RimM and RbfA Are Essential for Efficient Processing of 16S rRNA in Escherichia coli

Abstract

The trmD operon is located at 56.7 min on the genetic map of the Escherichia coli chromosome and contains the genes for ribosomal protein (r-protein) S16, a 21-kDa protein (RimM, formerly called 21K), the tRNA (m¹G37)methyltransferase (TrmD), and r-protein L19, in that order. Previously, we have shown that strains from which the rimM gene has been deleted have a sevenfold-reduced growth rate and a reduced translational efficiency. The slow growth and translational deficiency were found to be partly suppressed by mutations in rpsM, which encodes r-protein S13. Further, the RimM protein was shown to have affinity for free ribosomal 30S subunits but not for 30S subunits in the 70S ribosomes. Here we have isolated several new suppressor mutations, most of which seem to be located close to or within the nusA operon at 68.9 min on the chromosome. For at least one of these mutations, increased expression of the ribosome binding factor RbfA is responsible for the suppression of the slow growth and translational deficiency of a ΔrimM mutant. Further, the RimM and RbfA proteins were found to be essential for efficient processing of 16S rRNA.

The trmD operon, located at min 56.7 on the molecular genetic map of the Escherichia coli chromosome (23), contains the genes for ribosomal protein (r-protein) S16 (rpsP), RimM (a 21-kDa protein formerly called 21K) (rimM, previously called 21K and yfjA), the tRNA(m¹G37)methyltransferase (or TrmD) (trmD), and r-protein L19 (rplS), in that order (8) (Fig. 1). The RimM and TrmD proteins are found in 12- and 40-fold-lower amounts, respectively, than the two r-proteins (47). This difference in expression is due to translational-level regulation (9, 47) by sequestering of the Shine-Dalgarno sequences and start codons of the rimM and trmD genes in mRNA secondary structures that prevent access of the translational-initiation regions to the ribosomes (48, 50).

FIG. 1.

Genetic organization of the trmD operon region of the chromosome of the ΔrimM-2 mutant MW37 and the congenic rimM⁺ strain MW38. P and T indicate the promoter and terminator, respectively, of the trmD operon. The arrows above and below the genes for Ffh (named Ffh for fifty-four homolog of the 54-kDa protein of the signal recognition particle) and a 16-kDa protein designated 16K, respectively, show the orientations of the transcripts and not their actual sizes. The Km^r gene derived from transposon Tn903 was previously inserted into the gene for the nonessential 16K protein (33).

All the genes in the trmD operon encode proteins that are involved in translation. Strains from which the chromosomal rpsP gene copy (for S16) has been deleted are nonviable in the absence of complementing gene copies (33), possibly because S16 is essential for the assembly of the 30S ribosomal subunits (13). Recently, S16 has been found to be an endonuclease also (30). r-protein L19 is essential for the viability of wild-type E. coli cells (33); however, compensatory mutations can rescue L19-lacking mutants (33, 42). Also, L19 seems important for ribosome assembly, since a reduction in the synthesis of L19 due to a polar insertion in rimM resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit (33). The tRNA(m¹G37)methyltransferase modifies the guanosine in position 37 next to the anticodon of a subset of the tRNAs in E. coli and Salmonella typhimurium (see references 3 and 4), and the modification is important for maintaining the correct reading frame during translation (5, 12). In addition, strains from which the trmD gene has been deleted have at least a fivefold-reduced growth rate in rich medium (33). Similarly, strains lacking the RimM protein show a five- to sevenfold-reduced growth rate, depending on the growth medium used (7, 33). Recently, it has been found that mutants lacking the RimM protein show reduced translational efficiency. In agreement with a role in translation, the RimM protein shows affinity for the 30S ribosomal subunits (7). The slow growth and translational deficiency of a ΔrimM mutant can be partially suppressed by mutations in rpsM, which encodes r-protein S13 (7). In the present study we have isolated and characterized 26 additional suppressor mutations that increase the growth rate of a ΔrimM mutant, 23 of which seem to be located within or close to the nusA operon at 68.9 min on the chromosome. We demonstrate that at least one of these suppressor mutations increases expression of the ribosome binding factor RbfA (10). Moreover, the ΔrimM mutation was suppressed by an increased gene dosage of rbfA, suggesting that the mechanism behind the suppression was an increased synthesis of RbfA. Furthermore, mutants lacking either RimM or RbfA showed reduced efficiency in the processing of 16S rRNA, implying that both proteins are important for the maturation of the ribosomal 30S subunits.

MATERIALS AND METHODS

Strains, phages, and plasmids.

Strains, phages, and plasmids used are listed in Table 1. Strain GOB162 was constructed by transducing strain MW100 with phage P1 grown on strain CD28 and selecting for Km^r.

TABLE 1.

