Regulation of Ribonuclease J Expression in Corynebacterium glutamicum

ABSTRACT

RNase J exerts both 5′–3′ exoribonuclease and endoribonuclease activities and plays a major role in ribonucleotide metabolism in various bacteria; however, its gene regulation is not well understood. In this study, we investigated the regulation of rnj expression in Corynebacterium glutamicum. rnj mRNA expression was increased in a strain with an rnj mutation. Deletion of the genes encoding RNase E/G also resulted in increased rnj mRNA levels, although the effect was smaller than that of the rnj mutation. rnj mRNA was more stable in the rnj mutant strain than in wild-type cells. These results indicate that RNase J regulates its own gene by degrading its mRNA. The growth of rnj and pnp mutant cells was impaired at cold temperatures. The expression of rnj mRNA was transiently induced by cold shock; however, this induction was not observed in the rnj mutant strain, suggesting that autoregulation by self-degradation is responsible for inducing of rnj expression under cold-shock conditions.

IMPORTANCE Corynebacterium glutamicum harbors one RNase E/G-type enzyme and one RNase J-type enzyme which are major ribonucleases in various bacteria. However, little is known about these gene regulations. Here, we show that RNase J autoregulates its own gene expression and RNase E/G is also involved in the rnj mRNA degradation. Furthermore, we show that transient induction of the rnj mRNA in the cold-shock condition is dependent on RNase J autoregulation. This study sheds light on the regulatory mechanism of RNase J in C. glutamicum.

INTRODUCTION

The degradation of mRNA is a key regulatory step in the control of gene expression. In Escherichia coli, most mRNA degradation is initiated through internal cleavage by RNase E and accelerated by the 5′-end monophosphate (1, 2). After the initial cleavage, further degradation is catalyzed by RNase E and exonucleases, such as PNPase, RNase II, and RNase R (3, 4). In Bacillus subtilis, RNase Y, which is an endoribonuclease, exerts an activity that is similar to that of RNase E despite being structurally distinct from RNase E (5). Bacillus subtilis has a 5′–3′ exonuclease that is RNase J, which also exhibits endoribonuclease activity (6). RNase J and 3′–5′ exonucleases, such as PNPase and RNase R, further degrade endonucleolytically cleaved RNA.

Bacillus subtilis contains two forms of RNase J, namely, RNase J1 and RNase J2; depletion of RNase J1 alters the abundance of many transcripts, indicating its role as a major RNA-degrading RNase (7). Bacillus subtilis strains lacking RNase J1 but possessing RNase J2 show slow growth and a cold-sensitive phenotype (8). A cold-sensitive phenotype has also been reported in an RNase J-depleted Staphylococcus aureus strain (9). Regulation of rnj expression is not clearly understood. In one study, deletion of rnj2 resulted in a slight increase in rnj1 expression, and the expression of rnj1 and rnj2 was autoregulated by 1.4-fold (10); however, the mechanism of autoregulation or gene regulation under cold-shock conditions remains unclear.

Corynebacterium glutamicum is a Gram-positive soil bacterium with high guanine and cytosine contents in its DNA and is widely used in the industrial production of amino acids (11, 12). It is also used to efficiently produce lactate and succinate from sugar (13); therefore, the molecular basis of its gene regulation is of great interest for the development of new bioprocesses. Regarding the major endoribonucleases, C. glutamicum harbors one RNase E/G-type enzyme and one RNase J-type enzyme in addition to an RNase III-type enzyme (14). Corynebacterium glutamicum differs from well-analyzed model organisms such as E. coli, which harbors RNases E, G, and III but not RNase J; B. subtilis harbors RNases J1, J2, Y, and III but not RNase E. Corynebacterium glutamicum also contains exonuclease-type enzymes, namely, RNase J, PNPase, and RNase R (15). RNase E/G is involved in the maturation of rRNA and 4.5S RNA in C. glutamicum (15, 16). In addition, degradation of ribM mRNA and aceA RNA is mediated by RNase E/G (17, 18). RNase III is involved in regulating of MraZ, a transcriptional regulator that controls the expression of the cell division protein FtsEX (14). However, little is known about RNase J in C. glutamicum.

