Regulation of the Expression of De Novo Pyrimidine Biosynthesis Genes in Corynebacterium glutamicum.

Regulation of the Expression of De Novo Pyrimidine Biosynthesis Genes in Corynebacterium glutamicum Yuya Tanaka,a Haruhiko Teramoto,a Masayuki Inuia,b Research Institute of Innovative Technology for the Earth, Kizugawa, Kyoto, Japana; Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japanb

ABSTRACT

Expression of pyrimidine de novo biosynthesis is downregulated by an exogenous uracil in many bacteria. In this study, we show that a putative binding motif sequence of PyrR is required for uracil-mediated repression of pyrR-lacZ translational fusion. However, the uracil response was still observed in the strain with the pyrR gene deleted, implying the existence of a uracil response factor other than PyrR which also acts through the PyrR binding loop region. Deletion of rho, encoding the transcription termination factor Rho, resulted in an increase in the expression of pyrR-lacZ. Moreover, the strain with a double deletion of pyrR and rho showed elimination of the uracil-responsive downregulation of the pyrR-lacZ. Therefore, expression of the pyrimidine biosynthetic gene cluster in Corynebacterium glutamicum is controlled by two different mechanisms mediated by PyrR and Rho. IMPORTANCE

The pyr genes of C. glutamicum are downregulated in the presence of uracil in culture medium. The mRNA binding regulator PyrR represses the expression of pyr genes, as reported previously. However, the uracil response was still observed in the pyrR deletion strain. Deletion of rho in addition to pyrR deletion results in the elimination of the uracil response. Therefore, we identified the factors that are involved in the uracil response. Involvement of Rho in the regulation of pyrimidine de novo biosynthesis genes has not been reported.

P

yrimidine de novo biosynthesis starts with the synthesis of a pyrimidine ring assembled from aspartate, bicarbonate, and glutamine (Fig. 1C). The pyrimidine ring reacts with phosphoribosyl pyrophosphate (PRPP) to generate orotidine 5=-monophosphate (OMP). Subsequently, decarboxylation of OMP results in the formation of UMP, which is converted to other essential pyrimidine nucleotides. Carbamoyl phosphate is also used for biosynthesis of arginine, connecting the pyrimidine biosynthesis with arginine biosynthesis. The pyrimidine-biosynthetic pathway consists of six enzymatic steps, and the genes coding for these enzymes (carA, carB, pyrB, pyrC, pyrD, pyrE, and pyrF) are widely conserved in bacteria (1). Regulation of the expression of pyrimidine de novo biosynthesis genes has been well studied in Escherichia coli and Bacillus subtilis. Many regulatory mechanisms that are not dependent on DNA-binding transcriptional regulators have been demonstrated. Expression of the pyrBI operons in E. coli is proposed to be controlled by transcriptional attenuation (2–4). The regulation at the transcription initiation step has also been reported. In the presence of high concentrations of UTP, nascent transcript slips from the template DNA at the consecutive T residues, resulting in the reiterative addition of UTP to the transcript (5). In B. subtilis, pyrimidine de novo biosynthesis genes are clustered (pyr operon). Expression of the pyr operon is regulated by the transcriptional termination-antitermination mechanism involving the RNAbinding protein PyrR (6, 7). In the absence of PyrR, formation of the antitermination stem-loop prevents the formation of the terminator hairpin structure in the 5= untranslated region of the regulated gene and promotes the expression of the pyr operon. Binding of PyrR to the specific sequence in pyr mRNA (PyrRbinding loop sequence) inhibits the antiterminator stem-loop formation and results in termination of transcription. The mRNA

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binding activity of PyrR is stimulated by UMP/UTP and inhibited by GTP, explaining the regulation of the pyr operon in response to the availability of cytoplasmic pyrimidine. In Mycobacterium smegmatis, binding of PyrR to the PyrR-binding loop also depends on the concentrations of UMP (8). However, its regulatory mechanism is different from that in the case of the B. subtilis pyr operon. In M. smegmatis, binding of PyrR sequesters the Shine-Dalgarno sequence from ribosome to prevent the translation initiation of pyrR, which is the first gene in the pyr gene cluster. These results demonstrate the diversity of the regulation mechanism of the pyr gene cluster even between bacterial species using the same regulatory components. Corynebacterium glutamicum is a high-GC-content, Grampositive soil bacterium which is widely used for the industrial production of amino acids (9, 10). This bacterium is also used for efficient production of lactate and succinate from sugar (11–13). Therefore, the regulatory mechanisms of the genes involved in the cellular metabolic pathways such as carbohydrate metabolism