Bacterial strains, bacteriophages, and plasmids

Strain, phage, or plasmid	Genotype	Source or referencea
Strains
ORN103	thr-1 leuB thi-1 Δ(argF-lac)U169 malA1 xyl-7 ara-13 mtl-2 gal-6 rpsL tonA2 minA minB recA13 Δpil	32
CD28	F−ara Δ(gpt-lac)5 rbfA::kan	10
GOB007	Hfr P4X ΔrimM-2 16K::nptI sdr-43 truB2422::mini-Tn10Cm
GOB083	Hfr P4X sdr-43 truB2422::mini-Tn10Cm
GOB113	Hfr P4X sdr+ truB2422::mini-Tn10Cm
GOB162	Hfr P4X rbfA::kan
MW37	Hfr P4X ΔrimM-2 16K::nptI	33
MW38	Hfr P4X 16K::nptI	33
MW100	Hfr P4X	49
PW098	Hfr P4X ΔrimM-2 16K::nptI sdr-32
PW100	Hfr P4X ΔrimM-2 16K::nptI sdr-34
PW101	Hfr P4X ΔrimM-2 16K::nptI sdr-35
PW105	Hfr P4X ΔrimM-2 16K::nptI sdr-39
PW107	Hfr P4X ΔrimM-2 16K::nptI sdr-41
PW109	Hfr P4X ΔrimM-2 16K::nptI sdr-43
PW119	Hfr P4X ΔrimM-2 16K::nptI sdr-53
PW121	Hfr P4X ΔrimM-2 16K::nptI sdr-55
Bacteriophages
λ439/λ22D7	rimM+	18
λ439ΔrimM-2	ΔrimM-2 16K::nptI	33
P1vir		Laboratory stock
Plasmids
pCL1921	Strr Spcr	20
pGOB3	pCL1921-′infB rbfA+ truB2422::mini-Tn10Cm rpsO+ pnp+
pGOB7	pCL1921-′truB rpsO+ pnp+
pGOB8	pCL1921-′infB rbfA+ truB2422::mini-Tn10Cm’
pGOB18	pCL1921-′infB rbfA+
pGOB19	pCL1921-′infB rbfA+
pGOB22	pCL1921-′infB rbfA+

^aUnless otherwise noted, the source was this study. 

Plasmid constructions.

Plasmid pGOB3 was constructed by cloning KpnI-digested chromosomal DNA isolated from strain GOB083 (sdr-43 truB2422::mini-Tn10Cm) into the low-copy-number vector pCL1921 and selecting for Cm^r conferred by the mini-Tn10Cm linked to sdr-43. Plasmids pGOB7 and pGOB8 were constructed by digesting pGOB3 with BamHI and EcoRI, respectively, and religating (see Fig. 3). To construct pGOB18, a fragment carrying the rbfA gene and its tentative promoter was amplified from strain GOB083 by PCR using the oligonucleotides 5′-TTTTGTCGACAGAACTACAACGACGTCC-3′ and 5′-TTTTGGATCCTGAGGTTTATCCAGCAAC-3′ (containing restriction sites for SalI and BamHI, respectively), digested with SalI and BamHI, and cloned into pCL1921. Plasmid pGOB19 was constructed in a similar way except that the SalI site included in the first oligonucleotide was replaced by an EcoRI site.

FIG. 3.

Suppression of the slow growth of a ΔrimM-2-containing mutant by plasmids that carry different parts of the nusA operon. The suppression level was judged after single-cell outstreaks on rich-medium plates. Plasmid pGOB18 conferred stronger suppression in the presence of 0.25 mM IPTG than in the absence of IPTG. IF2, translational initiation factor IF2; TruB, tRNA Ψ55 synthase; PNP, polynucleotide phosphorylase. Restriction sites used in the plasmid constructions: B, BamHI; E, EcoRI; K, KpnI; S, SalI. +, suppression; −, no suppression.

Media and growth conditions.

The minimal medium used was morpholinepropanesulfonic acid (MOPS) (28) supplemented with 0.4% glucose. Rich medium was either rich MOPS (27) or Luria-Bertani (LB) medium (2) supplemented with medium E plus thiamine (45) and 0.4% glucose. Cultures were grown at 37°C, and the growth was monitored either on a Zeiss PMQ3 spectrophotometer at 420 or 600 nm or on a Klett-Summerson colorimeter equipped with a red filter.

P1 and λ transduction.

Standard procedures were used for generalized transduction with P1 (24). Transduction of different recipient strains to Km^r by λ439ΔrimM-2 was carried out mainly as described by Kulakauskas et al. (19).

Construction of Tn10 libraries.

Libraries of mini-Tn10Cm insertions were constructed as described elsewhere by using λNK1324 (17).

PCR amplification of chromosomal DNA and DNA sequencing.

Regions of the E. coli chromosome were amplified from colonies resuspended in H₂O by PCR (26, 37). Pfu DNA polymerase from Stratagene (La Jolla, Calif.) was used if the fragments obtained were to be cloned into plasmids, and Taq DNA polymerase from Boehringer Mannheim Scandinavia AB (Bromma, Sweden) was used in all other cases. Fragments obtained were separated on agarose gels, cut out, and purified with Gene Clean from Bio 101 Inc. (La Jolla, Calif.). DNA sequencing of PCR fragments was carried out with Thermo Sequenase as described by Amersham Life Science, Inc. (Cleveland, Ohio), whereas plasmid DNA sequencing was carried out with a T7 sequencing kit purchased from Pharmacia Biotech (Uppsala, Sweden).

Determination of polypeptide chain growth rate.

The polypeptide chain growth rate (cgr_p) of β-galactosidase was determined by measuring the time necessary for the first β-galactosidase activity to appear after induction (the delay time) with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) essentially as described by Schleif et al. (40). Cells were grown in MOPS medium to an optical density at 420 nm of 0.5, at which point IPTG was added. Samples (0.5 ml) were withdrawn at intervals of 5 or 10 s and transferred to tubes on ice containing 0.5 ml of Z buffer (24) supplemented with 200 μg of chloramphenicol/ml, 0.005% sodium dodecyl sulfate, and 50 μl of chloroform. The β-galactosidase activity in the samples was determined according to the method of Miller (24).

Northern blot analysis.

Total RNA was prepared according to the work of von Gabain et al. (46) and subjected to Northern blot analysis mainly as described by Sambrook et al. (38). Equal amounts of the RNA from the different strains were loaded onto Northern gels as determined both by spectrophotometric measurements at 260 nm and by ethidium bromide staining of aliquots of the RNA electrophoresed on agarose gels. DNA fragments used as probes were purified as described above and labeled with [α-³²P]dATP by using the Megaprime DNA labeling system from Amersham Life Science, Inc.