RESULTS

Effect of RNase mutations on rnj expression.

Corynebacterium glutamicum contains one copy of rnj encoding RNase J, which is located between dapA encoding an enzyme involved in lysine biosynthesis (19) and cgR_1798 encoding a hypothetical protein (Fig. 1). Corynebacterium glutamicum RNase J has an N-terminal domain of approximately 150 amino acids, which is not found in other bacteria (see Fig. S1). Interestingly, C. glutamicum RNase E/G also has an additional N-terminal domain (20). However, there is no sequence homology between these domains, and their functions are unknown.

FIG 1.

rnj gene cluster on the chromosome of C. glutamicum R. Open arrows represent the coding regions of rnj and neighboring genes. A putative transcriptional terminator is indicated by a stem-loop. Major 5′ termini of rnj mRNA determined by 5′-RACE are indicated by asterisks.

RNase expression is often posttranscriptionally regulated by mRNA degradation (21–25). Therefore, we determined whether the rnj transcript is self-regulated and whether it is a target of other RNases. To investigate its autoregulation, we constructed a mutant strain (H514F). The His514 residue is a component of the substrate 5′ phosphate-binding pocket (26) and is conserved among RNase J (see Fig. S1 in the supplemental material). To evaluate the effect of the rnj(H514F) mutation, we compared the rnj mutant and rnj knockout according to the level of several mRNAs. The tested genes were some of those affected and not affected by rnj knockout (see Fig. S2). The rnj(H514) mutant showed a very similar expression pattern to that of the rnj deletion strain, except for the rnj mRNA itself, as expected. Therefore, this mutation likely has considerably decreased RNase J activity.

We then determined the expression of rnj in wild-type and RNase mutants. Total RNA was extracted from exponentially growing cells at 33°C, and the expression levels of rnj mRNA were determined using quantitative reverse transcription-PCR (qRT-PCR) (Fig. 2A). The level of the rnj transcript increased by ∼10-fold in the rnj(H514) mutant strain compared to that in the wild-type strain. rneG deletion resulted in a 1.7-fold increase in rnj mRNA levels, with no significant increase in rnj mRNA levels in either pnp, rnc, or rnr deletion strains. These results suggest that RNase J autoregulates its expression and that other RNases have minor effects on rnj mRNA levels under conventional culture conditions.

FIG 2.

Effect of RNase mutations on rnj expression. (A) qRT-PCR analysis of rnj mRNA levels in various RNase mutants. Total RNA was prepared from cells grown in nutrient-rich A medium supplemented with 2.0% (wt/vol) glucose at 33°C with an OD₆₁₀ of 4.0. The transcription expression levels of rnj were determined by using qRT-PCR. The relative mRNA level is the ratio of mRNA level against that of WT cells. Statistical values are presented as means and standard deviations across three independent experiments. (B) Northern blot analysis of rnj mRNA expression. WT, rnj H514F mutant, and ΔrneG strains were grown in nutrient-rich A medium with 2% (wt/vol) glucose, and dapA and rnj mRNAs were detected by Northern blotting. Major RNA products observed with rnj probe is marked with arrowheads. The position of the RNA size marker (DynaMarkers, Inc., Prestain Ladder Marker for RNA High) is indicated on the left. (C) Reprobing the membrane used in Fig. 2B with 16S rRNA probe. (D) Immunoblot analysis of RNase J expression. WT, rnj H514F mutant, and ΔrneG strains were grown in nutrient-rich A medium with 2% (wt/vol) glucose, and RNase J proteins were detected using immunoblot analyses with an anti-RNase J antibody. Representative data from three independent experiments are shown.