Received 28 May 2015 Accepted 4 August 2015 Accepted manuscript posted online 10 August 2015 Citation Tanaka Y, Teramoto H, Inui M. 2015. Regulation of the expression of de novo pyrimidine biosynthesis genes in Corynebacterium glutamicum. J Bacteriol 197:3307–3316. doi:10.1128/JB.00395-15. Editor: R. L. Gourse Address correspondence to Masayuki Inui, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JB.00395-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.00395-15

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FIG 1 (A) pyr gene cluster on the chromosome of C. glutamicum R. Arrowheads represent the coding regions of the pyr genes and neighboring genes. The transcription start site is represented by an arrow. (B) Nucleotide sequence between cgR_1663 and pyrR. The transcription start sites are shown by arrows. The ⫺10, Shine Dalgarno (SD), and anti-SD regions are underlined. The translation initiation codons and the stop codon are boxed. The PyrR-binding loop region is depicted with bold letters. The region that was deleted in the PyrR binding-loop or the translation initiation region of the leader ORF for construction of the respective mutants is indicated by dashed underlines. (C) Pyrimidine de novo biosynthesis pathway. Enzymes (genes): 1, carbamoylphosphate synthetase (carA and carB); 2, aspartate carbamoyl transferase (pyrB); 3, dihydroorotase (pyrC); 4, dihydroorotate dehydrogenase (pyrD); 5, orotate phosphoribosyltransferase (pyrE); 6, OMP decarboxylase (pyrF). (D) Predicted mRNA secondary fold model of the PyrR-binding loop. SD and anti-SD regions are indicated with lines. The translation initiation codon (AUG) is boxed.

have been intensely studied for the improvement of the production of valuable fuels and chemicals (14, 15). Pyrimidine nucleotides are de novo synthesized from PRPP, which is derived from the pentose phosphate pathway, with aspartate and glutamine (Fig. 1C). Thus, the regulation of pyrimidine biosynthesis genes should be finely controlled for preventing the wasteful utilization of these metabolites. In C. glutamicum, the regulation of uridine utilization genes by LacI/GalR-type transcriptional regulator UriR was reported (16). However, regulation of the pyrimidine de novo biosynthesis genes has remained unknown. Therefore, we investigated how these genes are regulated in response to addition of pyrimidine to the culture medium in C. glutamicum. In C. glutamicum, a set of pyrimidine de novo biosynthesis genes are clustered with a putative regulatory gene, pyrR (Fig. 1A). In the upstream region of pyrR, a putative PyrR-binding loop

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motif is present. This gene structure resembles that of M. smegmatis, suggesting the PyrR-dependent regulation of the pyr gene cluster. In this study, we show that expression of the pyr gene cluster is controlled by PyrR. Deletion studies conducted with the strains carrying the pyrR upstream region fused to the lacZ reporter gene indicated that PyrR exerts its effect through the PyrR-binding loop motif. In addition, we show evidence that uracil-dependent downregulation of the pyr gene cluster is mediated by not only PyrR but also a transcriptional termination factor, Rho. MATERIALS AND METHODS Media and growth conditions. C. glutamicum R was grown aerobically at 33°C in nutrient-rich A medium or minimum BT medium (17) supplemented with 2% (wt/vol) glucose with or without uracil. Bacterial growth was monitored by determining the optical density at 610 nm.