Analysis of proteins by 2D gel electrophoresis.

Steady-state cultures of bacterial cells were grown at 37°C to an optical density at 600 nm of 0.25 and then shifted to 15°C. Just prior to the shift, 1-ml aliquots of the cultures were labeled for 15 min with 250 μCi of [³⁵S]methionine each (>1,000 Ci/mmol) and chased with 0.167 ml of 0.2 M methionine for 3 min. Similarly, 1-ml aliquots withdrawn 30 min after the temperature shift were labeled for 30 min and chased for 6 min. Extracts were prepared mainly as described previously (44). O’Farrell two-dimensional (2D) polyacrylamide gels (31) were used to analyze the protein expression pattern. One million counts per minute was loaded onto each first-dimension isoelectric focusing gel containing ampholines 3 to 10 and Duracryl acrylamide from Oxford Glycosystems. The first dimension was run as described by Millipore Intertech (Bedford, Mass.), whereas the second dimension was 10 to 17.5% gradient polyacrylamide slab gels containing sodium dodecyl sulfate. The gels were dried and exposed to X-ray film, and the autoradiographs obtained were analyzed by the Bio Image 2-D Analyzer, version 6.0.3, from B.I. Systems Corporation.

Expression of rbfA in minicells.

The minicell-producing strain AA10 was transformed with plasmid pGOB18 containing the rbfA gene. Preparation and labeling of plasmid-containing minicells were carried out essentially as described previously (16, 43).

Primer extension on rRNA.

Total RNA was prepared either with the Total RNA Kit from Qiagen GmbH (Hilden, Germany) or according to the work of von Gabain et al. (46). Two micrograms of the RNA was subjected to primer extension with 2 pmol of a ³²P-end-labeled primer specific for 5S rRNA (5′-GGCGTTTCACTTCTGAG-3′), 16S rRNA (5′-CGACTTGCATGTGTTAGG-3′), or 23S rRNA (5′-CGTCCTTCATCGCCTCTG-3′) by using avian myeloblastosis virus reverse transcriptase from Pharmacia Biotech (Sollentuna, Sweden). Small aliquots of the reaction mixtures (1:100 to 1:500) were run next to a DNA sequencing ladder on 6% polyacrylamide gels containing 8 M urea. The gels were dried and exposed to Hyperfilm-MP from Amersham International plc (Buckinghamshire, England). The autoradiographs obtained were scanned with a ScanMaker III from Microtek International, Inc., and processed with Adobe Photoshop 3.0. The gels were also exposed to a phosphor screen, and the amounts of radioactivity in the primer extension products were quantified by using ImageQuant from Molecular Dynamics, Inc.

RESULTS

New suppressor mutations increase the growth rate of the ΔrimM-2 mutant up to fourfold.

The growth rate of the ΔrimM-2 mutant MW37 is 4.4-fold lower in LB medium and 7-fold lower in MOPS minimal medium containing 0.4% glucose than that of the congenic rimM⁺ strain MW38 (7). In an attempt to isolate mutations that suppressed the slow growth of strain MW37, 19 single colonies of strain MW37 were grown in LB overnight at 37°C, reinoculated from the overnight cultures into fresh medium, and incubated again. This procedure was repeated for 3 to 5 days, and samples were withdrawn from each overnight culture and streaked onto rich-medium plates to examine if any faster-growing derivatives had arisen. In this way, 29 independent suppressor-containing mutants (PW093 to PW121) were isolated; from some of the original cultures, more than one class of mutants was obtained, as judged by differences in their growth rates. That the isolated clones indeed were derivatives of the ΔrimM-2 mutant was confirmed by PCR analysis using primers specific for the DNA sequences flanking the deletion (data not shown). Three of the mutations were shown to be in rpsM, encoding r-protein S13, as presented in a separate report (7).

To quantify the efficiency of the suppressor mutations, the steady-state growth rate in rich medium was determined for 28 of the isolated suppressor-containing clones. The strongest suppressor mutation, sdr-43 (named sdr for suppressor of deletion of rimM), increased the growth rate 2.6-fold (strain PW109), resulting in a growth rate which was 60% of that of the rimM⁺ strain MW38, while the weakest suppressor, sdr-41 (strain PW107), increased the growth rate 1.4-fold (Fig. 2). Further, the growth rate of strain PW109 in MOPS minimal medium containing 0.4% glucose was fourfold higher than that of strain MW37 (data not shown).

FIG. 2.

Relative cell yields and growth rates for wild-type and mutant strains. The cell yields for the different strains grown in MOPS minimal medium were calculated as ΔA₄₂₀/Δ(glucose concentration, expressed as a percentage). The glucose concentrations used were 0.005 to 0.1%. The growth rates are for growth in LB medium. Both the cell yield values and the growth rates have been normalized to those for strain MW38.

The suppressor mutations improve the energy utilization efficiency and translational-elongation rate of the ΔrimM-2 mutant.

