tRNAPhe. The processing of 16S rRNA, 5S rRNA and tRNAPhe were analyzed by agarose-Synergel electrophoresis or Northern hybridization using total RNAs isolated from rne+, rne-1 and rne-1 hfq::cat strains. In the rne+ control, all of these RNA molecules were completely processed in both analyses. In the rne-1 strain, a small amount of 16S rRNA precursor 16.5S was detected at 40.5 °C and significantly more accumulated at 42 °C. Introduction of the hfq::cat mutation hardly affected 16S rRNA maturation (Fig. 3A). In the case of 5S rRNA, the precursor molecules were detected in the rne-1 mutant strain at 40.5 and 42 °C. The amount of these precursor molecules was not decreased but was actually slightly increased by the introduction of the hfq::cat mutation (Fig. 3B). The effect of the hfq::cat mutation on the maturation of tRNA was also investigated. A precursor transcript of tRNAPhe accumulated at both 40.5 and 42 °C in the rne-1 strain, and became more evident with the introduction of the hfq::cat mutation (Fig. 3C). These results showed that hfq::cat did not rescue the defective maturation process of 16S rRNA, 5S rRNA and tRNAPhe caused by the rne-1 mutation. It is noteworthy that a significant amount of mature molecules of these RNAs were still present in the rne-1 or rne-1 hfq::cat mutant cells.



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  		Figure 3  Effects of the hfq::cat mutation on maturation of functional RNA molecules. Total cellular RNA was isolated from the cells grown at 30, 40.5 and 42 °C for 3 h. (A) 16S rRNA was analyzed by agarose-Synergel electrophoresis as described in the Experimental procedures. The shadow band seen above 16S rRNA in the first three lanes is 17S rRNA precursor. (B) 5S rRNA and (C) tRNAPhe were analyzed by Northern hybridization as described in the Experimental procedures. (1) HAT100 (rne+), (2) HAT103 (rne-1), (3) HAT113 (rne-1 hfq10::cat).

 
Effects of the hfq::cat mutation on the processing and translation of ftsZ mRNA

It was reported that the polycistronic ftsA-ftsZ transcripts were cleaved by RNase E and that this cleavage affected the decay of ftsA and ftsZ mRNAs (Cam et al. 1996). It was also suggested that this cleavage was required for the efficient expression of the ftsZ gene (Flärdh et al. 1997). We reported that the hfq::cat mutant strain overproduced FtsZ (Takada et al. 1999). Therefore, we first surmised that the Hfq protein participated in the RNase E-mediated regulation of FtsZ synthesis. We thus analyzed the fate of ftsZ transcripts in rne+, rne-1 and rne-1 hfq::cat strains by Northern hybridization. Unprocessed precursor transcripts were detected in the rne-1 mutant strain at both 40.5 and 42 °C. Introduction of the hfq::cat mutation did not recover the processing of ftsZ mRNA, as in the case of the maturation of 16S rRNA, 5S rRNA and tRNAPhe (Fig. 4A). We then examined the synthesis of FtsZ by Western blotting using anti-FtsZ antibody. In the rne-1 mutant strain, the amount of FtsZ protein decreased markedly at 40.5 and 42 °C. The rne-1 mutant strain formed elongated cells at these temperatures (data not shown) as reported previously (Goldblum & Apirion 1981), indicating that cell division was inhibited. This result correlates well with the previous report that the cell length began to increase when the FtsZ level had decreased by 30–40% from the wild-type level (Dai & Lutkenhaus 1991). On the other hand, almost similar levels of FtsZ as in the wild-type cells were detected in the rne-1 hfq::cat mutant strain (Fig. 4B). These results indicate that the hfq::cat mutation enabled translation of the FtsZ protein from unprocessed precursor transcripts without affecting the processing efficiency of ftsZ mRNA.



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  		Figure 4  Effects of the hfq::cat mutation on the processing and translation of ftsZ mRNA. (A) Total cellular RNA was isolated from cells grown at 30, 40.5 and 42 °C for 3 h. ftsZ mRNA was analyzed by Northern hybridization as described in the Experimental procedures. The positions of 16S and 23S rRNAs are indicated on the left side. (B) Total cell lysates were analyzed by Western blotting using anti-FtsZ antibody. The amount of FtsZ protein was estimated by using NIH-image software, and the ratios of wild-type vs. mutants at each temperature are shown beneath the lines. (1) HAT100 (rne+), (2) HAT103 (rne-1), (3) HAT113 (rne-1 hfq10::cat).