We then examined rnj expression by Northern blotting analysis (Fig. 2B). Total RNA was extracted from exponentially grown cells, and Northern blotting was performed. A band that migrated slightly more slowly than a 2,000-base RNA marker was detected with a probe specific for rnj mRNA (band A). This band was increased in the rnj mutant strain but was not detected in the rneG mutant, suggesting that this band is a result of RNase E/G endonucleolytic cleavage, which leaves a free 5′ end that is degraded exonucleolytically by RNase J. Several additional bands were detected in the rnj and rneG mutant strains. We also performed Northern blotting using a probe specific for the first half of dapA. Unlike the results obtained using the rnj probe, band A was not detected using a dapA probe, suggesting that this mRNA product does not contain the first half of dapA. Other long RNA products of approximately 3000 bases or more were detected using dapA and rnj probes in rnj and rneG mutant strains (band B), suggesting that this product is transcribed as an operon that includes both dapA and rnj. We also performed Northern blot analysis using a probe specific for cgR_1798 gene, which is downstream of rnj, but only faint bands were observed (data not shown). A stem-loop structure of 35 nucleotides (nt) that is not followed by a run of U residues (ΔG = −27.9 kcal/mol) is present downstream of rnj (Fig. 1), which may act as a transcriptional terminator.

We further examined the effect of RNase mutations on the protein expression of RNase J using Western blot analysis. RNase J protein expression was increased by (3.5 ± 1.1)-fold in the rnj mutant strain compared to in the wild-type cell (Fig. 2D; see also Fig. S3 for long exposure data), confirming autoregulation of rnj expression in C. glutamicum. We also observed a (1.8 ± 0.7)-fold increase in RNase J levels in the rneG mutant, supporting the involvement of RNase E/G in RNase J expression.

rnj mRNA is stabilized upon inactivation of RNase J.

To determine whether RNase J is autoregulated by digesting its own mRNA, we evaluated the stability of rnj mRNA (Fig. 3; a semilog plot of the band intensity is shown in Fig. S4). Exponentially growing cells cultured at 33°C were treated with 0.2 mg/mL rifampicin to prevent further initiation of transcription (27). Total RNA was isolated at various times after adding rifampicin, and the decay rate of rnj mRNA was determined. The band A rnj transcript was degraded with a half-life of 1.7 ± 0.68 min in the wild-type strain but showed prolonged half-life of 11 ± 2.6 min in the rnj H514F mutant. The band B rnj transcript was degraded with a half-life of 4.0 ± 0.81 min in the rnj mutant, with a similar half-life in rneG deletion cells (3.4 ± 0.82 min), while it was undetectable in the wild type (WT). These results indicate that RNase J plays a major role in the degradation of rnj RNA and that RNase E/G is also involved.

FIG 3.

Determination of rnj mRNA decay rate. The half-lives of rnj mRNA in wild-type, rnj H514F mutant, and ΔrneG strains were measured. Each C. glutamicum strain was grown in nutrient-rich A medium supplemented with 2.0% (wt/vol) glucose at 33°C. Rifampicin was added to the cultures at an OD₆₁₀ of 4.0, and the total RNA was isolated at the indicated times. rnj mRNA expression was analyzed by Northern blotting. The Northern blot membranes were then reprobed with a 16S rRNA probe. Representative data from three independent experiments are shown.

Mapping of 5′ ends of rnj mRNA.

To determine the 5′ end of rnj mRNA, we performed 5′ rapid amplification of cDNA end (RACE) analysis. For each strain, at least 30 clones were sequenced for each 5′ RACE-PCR product. The results are summarized in Fig. 4. In the WT strain, 44% of the 5′ end was located at 219 nt upstream of the translation initiation codon of rnj, while 12% of the 5′ ends mapped at 908 nt upstream of rnj, consistent with bands A and B in the Northern blot. A similar result was observed for the rnj mutant strain, with 33% located 219 nt upstream and 13% located 908 nt upstream of the rnj initiation codon. In the rneG mutant strain, 67% of the 5′ end was mapped 908 nt upstream of rnj, but no 5′ end was mapped 219 nt upstream, suggesting that RNase E/G is involved in generating the 5′ end 219 nt upstream of rnj. The position 908 nt upstream of rnj is identical to that of the initiation codon of dapA, indicating that this transcript contains both dapA and rnj.