Previously, we have shown that strains lacking RimM seem to have reduced energy utilization efficiency, as demonstrated by a lower stationary-phase cell density at a given concentration of carbon source in the growth medium, and that mutations in rpsM, coding for r-protein S13, suppress this deficiency (7). To see if some of the other suppressor mutations isolated here also altered the energy utilization efficiency, the stationary-phase cell density was determined for eight of the suppressor strains grown in MOPS minimal medium containing different amounts of glucose (Fig. 2). All of them had a higher ratio of cell yield to carbon source concentration than the suppressor-free strain MW37, suggesting that the new suppressor mutations increased the energy utilization efficiency. Since translation is the single most energy-consuming process in the cell, these results suggested that the suppressor mutations increased the translational proficiency of the ΔrimM-2 mutant. To test this, we measured the cgr_p of β-galactosidase for two of the ΔrimM-2-containing suppressor strains. As shown in Table 2, strains PW098 (ΔrimM-2 sdr-32) and PW109 (ΔrimM-2 sdr-43) had a higher cgr_p of β-galactosidase than did strain MW37 (ΔrimM-2). In conclusion, at least two of the mutations isolated here as suppressors of the slow growth of the ΔrimM-2 strain MW37 suppressed the translational deficiency of the ΔrimM-2 mutant.

TABLE 2.

β-Galactosidase cgr_p’s of mutant and wild-type strains

Strain	Relevant marker(s)	cgrp (no. of amino acids per s)a
MW38	rimM+	12.0 (11.1–12.8)
MW37	ΔrimM-2	8.6 (8.5–8.7)
PW098	ΔrimM-2 sdr-32	10.4 (9.7–11.1)
PW109	ΔrimM-2 sdr-43	10.9 (10.7–11.1)

^aThe cgr_p of β-galactosidase in MOPS minimal medium supplemented with 0.4% glucose was measured as described by Schleif et al. (40). 

The slow growth of the ΔrimM-2 mutant MW37 is suppressed by increased gene dosage of rbfA.

In order to localize one of the suppressor mutations, a library of mini-Tn10Cm insertions was constructed on the sdr-43-containing strain PW109 and several clones with a mini-Tn10Cm linked to sdr-43 were identified by transducing strain MW37 (ΔrimM-2) with phage P1 grown on the library, selecting for Cm^r, and screening for growth faster than that of strain MW37. One clone that had a mini-Tn10Cm 95% linked to sdr-43, as demonstrated by backcrosses to MW37, was used in further studies (strain GOB007). The cotransduction frequency implied that sdr-43 was approximately 1.6 kb from the mini-Tn10Cm. Therefore, the mini-Tn10Cm was cloned together with the flanking chromosomal DNA into the low-copy-number vector pCL1921, selecting for Cm^r. The resultant clone, pGOB3, was shown by DNA sequencing to contain genes from the 68.9-min region of the chromosome. The sequence immediately upstream from the mini-Tn10Cm corresponded to codon 170 of truB (Fig. 3), and the insertion is hereafter designated truB2422::mini-Tn10Cm. Plasmid pGOB3 was found to suppress the slow growth of the ΔrimM-2 mutant (Fig. 3). By subcloning, it appeared that the rbfA gene (encoding ribosome binding factor A) just upstream of truB was responsible for the observed suppression (pGOB8). The rbfA gene is the fifth gene in the polycistronic nusA operon, starting with the metY gene, and has been suggested to be expressed from an internal promoter located 170 bp upstream of rbfA (39). The region containing rbfA and its tentative promoter was PCR amplified from strain GOB083 (sdr-43 truB2422::mini-Tn10Cm) and cloned into the low-copy-number vector pCL1921. The resultant plasmid clone, pGOB18, with rbfA in the same orientation as the lac promoter of the vector, suppressed the slow growth of the ΔrimM-2 mutant, whereas plasmid pGOB19, with rbfA in the opposite orientation, did not mediate suppression (Fig. 3). The suppression by pGOB18 was stronger when IPTG was added to the medium, which indicated that expression of rbfA from the lac promoter in the plasmid was responsible for the observed suppression. Further, these findings also imply that the suggested internal promoter for rbfA is not strong enough to mediate suppression. To see if there was any mutation in rbfA that could explain the observed suppression, the chromosomal region corresponding to the insert in pGOB18 was sequenced from sdr-43 as well as sdr⁺ strains after PCR amplification and cloning into pUC119. To our surprise, there was no mutation in that part of the chromosome. Therefore, we cloned the similar fragment from the wild-type strain MW100 into pCL1921 and found that the resulting plasmid, pGOB22, also suppressed the slow growth of the ΔrimM-2 mutant in an IPTG-dependent manner (data not shown). These results strongly suggest that the suppression observed for the plasmid-containing strains was due to increased expression of the wild-type rbfA gene.

To see if any of the suppressor mutations other than sdr-43 was linked to rbfA, the ability of the regions flanking truB2422::mini-Tn10Cm in the suppressor-free strain GOB113 to cross out the other suppressor mutations was examined. Phage P1 was grown on strain GOB113, and the different suppressor strains (PW093 to PW121) were transduced with the obtained lysate, selecting for Cm^r. The growth of the obtained transductants was examined by single-cell outstreaks on rich-medium plates. For 23 of the 29 strains tested, a majority of the transductants showed the slow-growth phenotype characteristic for the suppressor-free ΔrimM-2 mutant, indicating that the regions containing the suppressor mutations had been replaced by the corresponding wild-type region from the donor strain. Thus, this result demonstrates that the suppressor mutations in 23 of the suppressor strains were tightly linked to rbfA.

The suppressor mutation sdr-43 increases the amount of rbfA-specific mRNA.