 
Overproduction of Hfq protein caused inhibition of cell division

We then examined the effect of overproduction of Hfq on the expression of FtsZ. The Hfq-expression plasmid pHFQ701, in which expression of hfq was under control of the lac promoter, was introduced into JM109. JM109/pHFQ701 could not form colonies on plates containing 0.1 mM IPTG (Fig. 5A). Growth inhibition could be observed without antibiotic selection of plasmid in spite of its negative effect on cell growth. This is probably because plasmid-less cells were rarely produced since inhibition of cell division was caused soon after induction of Hfq. In the presence of 0.1 mM IPTG, filamentous cells with evenly distributed nucleoids were induced (Fig. 5B), indicating that cell division was inhibited. Under these conditions, the amount of intracellular FtsZ decreased to about one-quarter of the wild-type level (Fig. 5C). This means that the overproduction of Hfq inhibited the synthesis of FtsZ.



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  		Figure 5  Inhibition of cell division by overproduction of Hfq protein. (A) Serial dilutions of overnight cultures of JM109 and JM109/pHFQ701 were spotted on L agar plates with or without 0.1 mM IPTG and the plates were incubated at 30 °C for 24 h. (1, 3) JM109, (2, 4) JM109/pHFQ701. (B) Exponentially growing cultures of JM109 and JM109/pHFQ701 were treated with or without 0.1 mM IPTG for 2 h, and cells were observed by fluorescence phase-contrast combined microscopy after DAPI staining. (1) JM109 without IPTG, (2) JM109/pHFQ701 without IPTG, (3) JM109 with 0.1 mM IPTG and (4) JM109/pHFQ701 with 0.1 mM IPTG. (C) Total cell lysates of (B) were analyzed by Western blotting using anti-FtsZ antibody. The amount of FtsZ protein was estimated by using NIH-image software and the ratios of JM109 vs. JM109/pHFQ701 with or without IPTG are shown beneath the lines.

 
Artificial expression of FtsZ protein can suppress temperature-sensitivity of the rne-1 mutant

We demonstrated heretofore that (1) the temperature-sensitive colony forming ability of the rne-1 mutant strain was partially suppressed by the hfq::cat mutation; (2) the hfq::cat mutation restored wild-type levels of FtsZ protein in the rne-1 mutant cells without affecting processing of ftsZ mRNA precursors; (3) overproduction of the Hfq protein caused inhibition of cell division because of decreased expression of FtsZ. Considering these results together, we surmised that inviability of the rne-1 mutant strain is largely due to a decreased level of FtsZ protein. To confirm this, we constructed an artificial FtsZ expression plasmid, in which a portion of the ftsZ gene 3' to the RNase E-cleavage site was fused to the lacUV5 promoter (Fig. 6A). In this plasmid, expression of the FtsZ protein is under the control of the lacUV5 promoter and is expected to be unaffected by RNase E. The constructed plasmid pFZ1 suppressed the temperature-sensitive growth phenotype of the ftsZ84 mutant strain JEFZ1 at the restrictive temperature 42 °C, indicating that the plasmid expressed a functional FtsZ protein. The colony-forming ability of the rne-1 mutant strain was substantially recovered by pFZ1 at 40.5 °C and only slightly at 42 °C (Fig. 6B). Again, pFZ1 could not eliminate the growth rate defect of the rne-1 mutant strain in liquid medium as in the case of hfq::cat (data not shown). The increased synthesis of FtsZ protein in the rne-1 mutant cells by pFZ1 was confirmed by Western blotting using anti-FtsZ antibody (Fig. 6C). JM109/pFZ1 was not able to accept the rne{Delta}1018::bla mutation (data not shown).