Regulation of the pyr Genes in C. glutamicum

TABLE 1 Strains used in this study Strain

Description

Reference

R YT195 YT246 YT593 YT151 YT459 YT310 YT252 YT596 YT467 YT248 YT487 YT264 YT483 YT284 YT286 TK21 YT302 YT578

JCM 18229 wild-type strain R with deletion of pyrD R with pyrR-lacZ R with pyrR-pyrB-lacZ R with gnd-lacZ YT246 with deletion of pyrR YT151 with deletion of pyrR YT246 with deletion of the PyrR-binding motif in pyrR-lacZ YT593 with deletion of the PyrR-binding motif in pyrR-pyrB-lacZ YT252 with deletion of pyrR YT246 with mutation at the translation start region of the leader ORF YT248 with deletion of pyrR YT246 with mutation at the translation initiation codon of the leader ORF which was changed to ATG YT264 with deletion of pyrR YT248 with deletion of the PyrR-binding motif in pyrR-lacZ YT264 with deletion of the PyrR-binding motif in pyrR-lacZ R with deletion of rho TK21with pyrR-lacZ YT302 with deletion of pyrR

22 This study This study This study 29 This study This study This study This study This study This study This study This study This study This study This study This study This study This study

Bacterial strains and plasmids. The strains used in this study are listed in Table 1. C. glutamicum R (JCM 18229) was used as a wild-type strain. The strains with a deletion of pyrD or pyrR were constructed as follows. A suicide vector, pCRA725, carrying the sacB gene was used for construction of deletion strains (18). Oligonucleotide primers used for gene disruption are summarized in Table S1 in the supplemental material. Briefly, DNA fragments that contain the pyrD or pyrR gene were PCR amplified and cloned into pCRA725. An internal segment of pyrD or pyrR was removed by inverse PCR. The resultant plasmids were introduced into C. glutamicum, and single-crossover cells were isolated based on kanamycin resistance. The isolated cells were cultivated in medium supplemented with 10% sucrose, and double-crossover cells were isolated. The gene modifications were confirmed by DNA sequencing of the PCR products around the modified region. The strain with a deletion of rho was constructed as described previously (19). The YT246 strain having the pyrR promoter-lacZ fusion gene (pyrRlacZ) and YT593 having the pyrR-pyrB-lacZ fusion was constructed as described in a previous paper (20). The 5= upstream region of pyrR was amplified by PCR using primers EcoRV-pyrR-680F and EcoRV-pyrR-15R (see Table S1 in the supplemental material). For pyrR-pyrB-lacZ, DNA amplification was performed using primers DraI-pyrR-680F and DraIpyrB-15R (see Table S1). The amplified fragment was digested with EcoRV for pyrR-lacZ and with DraI for pyrR-pyrB-lacZ and cloned into the DraI site of the pCRA741 reporter plasmid (17). A deletion mutant of the PyrR-binding loop sequence and the ribosome-binding region of the leader open reading frame (ORF) was conducted as follows. The plasmid containing pyrRlacZ or pyrR-pyrB-lacZ was used as a template for inverse PCR using the following primer sets (see Table S1 in the supplemental material): for the PyrR-binding loop sequence, NheI-pyrR-34R and NheI-pyrR-10F; for the ribosome-binding region of the leader ORF, BglII-pyrR-280R and BglII-pyrR-269F. The amplified fragment was digested with NheI or BglII and self-ligated. Construction of the mutant with a point mutation at the translation initiation codon of the leader ORF (substitution of GTG by ATG) was conducted as follows. The mutation was introduced by PCR using primer sets listed in Table S1 in the supplemental material (EcoRVpyrR-680F, pyrR-leaderATGR, pyrR-leaderATGF, and EcoRV-pyrR15R). The amplified fragment was digested with EcoRV and cloned into the DraI site of the pCRA741 reporter plasmid. The resultant plasmids were used to transform C. glutamicum R, and a recombinant cell with a kanamycin resistance marker was selected. Insertion of the promoter-lacZ