The observed multicopy suppression by rbfA implied that the chromosomally located mutation sdr-43 of strain PW109 suppressed slow growth and translational deficiency by increasing expression of rbfA. Since there was no mutation in rbfA or in the 239 bp preceding rbfA in strain PW109, we reasoned that the suppressor mutation was not likely to have increased the translational efficiency of rbfA but would be a mutation increasing the synthesis or stability of the rbfA mRNA. Therefore, the amounts of rbfA mRNA in different strains were measured by Northern blot analysis using a probe corresponding to the rbfA gene (probe B in Fig. 4A). Evidently, strains PW109 (sdr-43 ΔrimM-2) and GOB083 (sdr-43 rimM⁺) had severalfold-increased levels of one rbfA-specific mRNA species of 2.6 to 3 kb (Fig. 4B). The size of this mRNA species was difficult to assess due to the proximity on the gels of the abundant 23S rRNA, which distorted the migration pattern. Similar results were obtained when a DNA fragment specific for the 3′ half of the rbfA gene was used as a probe (data not shown). Two mRNA species showed increased levels in the sdr-43-containing strains PW109 and GOB083 relative to those in the sdr⁺ strains (Fig. 4C) when the mRNA was probed with a fragment corresponding to the region upstream from a transcriptional terminator preceding the rbfA gene (probe C in Fig. 4A). The size of the longer of these two mRNAs corresponded to that of the mRNA also detected with probe B, whereas the shorter mRNA (approximately 900 nucleotides [nt]) was specific for probe C. These findings suggest that both mRNAs have their 5′ ends within the infB gene preceding rbfA and that the longer mRNA covers the rbfA gene, while the shorter is the result of termination at the transcriptional terminator just upstream of rbfA. When the mRNA was probed with the region corresponding to p15a, the second gene of the nusA operon (probe D in Fig. 4A), two mRNA species of approximately 6.7 and 4.8 kb were detected (Fig. 4D). Note that the exposure times in Fig. 4B and C were shorter than that in Fig. 4D in order to avoid overexposure of the two bands corresponding to the mRNAs which had dramatically increased levels in strains PW109 and GOB083. With longer exposure times, the 4.8-kb mRNA was also detected with probe C, and the 6.7-kb mRNA was detected with both probes B and C (data not shown). Probably, both the 6.7- and the 4.8-kb mRNAs start at the RNase III processing site upstream of p15a. The apparent lengths of the mRNAs and the results with probes B and C suggest that the shorter mRNA terminates at the transcriptional terminator preceding rbfA, while the longer also contains the rbfA and truB genes. The amounts of the 6.7- and 4.8-kb mRNAs were not higher in strains containing sdr-43 (PW109 and GOB083) than in the sdr⁺ strains (Fig. 4D), indicating that sdr-43 increased the amounts of only promoter-distal parts of the operon mRNA. Interestingly, strain PW100, which contains sdr-34 (one of the other suppressor mutations that are tightly linked to the rbfA gene) showed higher levels of the 6.7-kb, and possibly also of the 4.8-kb, mRNA species than strain PW109 (Fig. 4D). These two strains have similar growth rates, so the comparison seems relevant, whereas a comparison to the other strains might be obscured by secondary effects caused by their growth rate differences. (Strain MW37 grows threefold slower and strains MW38, GOB083, and GOB113 grow twofold faster than PW100 and PW109.) The 6.7-kb transcript was not detected in strains GOB083 and GOB113 because the mini-Tn10Cm insertion in truB probably results in premature termination of transcription.

FIG. 4.

Northern blot analysis of nusA operon mRNA. (A) Genetic organization of the nusA operon. Abbreviations: B, C, and D, probes used in the experiments for which results are shown in panels B, C, and D, respectively; R III and R E, processing sites for RNase III (34, 35) and RNase E (25, 36), respectively; (B through D) P, promoter; T, terminator. Five micrograms of total RNA was subjected to electrophoresis in an agarose gel containing formaldehyde, transferred to a Hybond N filter, and probed with a radiolabeled PCR fragment (probe D). The probe was removed by washing, and the filter was reprobed twice (probes C and B). The exposure times in the experiments for which results are shown in panels B and C were shorter than those in the experiment for which results are shown in panel D in order to avoid overexposure of the bands in strains PW109 and GOB083. The sizes of the γ-³²P-labeled ATP kinase-treated fragments of the 1-kb DNA ladder from GIBCO BRL Life Technologies Inc. (Gaithersburg, Md.) are indicated. The strains used (with the relevant genetic markers in parentheses) were MW38 (rimM⁺), MW37 (ΔrimM-2), PW109 (ΔrimM-2 sdr-43), PW100 (ΔrimM-2 sdr-34), GOB113 (rimM⁺ sdr⁺), and GOB083 (rimM⁺ sdr-43).

In summary, two mutations that increase the translational proficiency of the ΔrimM-2 mutant MW37 and are tightly linked to the rbfA gene increase the amounts of rbfA-specific mRNA species. One of the mutations, sdr-43, increases the amount of a 2.6- to 3-kb mRNA that starts within infB, whereas the other mutation, sdr-34, increases the amount of a 6.7-kb mRNA that probably starts upstream of p15a. Since both an increased gene copy number of rbfA and an increased amount of its mRNA were found to suppress the slow growth and translational deficiency of the the ΔrimM-2 mutant, it seems likely that increased synthesis of the RbfA protein was responsible for the suppression.

The ΔrimM-2 mutant MW37 has a normal level of the RbfA protein.