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  		Figure 6  Suppression of temperature-sensitive colony formation of the rne-1 mutant by artificially expressed FtsZ protein. (A) Construction of the ftsZ expression plasmid. Zp4, p3 and p2 are ftsZ promoters and plac indicates the lacUV5 promoter. An open triangle represents the RNase E-cleavage site. (B) Suppression of temperature-sensitivity of ftsZ84 and rne-1 mutant strains. Serial dilutions of cultures of (1) JE1011 (ftsZ+) (2) JEFZ1 (ftsZ84) (3) JEFZ1/pMW218 (4) JEFZ1/pFZ1 (5) HAT100 (rne+) (6) HAT103 (rne-1) (7) HAT103/pMW218 (8) HAT103/pFZ1 were spotted onto L agar plates and the plates were incubated at 30, 40.5 and 42 °C for 24 h (1, 2, 3, 4) or 72 h (5, 6, 7, 8). (C) Expression of FtsZ protein from pFZ1 in the rne-1 mutant cells. Total cell lysates were analyzed by Western blotting using anti-FtsZ antibody. Lysates were prepared from (5) HAT100 (rne+) (6) HAT103 (rne-1) (7) HAT103/pMW218 and (8) HAT103/pFZ1, grown at 30, 40.5 and 42 °C for 3 h.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
RNase E is involved in cellular RNA metabolism such as maturation of rRNA and tRNA and degradation of many mRNAs and is essential for E. coli cell growth. In vivo experiments with temperature-sensitive mutant strains carrying the rne-1 and rne-3071 mutations suggested that the inviability associated with the inactivation of RNase E is due to a defect in either 5S rRNA maturation or mRNA decay (Ghora & Apirion 1978; Arraiano et al. 1988; Mackie 1991; Régnier & Hajnsdorf 1991; Babitzke et al. 1993). Recently it has been suggested that tRNA processing is the likely reason for the essentiality of RNase E (Ow & Kushner 2002), but the opposite conclusion was also reported by another group (Deana & Belasco 2004).

Here, we demonstrated that temperature-sensitive colony formation by the rne-1 mutant strain was partially suppressed by introduction of the hfq::cat mutation. It could be argued that the hfq::cat mutation affected the expression of the rne-1 mutant gene, that is, the increased mutant-type RNase E, which might have a residual activity even at the restrictive temperature, could support the cell growth. However, this possibility is low because the hfq::cat mutation did not suppress the deficiency in the maturation process of the 16S rRNA, 5S rRNA and tRNAPhe in the rne-1 mutant. Rather, the amount of precursor molecules in the rne-1 mutant increased with the introduction of the hfq::cat mutation.

It was reported that the polycistronic ftsA-ftsZ transcripts were cleaved by RNase E and that this cleavage affected the decay of ftsA and ftsZ mRNAs (Cam et al. 1996). It was also suggested that this cleavage was required for the efficient expression of the ftsZ gene (Flärdh et al. 1997). In this study, we demonstrated that in the rne-1 mutant strain, actually, the amount of FtsZ protein decreased at the restrictive temperatures (Fig. 4). On the other hand, we reported that the hfq::cat mutant strain overproduced FtsZ protein (Takada et al. 1999). Therefore, we assumed that the Hfq protein is involved in the regulatory function of RNase E on FtsZ production. In the rne-1 hfq::cat double mutant strain, however, unprocessed transcripts were detected at levels as high or even higher than those in the rne-1 mutant. This means that the introduction of the hfq::cat mutation did not rescue the processing of ftsZ mRNA as in the case of maturation of functional RNAs. Western-blot analysis with anti-FtsZ antibody suggested that Hfq plays a role in FtsZ expression at the translational level. In the rne-1 mutant strain, the amount of FtsZ protein decreased markedly at the restrictive temperatures, 40.5 and 42 °C, while it was recovered to levels quite similar to wild-type cells in the rne-1 hfq::cat double mutant strain. These results suggested that the hfq::cat mutation enabled translation of the FtsZ protein from unprocessed precursor transcripts. Thus it was suggested that the inviability of the rne-1 mutant was caused by a decreased level of FtsZ. To confirm this, we examined whether the artificial expression of FtsZ could suppress the temperature-sensitive growth phenotype of the rne-1 mutant strain. As expected, the temperature-sensitivity of the rne-1 mutant was suppressed by introduction of pFZ1, in which expression of the ftsZ gene was under control of the lacUV5 promoter. From these results, we concluded that a decreased level of FtsZ is, at least in part, responsible for inviability of rne-1 mutant cells.

Hfq often regulates gene expression by modulating mRNA stability and/or translation efficiency via binding to small non-coding RNAs. Since it has been reported that Hfq binds to DicF non-coding RNA, which is known to be an inhibitor of FtsZ expression (Zhang et al. 2003), it is possible that Hfq acts on ftsZ mRNA via DicF RNA. However, this possibility is low because the maturation of DicF RNA itself is RNase E-dependent (Faubladier et al. 1990). In the rne-1 mutant cells, functional DicF RNA is not produced.