fusion gene between cgR_0734 and cgR_0735 was confirmed by PCR using primers LlacZLR-4354F and Ind7insert-checkR or primers LlacZLR6425R and Ind7insert-checkF. Construction of recombinant plasmid pCRC807 and pCRC808, carrying the leader ORF and the FLAG-tagged leader ORF gene, respectively, was carried out as follows. The region including the leader ORF was amplified from C. glutamicum R genomic DNA by PCR using primers EcoRIpyrR-680F and SalI-pyrR-3R (see Table S1 in the supplemental material). For the addition of the FLAG tag at the 3= terminus of the leader ORF, DNA was amplified using the primer set EcoRI-pyrR-680F and SalI leader-FL-R. The amplified DNA fragment was digested with EcoRI and SalI and cloned into the corresponding sites on pCRB1 (18, 21) to construct pCRC807 and pCRC808. Mapping of transcription initiation sites by 5= RACE-PCR. Transcription initiation sites were determined by using the SMART RACE (rapid amplification of cDNA ends) cDNA amplification kit (Clontech). 5= RACE-PCRs were carried out as recommended by the supplier with 1 ␮g of total RNA and gene-specific primer (see Table S1 in the supplemental material). The resulting PCR product was cloned into a pGEM-T Easy vector (Promega). Twelve clones were sequenced. Four clones mapped to the position 320 bp upstream and eight clones mapped to the position 317 bp upstream of the translation initiation start site of pyrR. ␤-Galactosidase assay. C. glutamicum R was grown aerobically in nutrient-rich A medium. The cells from 1 ml of culture were harvested by centrifugation and dissolved in 1 ml of Z buffer (Na2H/NaH2PO4 [pH 7.0], 10 mM KCl, 1 mM MgSO4, and 50 mM ␤-mercaptoethanol) with 2% toluene to permeabilize the cell. ␤-Galactosidase activity was determined with permeabilized cells as described previously (20). Real-time RT-PCR. Total RNA was isolated from exponentially growing cells (optical density at 610 nm [OD610] of 2.0) using NucleoSpin RNA (Macherey-Nagel). Quantitative reverse transcriptionPCR (qRT-PCR) was performed under the following experimental conditions. Each real-time RT-PCR mixture (20 ␮l) contained a 500 nM concentration of a primer set, 10 ␮l of Power SYBR green PCR master mix, eight units of RNase inhibitor, five units of murine leukemia virus (MuLV) reverse transcriptase (Applied Biosystems), and total RNA (20 ng for the leader ORF and 0.4 ng for 16S rRNA). The primers used in these reactions are listed in Table S1 in the supplemental material. The RT-PCR was performed using ABI 7500 Fast realtime PCR system (Applied Biosystems) with the following cycle pa-


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FIG 2 Effects of deletion of the pyrR and/or the PyrR-binding loop sequence on the expression of pyrR-lacZ. Growth of the wild type and the pyrD mutant strain in the nutrient-rich A medium (A) and in minimum BT medium (B) supplemented with uracil or UMP is shown. Squares, wild type without pyrimidine supplementation; exes, pyrD strain without pyrimidine supplementation; open triangles, pyrD strain with 10 ␮⌴ uracil; open diamonds, pyrD strain with 100 ␮M uracil; open circles, pyrD strain with 1 mM uracil; closed triangles, pyrD strain with 10 ␮M UMP; closed diamonds, pyrD strain with 100 ␮M UMP; closed circles, pyrD strain with 1 mM UMP. Similar results were obtained from independent experiments, and representative results are shown. (C) Schematic representation of the pyrR-lacZ reporter system. The construct involves the promoter, the leader ORF, the PyrR-binding loop region, the pyrR SD, and the first five codons of pyrR fused in frame to lacZ. A deleted region is depicted as a dotted line. The pyrR-lacZ reporter construct was integrated into the indel 7 region of the chromosome of C. glutamicum R. (D) Effects of the pyrR deletion on the expression of pyrR-lacZ. The C. glutamicum strain carrying pyrR-lacZ on the background of the wild-type strain (WT) and the pyrR deletion mutant strain (⌬pyrR) was cultured in nutrient-rich A medium with or without 1 mM uracil supplementation, and the ␤-galactosidase activity was monitored. The values are the means from three independent experiments, and standard deviations are indicated. (E) The C. glutamicum strain carrying the mutated pyrR-lacZ fusion that lacks the PyrR binding motif (⌬PyrR-binding loop) on the wild-type or the pyrR deletion mutant background (⌬pyrR) was cultured in nutrient-rich A medium with or without uracil supplementation, and the ␤-galactosidase activity in the cell was measured. The values are the means from three independent experiments, and standard deviations are indicated. rameters: one cycle of 50°C for 30 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 15s. The result of 16S rRNA was used as an internal control. Western blotting. A 10-ml aliquot of cell cultures grown to an OD610 of 2.0 was collected by centrifugation. The pellets were mixed with glass beads and 0.5 ml of buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 1 mM EDTA). Cells were disrupted by vigorous vortexing. The supernatant after centrifugation at 10,000 ⫻ g for 2 min was used as the protein extract. The extract containing 10 ␮g of proteins was subjected to 0.1% (wt/vol) SDS–12% (wt/vol) polyacrylamide gel electrophoresis. Separated proteins were blotted to the polyvinylidene difluoride membrane (Immobilon-P; Millipore) and reacted with monoclonal anti-FLAG M2 antibody (Sigma) and horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare). Chemiluminescence reaction was done using ChemiLumi One Super (Nacalai Tesque). The signal was scanned by a luminescent image analyzer (Fuji model LAS-3000).