FIG 4.

Identification of the 5′ terminus of rnj mRNA. The 5′ end of rnj mRNA was mapped. A 5′-RACE analysis was performed on WT, rnj H514F mutant, and ΔrneG strains. The proportion of the 5′ end according to the strain is shown. The translation initiation codon of rnj gene is defined as position +1.

RNase J and PNPase affect growth at cold temperatures.

A cold-sensitive phenotype was observed in B. subtilis and S. aureus strains lacking RNase J (8, 9, 28). We tested whether C. glutamicum RNase J is also required for growth by incubating cells plated at a low temperature (Fig. 5). Indeed, impaired growth was observed in the rnj mutant incubated at 15°C. Growth was also impaired in the pnp deletion strain, whereas growth of the strain with deleted rnr, which encodes another 3′–5′ exonuclease, was similar to that of the WT cell. Deletion of rneG or rnc encoding RNase E/G or RNase III resulted in reduced cell growth at low temperatures; however, the effect was minor compared to that of depletion of the genes encoding RNase J or PNPase. The growth of strains with deleted cspA1 or cspA2, which encode RNA chaperones, was not impaired. These results indicate that RNase J and PNPase, as 5′- and 3′-exonucleases, respectively, are important for the proper growth of C. glutamicum under cold temperature conditions.

FIG 5.

Effect of RNase mutation on growth under cold temperatures. Overnight cultures of WT and RNase mutant C. glutamicum strains grown in nutrient-rich A medium with 2% (wt/vol) glucose at 33°C were serially diluted with A medium and spotted onto A medium plates with 2% (wt/vol) glucose. The plates were incubated at 33 and 15°C.

Expression of rnj under cold-shock conditions.

Although RNase J is important for growth under low-temperature conditions, the regulation of rnj expression has not been reported. We examined the expression of rnj under cold-shock stress conditions. Wild-type and RNase mutant C. glutamicum were aerobically pregrown in nutrient-rich A medium at 33°C and then aerobically incubated at 15°C for 1 h. The total RNA was extracted, and the expression levels of rnj mRNA were determined using qRT-PCR (Fig. 6A). In WT cells, the rnj transcript level increased by ∼3.7-fold at 1 h after cold-shock stress. In the rnj H517F mutant, a 14-fold increase was observed under cold-shock conditions compared to the WT strain at 33°C. This represents a 1.4-fold increase compared to the 10-fold autoregulatory effect seen in this strain at 33°C. In other RNase mutants, a similar level of increase was observed compared to in WT cells. We then examined the temporal response of rnj expression after decreasing the temperature. In WT cells, rnj mRNA levels temporally increased: rnj mRNA levels peaked at 1 h after cold shock, and rnj expression level gradually decreased (Fig. 6B). In rnj mutant cells, rnj expression levels slightly increased during growth at low temperatures, and no temporal induction pattern was observed (Fig. 6B). These results indicate that RNase J catalytic activity is necessary for the temporal induction of rnj expression under cold-shock conditions. The half-life of rnj mRNA was measured under cold-shock conditions. Corynebacterium glutamicum cells pregrown at 33°C were incubated at 15°C for 1 h. Rifampicin was added to the medium, and the decay rate of rnj mRNA was determined (Fig. 6C). The half-life of the band A rnj transcript was increased under cold-shock conditions and was 29 ± 5.5 min in the WT and more than 60 min in the rnj mutant. The band B rnj transcript in the WT showed a half-life of 9.4 ± 2.2 min, whereas those in the rnj and rneG mutant strains were 25 ± 8.4 and 15 ± 4.4 min, respectively. These results indicate that the stability of the rnj transcript was increased in all tested strains, as expected (29) and suggest that at least part of the induction of rnj expression under cold-shock conditions is mediated by mRNA stabilization.