The amount of FtsZ was decreased by inactivation of RNase E in the hfq+ cells, or by overproduction of Hfq in the rne+ cells. The imbalance of RNase E activity and Hfq activity may cause inhibition of translation of ftsZ mRNA. That is, Hfq inhibits the translation of ftsZ mRNA, for example, by competing with the 30S ribosome binding to the SD sequence (Moll et al. 2003), while RNase E-mediated processing of ftsZ mRNA enhances translation. In wild-type cells, these processes are balanced (see Fig. 7).



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  		Figure 7  Possible mechanism of the regulation of ftsZ expression by Hfq and RNase E. Hfq inhibits the translation of ftsZ mRNA, for example, by competing with the 30S ribosome binding to the SD sequence, while RNase E-mediated processing of ftsZ mRNA enhances translation. In wild-type cells, these processes are balanced. Decrease of RNase E activity or increase of Hfq activity may cause inhibition of translation of ftsZ mRNA.

 
Suppression of the temperature-sensitivity of the rne-1 mutant strain by introduction of the hfq::cat mutation or pFZ1 was only partial at 42 °C. In addition, hfq::cat, as well as pFZ1, did not restore the growth rate to wild-type levels in the rne-1 mutant. This is probably because of a defect in the maturation of functional RNAs such as rRNAs and tRNAs, which was not suppressed by the hfq::cat mutation. It is likely that processing of functional RNAs is also an essential reaction catalyzed by RNase E in E. coli. In fact, complete loss of RNase E activity cannot be suppressed by the hfq::cat mutation or by pFZ1. Therefore, the results demonstrated here indicate that the synthesis of FtsZ protein from its mRNA is one of the most sensitive reactions for viability when RNase E activity is decreased by the rne-1 mutation.


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

E. coli K-12 strain HAT100 (F–ara{Delta}(lac-pro) thi zce-726::Tn10) and its derivatives HAT103 (the same as HAT100 but rne-1), HAT110 (the same as HAT100 but hfq10::cat) and HAT113 (the same as HAT100 but rne-1 hfq10::cat) and JE1011 (F–thr leuB trp his thy thi ara lac gal xyl mtl str tonA) and its derivative JEFZ1 (the same as JE1011 but leuB+ftsZ84) (Jung et al. 1989) were used in this study. HAT100 and HAT103 were constructed by P1 phage mediated transduction using CSH26 [F–ara{Delta}(lac-pro) thi] (Miller 1972) as the recipient and GW20 (rne-1 zce-726::Tn10) (Wachi et al. 1997) as the donor. HAT113 was constructed using HAT103 as the recipient and HAT10 (the same as CSH26 but hfq10::cat) (Wachi et al. 1999b) as the donor. The Hfq-overproducing strain JM109/pHFQ701 was obtained from A. Ishihama (National Institute of Genetics, Mishima, Japan) and the rne deletion strain SK9206 (F–thyA715 {lambda}–rph-1 rne{Delta}1018::bla/pQLK26 rne+; Ow et al. 2000) from S. R. Kushner (University of Georgia, Athens, GA, USA). A 360 bp DNA fragment containing the coding region of hfq and 26 bp 5'- and 28 bp 3'-flanking sequences was cloned into the EcoRI—HindIII site of the expression vector pKK223-3 (a high copy number plasmid with a lac promoter and an ampicillin resistance gene, Pharmacia, Uppsala, Sweden) to produce pHFQ701. In JM109/pHFQ701, expression of Hfq was induced by 0.1 mM IPTG. Cells were grown in L broth containing 1% Bactopeptone, 0.5% yeast extract, 0.5% NaCl and 0.1% glucose (pH 7.0) supplemented with 20 µg/mL thymine. Appropriate antibiotics were added for culturing cells carrying plasmids.

To test for temperature-sensitivity, fully grown E. coli cultures were serially diluted with 0.85% NaCl and spotted onto L agar plates (1.5% agar). Plates were incubated at 30, 40.5 and 42 °C. Cell growth in liquid culture was monitored by measuring OD660. Precultures grown to late log phase in L medium at 30 °C were diluted 1 : 50 into prewarmed L medium at each temperature, and the subsequent increase in turbidity was monitored.