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RESULTS

Effects of pyrR deletion on the expression of pyrR-lacZ. Five genes (from cgR_1661 to cgR_1657) in the genome of C. glutamicum R (22) are annotated as de novo pyrimidine biosynthesis genes, i.e., pyrB, pyrC, carA, carB, and pyrF, which code for aspartate carbamoyltransferase, dihydroorotase, carbamoyl phosphate synthase small subunit, carbamoyl phosphate synthase large subunit, and orotidine-5=-phosphate decarboxylase, respectively (Fig. 1A and C). These genes are clustered with a putative regulator gene pyrR (cgR_1662). cgR_1571 (annotated as pyrD) and cgR_2670 (annotated as pyrE) encode dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, respectively, and these two genes are distantly located from the other pyrimidine de novo biosynthesis genes.




FIG 3 Expression of the pyrR-pyrB-lacZ reporter gene. The C. glutamicum strain carrying the pyrR-pyrB-lacZ fusion was cultured for 6 h in nutrient-rich A medium with or without 1 mM uracil supplementation, and the ␤-galactosidase activity was measured. The values are the means from three independent experiments, and standard deviations are indicated.

The transcription start site of the pyr gene cluster was determined by the 5=-RACE method (Fig. 1B). The two sites were detected near positions located 320 and 317 bp upstream of the translation initiation start site of pyrR. The putative ⫺10 (TAA AAT) region that matches the consensus of C. glutamicum SigAdependent promoters (TANANT) is found in the pyrR 5= upstream region. The ⫺35 sequence is not obvious for the pyrR promoter, which is often observed in C. glutamicum (10). A sequence exhibiting 77% identity to the PyrR-binding loop of M. smegmatis (8) is present adjacent to the pyrR gene (Fig. 1D). In addition, we found a possible open leading frame for a 78-aminoacid polypeptide upstream of the pyrR gene, which has not been reported in other bacteria, including M. smegmatis. The intergenic region between the leader ORF and pyrR is 34 bp long. We tested the growth of the wild type and the pyrD deletion strain in nutrient-rich A medium or BT minimum medium with varied concentrations of uracil or UMP. Disruption of pyrD gene resulted in the growth defect without the addition of pyrimidine which was partially recovered by the addition of uracil at 100 ␮M in nutrient-rich A medium, and addition of 1 mM uracil completely recovered growth. In the BT minimum medium, addition of 10 ␮M uracil slightly recovered growth and addition of 100 ␮M uracil further supported growth. In contrast, addition of UMP did not support growth. These data indicate that C. glutamicum can utilize external uracil for growth but not UMP. Nutrient-rich A medium may contain a small amount of pyrimidine, which has the same effect as 10 ␮M uracil on the growth of the pyrD strain. This pyrimidine level is not enough for the pyrD strain to reach an OD610 of 1.0. To examine the role of PyrR on expression of the pyrR gene cluster, we constructed a translational fusion of pyrR with the lacZ reporter gene (pyrR-lacZ). The DNA fragment of pyrR that was fused corresponds to the region between bp ⫺360 and ⫹335 with respect to the transcription start site, containing five amino acids derived from the pyrR ORF (Fig. 2C). The gene fusion was integrated into the C. glutamicum genome at strain-specific island 7 (SSI7), which is distant from the pyr gene cluster, and effects of deletion of the pyrR gene on the expression of pyrR-lacZ were examined. Growth of the strain carrying pyrR-lacZ on the genetic