To distinguish between the possibility that the RimM protein was needed for the expression of rbfA and the possibility that increased expression of rbfA somehow compensated for the lack of the RimM protein, we decided to identify RbfA on 2D protein gels. Since RbfA is a cold shock protein (15), bacterial cultures were labeled with [³⁵S]methionine before or 30 min after a shift in temperature from 37 to 15°C, and prepared total protein extracts were separated on 2D gels. The levels of known cold shock-induced proteins, such as CspA, CspG, and H-NS, increased upon the shift to the lower temperature (Fig. 5). One protein spot that increased in intensity was putatively identified as RbfA based on a comparison of its position on our gels with that of a protein spot previously identified as RbfA (15). The protein spot was unambiguously identified as RbfA by addition of a [³⁵S]methionine-labeled minicell extract of a strain expressing rbfA from plasmid pGOB18 (Fig. 3) to a total protein extract of the rbfAKm^r mutant GOB162 labeled after a shift from 37 to 15°C (Fig. 6). In strains MW38 (rimM⁺) and MW37 (ΔrimM-2), the amount of RbfA at 37°C was below detection level; however, at 15°C, it seemed comparable in the two strains (compare Fig. 5B and 7C). In fact, the levels of RbfA relative to those of total protein were 0.56 and 0.45% in strains MW38 and MW37, respectively, as demonstrated by scanning and analysis of the autoradiographs shown (data not shown). This demonstrates that the RimM protein is not needed for the expression of rbfA. Moreover, in the suppressor strain PW109, the level of RbfA was severalfold higher than that in the suppressor-free strain MW37 at both 15 and 37°C (Fig. 7). The amount of RbfA at 15°C relative to that of total protein was found to be approximately sixfold higher in the suppressor strain PW109 than in strain MW37 (data not shown). In conclusion, the translational deficiency and slow growth of the ΔrimM-2 mutant MW37 do not result from reduced levels of RbfA; however, the observed suppression in strain PW109 results from overexpression of rbfA.

FIG. 5.

Syntheses of individual proteins at 37°C and after a shift in temperature to 15°C. Total cell extracts of strain MW38 (rimM⁺ rbfA⁺) labeled with [³⁵S]methionine just prior to (A) and 30 min after (B) the shift to 15°C were separated on 2D gels. The indicated proteins are as follows (protein labels shown in parentheses): proteins CspG (1), CspA (2), and H-NS (3) and r-proteins S6 (4 and 5), L12 (6), and L7 (7). The identities of these proteins were obtained by comparing our gels with those of Fang et al. (11) and Jones and Inouye (14). A protein putatively identified as RbfA is indicated by a circle.

FIG. 6.

Identification of RbfA on 2D gels. (A) A total cell extract of strain GOB162 (rbfA::Km^r) labeled with [³⁵S]methionine 30 min after a shift in temperature from 37 to 15°C was separated on 2D gels. (B) A [³⁵S]methionine-labeled minicell extract of strain AA10, expressing rbfA from plasmid pGOB18, was added to the total extract. The indicated proteins are as follows (protein labels shown in parentheses): proteins CspG (1), CspA (2), and H-NS (3) and r-proteins S6 (4 and 5), L12 (6), and L7 (7). The position of RbfA is indicated by a circle.

FIG. 7.

Syntheses of individual proteins at 37°C and after a shift in temperature to 15°C. Strains MW37 (ΔrimM-2) and PW109 (ΔrimM-2 sdr-43) were labeled with [³⁵S]methionine just prior to and 30 min after the shift to 15°C. (A) MW37 at 37°C; (B) PW109 at 37°C; (C) MW37 at 15°C; (D) PW109 at 15°C. The indicated proteins are as follows (protein labels shown in parentheses): proteins CspG (1), CspA (2), and H-NS (3) and r-proteins S6 (4 and 5), L12 (6), and L7 (7). The position of RbfA is indicated by a circle.

The ΔrimM-2 mutant MW37 is deficient in the processing of 16S rRNA.

RbfA has been shown to act as a multicopy suppressor of a mutation in the 5′-terminal helix of 16S rRNA, which causes cold sensitivity (10). RbfA has also been found associated with free 30S ribosomal subunits but not with 70S ribosomes and has been suggested to interact with the 5′-terminal helix of 16S rRNA during maturation of the 30S subunits (10). Therefore, we wanted to learn if the ΔrimM-2 mutant MW37 had a defect in the maturation of 16S rRNA. Primer extension reactions were run on total RNA from different strains by using primers binding downstream from the 5′ ends of mature 5S, 16S, and 23S rRNA. In the rimM⁺ strain MW38, most of the rRNA had 5′ ends corresponding to mature rRNA (Fig. 8). However, in the ΔrimM-2 mutant MW37, approximately 50% of the 16S rRNA molecules had not been processed completely (Fig. 8; Table 3) and were found in a precursor form corresponding to the product of cutting at the RNase III site 115 nt upstream of the 5′ end of mature 16S rRNA. We examined if in addition, RbfA was required for proper processing of rRNA. Evidently, the rbfA::Km^r mutant GOB162 showed the same dramatically elevated levels of the precursor form of 16S rRNA as did the ΔrimM-2 mutant MW37 (Fig. 8; Table 3). Thus, both RimM and RbfA seem important for the maturation of 16S rRNA. However, the increased expression of rbfA in the suppressor strain PW109 seemed to increase the efficiency of processing of 16S rRNA only slightly (Fig. 8; Table 3).

FIG. 8.

Primer extension analysis of the 5′ ends of 5S, 16S, and 23S rRNA in wild-type and mutant strains. Lanes 1, strain MW38 (rimM⁺ rbfA⁺); lanes 2, strain MW37 (ΔrimM-2 rbfA⁺); lanes 3, strain PW109 (ΔrimM-2 sdr-43 rbfA⁺); lanes 4, strain GOB162 (rimM⁺ rbfA::Km^r). The sizes of the primer extension products obtained were determined by comparing the mobilities with those of a known DNA sequencing ladder. (A) RIII indicates a primer extension product of 179 nt corresponding to pre-16S rRNA processed at the RNase III site 115 nt upstream of the 5′ end of mature 16S. M indicates a product of 64 nt corresponding to the 5′ end of mature 16S rRNA. (B) M indicates a primer extension product of 63 nt corresponding to mature 5S rRNA. (C) RIII indicates a primer extension product of 65 nt corresponding to pre-23S rRNA processed at the RNase III site 7 nt upstream of the 5′ end of mature 23S. M indicates a product of 58 nt corresponding to the 5′ end of mature 23S rRNA.