Plasmid construction

To construct pFZ1, DNA fragments containing the ftsZ genes were amplified by polymerase chain reaction (PCR) from chromosomal DNA of CSH26, using a set of primers, 5'-GATGATTACGGCCTCAGTCGACAGG-3' and 5'-CACAAAGAGCGTCGACACCCAAATTC-3', in which engineered SalI sites are underlined. The amplified fragments were digested with SalI and cloned into the SalI site of pMW218 vector (a low copy number plasmid with pSC101 ori and a kanamycin resistance gene, Nippon Gene, Toyama, Japan), generating pFZ1.

RNA analysis

Total cellular RNA was isolated and analyzed by modified agarose gel electrophoresis and Northern hybridization as previously described (Wachi et al. 1999a; Takada et al. 1999). Cells were incubated at 30, 40.5 and 42 °C for 3 h (OD660 = about 1). E. coli cultures were centrifuged and washed with ice-cold TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Cells were suspended in 100 µL of TE and directly extracted by vigorously vortexing with an equal volume of equilibrated phenol/chloroform. After centrifugation (10 000 g, 10 min), a portion of the upper layer was used for analysis. For 16S rRNA analysis, RNA samples were fractionated on a modified agarose gel containing 0.7% agarose and 0.9% Synergel gel modifier (Diversified Biotech, Boston, MA, USA) in 0.5 x TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0). rRNA bands were visualized by staining with 1 µg/mL ethidium bromide solution. For 5S rRNA and tRNA analysis, RNAs were fractionated on 6% polyacrylamide gels in TAE (40 mM Tris-acetate, 1 mM EDTA pH 8.0). For ftsZ mRNA analysis, RNAs were fractionated by formaldehyde-agarose gel electrophoresis (6.5% formaldehyde, 1 x MOPS buffer [20 mM 3-(N-morpholino)-propane sulfonic acid pH 7.0, 5 mM sodium acetate, 1 mM EDTA], 1% agarose). For Northern hybridization, RNAs were transferred onto positively charged nylon membranes (Hybond-N+, Amersham Bioscience, Piscataway, NJ, USA) by electroblotting (5S rRNA and tRNA) or capillary method (mRNA). To generate hybridization probes, DNA fragments were amplified by PCR using primer sets of 5'-ACGCCGAAGCTGTTTTGGCGGATGA-3' and 5'-TTGTCCTACTCAGGAGAGCGTTCAC-3' for 5S rRNA, 5'-ACATCGGCTGGCGGAAGATATCCTG-3' and 5'-CGCTTAAATCGTGGCGTCCTGAAAC-3' for tRNAPhe and 5'-GAACTTACCAATGACGCGGTGATTAAA-3' and 5'-TTAATCAGCTTGCTTACGCAGGAATGCTGG-3' for ftsZ. Hybridization was performed using a Gene Images Kit (Amersham Bioscience).

Western blotting

Cells suspended in sodium phosphate buffer (50 mM pH 7.0) were disrupted by sonication. Cell lysates were separated on SDS-polyacrylamide gels. Proteins were electroblotted on to nitrocellulose membranes (Hybond-C, Amersham Bioscience). FtsZ protein was detected by using polyclonal anti-FtsZ antiserum (mouse) as the primary antibody and alkaline phosphatase-conjugated anti-mouse IgG antibody (Bio-Rad Laboratories, Hercules, CA, USA) as the secondary antibody. Detection was carried out using a Gene Images Kit (Amersham Bioscience). The amount of FtsZ was estimated by measuring band intensity using the NIH-image software after two-fold serial dilutions.

Microscopic observation

Cells spread on a slide glass were treated with methanol for 5 min and covered with poly-L-lysine. The fixed cells were stained with a 4',6-diamidino-2-phenylindole (DAPI) solution (5 µg/mL in saline) and then observed by the fluorescence phase-contrast combined method through an Axioskop 2 microscope (Carl Zeiss Co., Ltd, Oberkochen, Germany).


   Acknowledgements
 
The authors thank Drs A. Ishihama and S. R. Kushner for providing bacterial strains. This work was supported in part by Grants-in-Aid for Scientific Research (B) (15380059 to M. W.), Grants-in-Aid for Encouragement of Young Scientists (17920007 to A. T.) from the Japan Society for the Promotion of Science, the Grant of the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science & Technology of Japan & the 2003 Research Grant from the Noda Institute for Scientific Research (Noda, Japan).


   Footnotes
 
Communicated by: Hiroji Aiba

* Correspondence: E-mail: mwachi{at}bio.titech.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 28 February 2005
Accepted: 13 April 2005




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