FIG 6.

rnj mRNA expression under cold-shock stress. (A) rnj mRNA expression in wild-type and various RNase mutant strains under cold-shock stress. WT and RNase mutant C. glutamicum strains pregrown at 33°C to an OD₆₁₀ of 4.0 were subjected to cold stress (15°C for 1 h). Total RNA was prepared, and rnj mRNA levels were determined using qRT-PCR. The relative mRNA level is the ratio of mRNA level against that of WT cells grown at 33°C. Statistical values are presented as the means and standard deviations across three independent experiments. (B) rnj expression after decreasing the temperature. WT (black circles) and rnj H514F mutant (open circles) strains pregrown at 33°C to an OD₆₁₀ of 4.0 were subjected to cold stress (15°C). Total RNA was prepared at the indicated times after decreasing the temperature, and rnj mRNA levels were determined using qRT-PCR. The relative mRNA levels are indicated as the ratio against cells grown at 33°C. Statistical values are presented as means and standard deviations across three independent experiments. (C) Half-lives of rnj mRNA under cold-shock stress conditions. WT and RNase mutant C. glutamicum strains pregrown at 33°C to an OD₆₁₀ of 4.0 were subjected to cold stress (15°C for 1 h). Rifampicin was added to the cultures, and the total RNA was isolated at the indicated times. The RNAs were subjected to Northern blot analysis. Representative data from three independent experiments are shown.

DISCUSSION

We investigated the regulation of rnj expression in C. glutamicum. The expression of rnj markedly increased in the strain with a mutation in the substrate 5′ phosphate-binding pocket of RNase J (Fig. 2). The stability of rnj mRNA was increased by the rnj mutation (Fig. 3), indicating that the regulation occurs at the level of rnj mRNA degradation. These results indicate that RNase J autoregulates its own gene expression in C. glutamicum. We also observed that RNase E/G is involved in rnj mRNA metabolism in C. glutamicum (Fig. 2). However, the effect was moderate compared to that of the rnj mutant. Considering the influence of RNase J autoregulation, the effect of RNase E/G on rnj expression is likely underestimated. Ideally, the effect of rneG deletion was tested in cells with an rnj mutant background; however, our attempts to construct the rnj rneG double mutant failed. Conditional inactivation of both RNases and a more detailed investigation of RNase E/G action on rnj mRNA are necessary. In addition, whether rneG expression is regulated by RNase J requires further analysis. Northern blot analysis using rnj and dapA probes indicated that the expression of dapA and rnj mRNA increased in the rnj and rneG mutant strains (Fig. 2B), further indicating that RNase E/G and RNase J affect dapA expression in addition to rnj. Understanding dapA regulation may be useful for the efficient industrial production of lysine by C. glutamicum.

We mapped the 5′ ends of rnj transcripts to 908 and 219 nt upstream of the rnj initiation codon (Fig. 4). Considering that the coding sequence of rnj is 2,157 bp, these 5′ end are consistent with the approximately 3- and 2-kb bands observed in the Northern blot analysis (Fig. 2B). The 5′ end located 908 nt upstream of rnj is identical to the initiation codon of dapA. The site was also identified as a transcription start site in native 5′ end RNA-sequencing transcriptome analysis (30), suggesting the existence of a promoter that drives dapA rnj leaderless transcript expression. A −10 like sequence (TAACCT) is present at this site. The 5′ end located 219 nt upstream of rnj was not detected by 5′ RACE analysis in the rneG mutant strain, and the 2-kb band was not detected in the rneG mutant strain by Northern blotting analysis. In the rnj mutant, the transcript levels of both the 3,000- and the 2,000-nt rnj transcripts were increased compared to in the WT strain. These results suggest that the 2,000-nt rnj transcript is generated from the full-length transcript by endonucleolytic cleavage by RNase E/G and that RNase J degrades both the 3,000- and the 2,000-nt rnj transcripts. The 3,000-nt band was not detected on the Northern blot in the WT strain. This is probably because the full-length transcript is subject to both RNase E/G cleavage and 5′-to-3′ degradation by RNase J. The combination of these two activities leaves undetectable levels of the full-length transcript. The region ∼219 nt upstream of rnj is not rich in AU residues, which are recognized and cleaved by RNase E-type enzymes. Determination of cleavage site specificity of RNase E/G in C. glutamicum is important to clarify the mechanism of rnj mRNA metabolism.