TABLE 3.

Efficiency of 16S and 23S rRNA processing in different strains

Strain	Amt of rRNAa
	16S						23S
	RIII (106 cpmb)		M (106 cpmb)		M/(RIII + M)		RIII (106 cpmb)		M (106 cpmb)		M/(RIII + M)
	Expt I	Expt II	Expt I	Expt II	Expt I	Expt II	Expt I	Expt II	Expt I	Expt II	Expt I	Expt II
MW38 (rimM+)	0.046	0.65	2.7	8.5	0.98	0.93	0.12	0.79	5.0	11.2	0.98	0.93
MW37 (ΔrimM-2)	0.46	2.9	0.79	2.2	0.63	0.43	0.18	3.6	2.3	7.4	0.93	0.67
PW109 (ΔrimM-2 sdr-43)	0.68	7.9	2.0	9.4	0.74	0.54	0.25	1.5	4.5	10.5	0.95	0.87
GOB162 (rbfA::Kmr)	3.0	ND	3.6	ND	0.54		0.38	ND	6.0	ND	0.94

^aThe amount of rRNA was determined by primer extension as described in Materials and Methods. 

^bThe amounts of radioactivity in the primer extension products were quantified with a PhosphorImager from Molecular Dynamics, Inc. See also the legend to Fig. 8. ND, not determined. 

DISCUSSION

In the present study we have isolated 29 fast-growing derivatives of a strain in which the rimM gene (formerly called 21K and yfjA) of the trmD operon in E. coli has been deleted. Twenty-three of the suppressor mutations were shown to be linked to the truB gene of the nusA operon at 68.9 min on the chromosome. All the suppressor mutations that were tested partially suppressed the translational deficiency of the ΔrimM mutant. In the process of localizing one of these mutations, we discovered that an increased gene dosage of the wild-type rbfA gene (coding for the ribosome binding factor RbfA), which precedes truB, partly suppressed the slow growth of a ΔrimM mutant. In agreement with this observation, the amount of the RbfA protein in strain PW109, which contains the suppressor mutation sdr-43, was found to be higher than that in suppressor-free strains. The suppressor mutation seems to affect the transcription or stability of the rbfA mRNA, since strains containing the suppressor mutation had severalfold-increased levels of the rbfA mRNA. However, there was no mutation in the region covering the promoter for the entire operon, in the region containing a tentative internal promoter for rbfA, or in the rbfA structural gene. Moreover, there was no increased amount of the promoter-proximal part of the operon mRNA. The processing of the nusA operon is complex and has been only partly characterized (25, 35). The cleavage by RNase III downstream from metY releases the tRNA-Metf2 from the primary transcript and starts the decay of the downstream parts of the operon mRNA (35). Further, RNase E cleaves the nusA operon mRNA upstream of a hairpin structure at a site located at position +200 of the infB part of the mRNA (25). In a temperature-sensitive RNase E mutant, the infB mRNA accumulates at nonpermissive temperature, indicating that processing by RNase E accelerates the decay of the mRNA downstream of the cleavage site, including the part corresponding to the rbfA gene (25). At present, it is unclear whether the sdr-43 mutation abolishes the processing at the RNase E site in infB, thereby increasing the stability of the rbfA part of the mRNA. However, at least the 900-nt mRNA species that had an increased level in sdr-43-containing strains (see Fig. 4C) was much too short to cover both the region hybridizing to the probe used and the region upstream of the RNase E site. Thus, if the sdr-43 mutation abolishes the processing at the RNase E site, then some additional processing further downstream must have occurred to explain the size of at least the 900-nt mRNA species. Possibly, the sdr-43 mutation alters the normal decay pathway of the nusA operon mRNA, resulting in the stabilization of the part of the mRNA corresponding to rbfA. In contrast, the other suppressor mutation, sdr-34, seems to have increased the synthesis or stability of a 6.7-kb transcript starting upstream of p15a and probably terminating downstream of truB.

The results of the measurements of the relative amounts of the precursor and mature forms of 16S rRNA in the different mutants seem contradictory. Both RimM and RbfA seem to be important for the processing of pre-16S rRNA, and an increased synthesis of RbfA suppresses the translational deficiency caused by the lack of the RimM protein. Therefore, one would expect the increased synthesis of RbfA to increase dramatically the ratio of mature 16S rRNA to the precursor form. However, the amount of mature 16S rRNA relative to the precursor form was only slightly higher in the RbfA-overproducing strain than in the ΔrimM mutant. Possibly, the absolute rate of rRNA synthesis in the suppressor strain is higher than that in the ΔrimM mutant, which implies that the absolute rate of production of mature 16S rRNA per unit of time would also be higher, allowing for a higher growth rate of the cells. Alternatively, increased synthesis of RbfA might improve the function of the ribosomes without affecting the processing of pre-16S rRNA.