background of a pyrR deletion mutant was similar to that of the strain on the wild-type background (data not shown). Because expression of the pyr genes is affected by the addition of uracil in M. smegmatis (8), we tested the effect of addition of 1 mM uracil to nutrient-rich A medium supplemented with glucose in the following experiments. The expression of pyrR-lacZ in exponentially growing cells was investigated by assaying ␤-galactosidase activity (Fig. 2D). Under low-uracil conditions, the pyrR-lacZ expression in the pyrR deletion mutant background was upregulated about 4-fold compared to that in the wild-type background. Deletion of pyrR had no effect on the ␤-galactosidase activity in the strain carrying gnd-lacZ, a translational fusion of gnd encoding 6-phosphogluconate dehydrogenase with the lacZ gene (see Fig. S1 in the supplemental material), indicating that the effect of pyrR deletion is dependent on the 5= upstream region of pyrR but not the region of the lacZ gene. We also tested the expression of pyrR-lacZ in the pyrD deletion strain. However, the pyrR-lacZ expression level was not significantly altered compared with that of the wild-type strain, with or without the addition of various amounts of uracil to the nutrient-rich A or BT medium. Therefore, we did not continue the pyrimidine depletion experiment with pyrD and focused on the downregulation of pyrR-lacZ in the presence of 1 mM uracil. These results confirmed the involvement of PyrR in the regulation of pyrR-lacZ. In the wild-type background, pyrR-lacZ was downregulated by the addition of uracil. The addition of uracil also resulted in a decrease in pyrR-lacZ expression in the pyrR deletion background, indicating the presence of a uracil-responsive regulatory system independent of PyrR. Effects of deletion of the PyrR binding motif on the expression of pyrR-lacZ. A sequence that resembles the PyrR-binding motif of M. smegmatis is present upstream of the pyrR gene (Fig. 1B). In M. smegmatis, binding of PyrR to the PyrR-binding motif prevents the translation initiation of pyrR by sequestering the Shine-Dalgarno sequence. We constructed the mutated pyrR-lacZ fusion that lacks the PyrR binding motif to test whether PyrR regulates the pyrR-lacZ expression through this sequence in C. glutamicum (Fig. 2C). The pyrR-lacZ strains with and without deletion of the PyrR-binding motif was cultivated under low- or high-uracil conditions, and the expression of pyrR-lacZ was investigated by assaying ␤-galactosidase activity. The expression level of the mutated pyrR-lacZ gene was markedly higher than that of the intact pyrR-lacZ gene (Fig. 2E). We assessed the stability of pyrR-lacZ mRNA by determining the mRNA level after addition of rifampin. The lacZ mRNA half-life was 1.0 ⫾ 0.3 min for intact pyrR-lacZ and 1.1 ⫾ 0.3 min for mutated pyrR-lacZ. These results indicate that deletion of the PyrR-binding loop sequence does not result in a change in the mRNA stability. Deletion of the pyrR gene in the strain carrying the mutated pyrR-lacZ gene showed an insignificant effect on the pyrR-lacZ expression compared to that in the parent strain, indicating that PyrR operates through the PyrRbinding motif, as expected. Interestingly, deletion of the PyrRbinding motif resulted in a higher level of pyrR-lacZ expression than the deletion of the pyrR gene (Fig. 2C and D). In addition, uracil had no effect on the mutated pyrR-lacZ expression (Fig. 2E). These results indicate that the PyrR-binding loop region affects the expression of pyrR-lacZ by both PyrR-dependent and PyrRindependent mechanisms and that this sequence is essential for the uracil response. We then investigated the role of the PyrR-binding motif in