Disruption of rnj results in a cold-sensitive phenotype in B. subtilis (8, 28) and S. aureus (9). At a low temperature, the rnj mutation also impaired the growth of C. glutamicum. We also observed growth impairment in the pnp mutant strain, indicating that RNase J and PNPase are required for proper growth at low temperatures. The expression of rnj was increased under cold-shock conditions at the mRNA level in WT cells (Fig. 6A) and peaked 1 h after cold shock, and then the rnj mRNA level returned to the basal level (Fig. 6B). Interestingly, rnj mRNA levels only moderately increased in the rnj mutant strain under cold-shock conditions compared to that at 33°C, and no transient induction pattern was observed (Fig. 6B). These results suggest that RNase J is involved in the temporal increase of rnj mRNA under cold-shock conditions; however, the stability of the rnj transcript was increased in the WT, rnj mutant, and rneG mutant strains, indicating that the inhibitory effect of cold shock on rnj mRNA degradation is not specific to RNase J or RNase E/G. The improved stability of the secondary structure of rnj mRNA at low temperatures may decrease the degradation rate of the rnj transcript and result in increased rnj mRNA levels. The effect of rnj mutation on the rnj mRNA level is small in the rnj mutant, likely because rnj mRNA is already stable at 15°C.

MATERIALS AND METHODS

Media and growth conditions.

Corynebacterium glutamicum R was grown aerobically at 33 and 15°C in a nutrient-rich A medium (31) supplemented with 2% (wt/vol) glucose. Bacterial growth was monitored by determining the optical density at 610 nm (OD₆₁₀).

Bacterial strains and plasmids.

The strains used in this study are listed in Table 1. Corynebacterium glutamicum R (JCM 18229) was used as the wild-type strain (32). Strains with markerless deletions of rnj, cspA, and cspA2 genes were generated as follows (33). The suicide vector pCRA725 carrying the sacB gene was used to construct the deletion strains. The oligonucleotide primers used for gene disruption are summarized in Table S1 in the supplemental material. A DNA fragment encoding rnj, cspA, or cspA2 was amplified using PCR, and an internal segment of the gene was removed by inverse PCR. The resultant plasmids were introduced into C. glutamicum, and single-crossover cells were isolated using kanamycin resistance. The isolated cells were cultivated in a medium supplemented with 10% sucrose, and double-crossover cells were isolated. The gene deletions were confirmed by DNA sequencing of the PCR products around the modified region.

TABLE 1.

Strains used in this study

Strain	Genotype	Reference
R	JCM 18229 WT strain	32
YT640	R with cspA deletion
YT650	R with cspA2 deletion
Δrnj	R with rnj deletion	15
ΔrneG	R with rneG deletion	17
Δrnc	R with rnc deletion	14
Δpnp	R with pnp deletion	14
Δrnr	R with rnr deletion	15
rnj(H514F)	R with rnj active site mutation

The strains with rnj mutation in the active site of RNase J was generated as follows. A DNA fragment encoding rnj was amplified using PCR with the primers rnj-mut2F1 and rnj-mut2R2 and cloned into a SacI restriction enzyme cut site of pCRA725. The mutation was introduced by inverse PCR with the primers rnj-mut2F2 and rnj-mut2R1. The resultant plasmid was introduced into C. glutamicum, and single-crossover cells were isolated using kanamycin resistance. The isolated cells were cultivated in a medium supplemented with 10% sucrose, and double-crossover cells were isolated. The rnj mutation was confirmed by DNA sequencing of the PCR products around the modified region.