RbfA has been shown to act as a high-copy-number suppressor of a cold-sensitive mutation in the 5′-terminal helix of 16S rRNA, and the growth of a strain lacking RbfA is cold sensitive (10). Recently, RbfA has been shown to be a cold shock protein (15). Strains that either have rbfA inactivated or contain the cold-sensitive mutation in the 5′-terminal helix of 16S rRNA show a constitutively induced cold shock response when shifted from 37 to 15°C and are unable to adapt their growth to the lower temperature (15). RbfA is associated with free 30S ribosomal subunits but not with 70S ribosomes and has been suggested to interact with the 5′-terminal helix region of 16S rRNA during a late step in maturation of the 30S subunits (10). Interestingly, the growth of ΔrimM mutants is slightly cold sensitive, and the RimM protein has affinity for the 30S ribosomal subunit (7); also, as demonstrated here, both RimM and RbfA seem to be important for the maturation of 16S rRNA. Evidently, increased expression of rbfA suppresses both a cold-sensitive mutation in 16S rRNA (10, 15) and the slow growth and translational deficiency of a ΔrimM mutant. It was proposed that the cold sensitivity caused by the mutation in the 5′-terminal helix of 16S rRNA results from the inability of RbfA to interact with the helix and that this deficiency is suppressed by increased expression of rbfA (10). We hypothesize that the reason why increased rbfA expression also suppresses the translational deficiency of the ΔrimM mutant is that in the absence of the RimM protein, RbfA might have a reduced ability to interact with the helix. However, the lack of the RimM protein has a much more profound effect than has the lack of the RbfA protein on the growth rate at 37°C (data not shown). Thus, the slow growth of a ΔrimM mutant cannot be attributed only to an inability of RbfA to interact with the ribosomal 30S subunits. This also explains why increased expression of rbfA only partially suppressed the lack of the RimM protein.

In the processing of rRNA, RNase III introduces a double cleavage in each of two stems that produce 17S and pre-23S rRNA (see reference 1a). The products formed are precursors that are further processed to complete the maturation process. In the case of the maturation of 16S rRNA, at least two enzymes are involved in the final processing steps. Since the rimM and rbfA mutants studied here both had increased levels of pre-16S rRNA produced by RNase III, RimM and RbfA might be two of these processing enzymes. However, neither of the two proteins seem responsible for the final processing step, since both the rimM and rbfA mutants produce mature 16S rRNA. Formally, the possibility exists that RimM and RbfA both possess the activity needed for the last processing step and that they can substitute for each other, since increased expression of rbfA suppressed the translational deficiency of the ΔrimM mutant. We find this explanation unlikely, since severalfold-increased expression of rimM from a plasmid could not suppress the slow growth of a rbfA::Km^r mutant at 37°C, whereas it completely complemented the slow growth of a ΔrimM mutant (data not shown). In fact, the plasmid-mediated expression of rimM dramatically impaired the growth of the rbfA::Km^r mutant (data not shown). Moreover, a rimM rbfA mutant grew slightly more slowly and formed colonies more heterogeneous in size on rich-medium plates than a ΔrimM mutant (data not shown). These findings suggest that the RimM and RbfA proteins have different functions in the cell, and they are consistent with a model in which the RimM protein is needed in a step prior to RbfA during the maturation of 16S rRNA.

The conversion of pre-16S rRNA produced by RNase III to mature 16S rRNA occurs within 30S subunits (see reference 29). It has been demonstrated that pre-16S rRNA is found only in 30S (or pre-30S) ribosomal subunits and not in 70S ribosomes both for wild-type cells (21, 22) and for 16S rRNA mutants with a reduced processing rate of pre-16S rRNA (41). Further, ribosomes containing pre-16S rRNA are inactive in translation, as demonstrated by reconstitution experiments (51). Thus, only after pre-16S rRNA has been processed to 16S rRNA can the 30S subunits participate in translation, probably because the secondary structure between the 5′ and 3′ ends of 16S rRNA within the pre-16S form is not present in the mature 30S subunits, where the 5′ and 3′ ends of 16S rRNA are far from each other (see reference 6). Thus, the reduced translational efficiency of the ribosomes in the ΔrimM mutant cannot be attributed to the 30S ribosomal subunits which contain pre-16S rRNA, since processing of pre-16S rRNA must precede function. This indicates that in the ΔrimM mutant, the ribosomes that contain mature 16S rRNA have a reduced function. Since correct 30S subunit assembly is a prerequisite for maturation of 16S rRNA, a deficiency in the assembly of the 30S subunits of the ΔrimM mutant might explain both a reduced function of ribosomes active in translation and a reduced efficiency in the processing of pre-16S rRNA. That the 30S subunits of ΔrimM mutants might be different from those in rimM⁺ strains is supported by the finding that alterations in the region of r-protein S13 that binds 16S rRNA partly suppress the translational deficiency of a ΔrimM mutant (7).

We propose that RimM and RbfA are part of the 30S subunits prior to or during the final step in the processing of 16S rRNA, since both proteins have been found associated with free 30S ribosomal subunits (7, 10). The two proteins are not necessarily the actual processing enzymes but could be some accessory proteins needed for efficient assembly of the 30S subunits. In recent years a number of reports have indicated that nonribosomal proteins might facilitate the correct assembly of the ribosomal subunits. For example, it has been implied that the chaperone protein DnaK and two ATP-dependent RNA helicases assist in ribosome assembly: strains with temperature-sensitive mutations in dnaK are deficient in ribosome assembly at high temperatures (1), whereas overproduction of the RNA helicases SrmB and DeaD can suppress some mutations in the rplX gene, which encodes r-protein L24 (29a), and in the rpsB gene, which encodes r-protein S2 (43a), respectively. Possibly, RimM and RbfA assist some of the r-proteins in their binding to 16S rRNA. Alternatively, the two proteins might stabilize RNA secondary structures needed for correct processing or help to refold the mature 16S rRNA after processing has been completed. In fact, since RbfA has been suggested to bind to the 5′-terminal helix of mature 16S rRNA, it might be involved in the formation of that structure. Also, the RimM protein might bind to 16S rRNA, since two different RNA binding motifs are present in the protein (7).

Experiments are in progress to elucidate what role the RimM protein plays in the maturation of 16S rRNA and the 30S ribosomal subunits and what the relation is between RimM and RbfA in those processes.