Total RNA purification.

Two volumes of RNA Protect bacterial reagent (Qiagen) were added directly to one volume of exponentially growing cultures at an OD₆₁₀ of ∼4 (in logarithmic phase) to stabilize cellular RNAs. The cells were harvested by centrifugation at 5,000 × g for 7 min at 25°C, and total cellular RNAs were isolated using NucleoSpin RNA (Macherey-Nagel) according to the manufacturer’s instructions.

Quantitative reverse transcription-PCR.

qRT-PCR was performed using the following experimental conditions. Each qRT-PCR mixture (20 μL) contained 500 nM a primer set, 10 μL of Power SYBR green PCR Master Mix, 8 U of RNase Inhibitor, 5 U of MuLV reverse transcriptase (Applied Biosystems), and total RNAs (20 ng for the rnj mRNA and 0.4 ng for 16S rRNA). The primers used in these reactions are listed in Table S1 in the supplemental material. qRT-PCR targets region from nt +1893 to +1951 (from the translation initiation codon) of rnj. qRT-PCR was performed using an ABI 7500 Fast real-time PCR system (Applied Biosystems) with the following cycle parameters: one cycle at 50°C for 30 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 15 s. 16S rRNA was used as an internal control.

Northern blot analysis.

Northern hybridization was performed as described previously (34). Digoxigenin (DIG)-labeled antisense RNA probes targeting the rnj, dapA, cgR_1798, or 16S rRNA gene were produced according to the manufacturer’s instructions (Roche Diagnostics). To generate a hybridization probe, specific DNA fragments were amplified by PCR using the primers RI-rnj-280F and HindIII-rnj-800R for rnj, RI-1800-1F and RI-1800-450R for dapA, EcoRI-1798-130F and EcoRI-1798-636R for cgR_1798, and EcoRI-r01-1F and EcoRI1-r01-500R for 16S rRNA. The amplified fragments were cloned into pSPT18 plasmid (Roche Diagnostics). The constructed plasmids were used as the templates for the synthesis of the DIG-labeled antisense RNA probes. Northern hybridization was performed according to the procedures described in the DIG Northern starter kit (Roche Diagnostics). 6.5 μg of total RNA was fractionated on 10% agarose gel. The hybridization temperature was 68°C. Positive hybridization bands were detected using CDP-Star reagent (Roche Diagnostics). The loading of equal amount of RNAs was confirmed by reprobing the membrane with the probe for 16S rRNA.

5′ RACE-PCR.

To map the 5′ ends of rnj mRNA, 5′ rapid amplification of cDNA ends (RACE) analysis was carried out using a SMARTer RACE cDNA amplification kit (Clontech). The cDNA was directly amplified from total RNA with universal primer A (supplied with the kit) and gene-specific primer 5′-ACGGTTGTTGTTGGAACGGTTGC-3′. The resulting PCR product was cloned into a pGEM-T Easy vector (Promega). For each strain, at least 30 clones of each 3′ RACE-PCR product was sequenced.

Immunoblot analysis.

An aliquot of cell cultures was collected by centrifugation, and pellets were mixed with glass beads and 1.0 mL of buffer (4% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol, 40 mM Tris-HCl [pH 6.8], and 0.1 mM EDTA). Cells were disrupted by vigorous vortexing, and the samples were centrifuged. Crude extract of 10 μg was loaded onto 0.1% (wt/vol) SDS–12% (wt/vol) polyacrylamide gels and electrophoresed. Western blotting was performed using monoclonal RNase J antibody and horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare). Chemiluminescence reactions were done by using an ECL Plus Western blotting detection system (GE Healthcare). The signal was scanned by a luminescent image analyzer (FUJI model LAS-3000). The band intensity of RNase J was analyzed by ImageJ.