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FIG 4 Role of the leader ORF translation in the expression of pyrR-lacZ. (A) Schematic representation of the pyrR-lacZ fusion. (B) The C. glutamicum strain carrying the wild-type or mutated pyrR-lacZ reporter in which the SD region and the translation initiation codon of the leader ORF were deleted (⌬leader) or in which the initiation codon of the leader ORF (GTG) was replaced with ATG was cultured in nutrient-rich A medium with or without uracil supplementation. The ␤-galactosidase activity in the cells was measured. The values are the means from three independent experiments, and standard deviations are indicated. (C) The C. glutamicum strain carrying the mutated pyrR-lacZ fusion that lacks the PyrR binding motif (⌬PyrR-binding loop) was cultured, and the ␤-galactosidase activity was measured as described for panel B. (D) Immunoblot analysis of the leader ORF product. Total proteins were extracted from C. glutamicum R transformed with a vector plasmid (pCRB1) or with a plasmid for expression of the FLAG-tagged leader ORF (pCRC808). Proteins were separated by SDS-PAGE and subjected to immunoblot analysis using anti-FLAG antibody. (E) Expression of the leader ORF mRNA. The C. glutamicum strain with pyrR-lacZ on the chromosome in the genetic background of the wild-type or the pyrR-deleted strain transformed with a vector plasmid (pCRB1) or a plasmid for expression of the leader ORF (pCRC807) was grown in nutrient-rich A medium supplemented with uracil. The mRNA levels of the leader ORF were determined by qRT-PCR. (F) Effect of the overexpression of the leader ORF on the expression of pyrR-lacZ. The C. glutamicum strain with pyrR-lacZ on the chromosome on the wild-type or the pyrR deletion mutant background harboring pCRB1 or pCRC807 was grown in nutrient-rich A medium supplemented with uracil, and the ␤-galactosidase activity was monitored.

expression of the pyrB gene, which is located downstream of pyrR (Fig. 1A). The DNA fragment including the pyrR 5= upstream region with or without the PyrR-binding motif, the entire pyrR gene, and a part of pyrB encoding the initial five amino acids was fused to the lacZ gene and integrated into the chromosome of C. glutamicum. The expression of the pyrR-pyrB-lacZ translational fusion was upregulated more than 2-fold by deletion of the PyrRbinding loop sequence (Fig. 3). Addition of uracil resulted in a decrease in pyrR-pyrB-lacZ expression, but the uracil effect was insignificant in the strain with a deletion of the PyrR-binding

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motif. The effect of deletion of the PyrR-binding motif was similar to, but much smaller than, that in the case of the pyrRlacZ fusion. Involvement of the leader ORF translation in the expression of pyrR-lacZ. In the upstream region of pyrR, a possible open reading frame is present (Fig. 1B). Interestingly, this ORF overlaps the PyrR-binding motif. It is hypothesized that in E. coli, tight coupling of the ribosome translating a leader ORF upstream of pyrB with RNAP prevents the formation of the transcriptional terminator structure which is involved in the transcription atten-




FIG 5 Effects of rho deletion on the expression of pyrR-lacZ. (A) The C. glutamicum strain carrying the pyrR-lacZ fusion in the genetic background of the rho deletion mutant (⌬rho) or the pyrR rho double deletion mutant (⌬pyrR ⌬rho) was cultured in nutrient-rich A medium with or without uracil supplementation, and the ␤-galactosidase activity in the cells was measured. The values are the means from three independent experiments, and standard deviations are indicated. (B) The C. glutamicum strain carrying the mutated pyrR-lacZ fusion that lacks the PyrR binding motif (⌬PyrR-binding loop) in the genetic background of the ⌬rho mutant or the ⌬pyrR ⌬rho mutant was cultured in nutrient-rich A medium with or without uracil supplementation, and the ␤-galactosidase activity in the cells was measured. The values are the means from three independent experiments, and standard deviations are indicated.

uation mechanism (1). Although the resemblance between the PyrR-binding motifs of C. glutamicum and M. smegmatis suggests that C. glutamicum PyrR acts at translation initiation (Fig. 1D), not at transcription termination, it is possible that translation of the leader ORF affects expression of the downstream gene. To test this possibility, we constructed a strain carrying the mutated pyrRlacZ gene that has a deletion of the ribosome binding region and the translation initiation codon of the leader ORF, thereby eliminating translation. Alternatively, the initiation codon (GTG) was replaced with ATG, which is expected to increase the translation efficiency of the leader ORF (Fig. 4A). Under low-uracil conditions, elimination of translation of the leader ORF resulted in a 40% decrease in pyrR-lacZ expression (Fig. 4B). In contrast, changing the translation initiation codon resulted in about a 3-fold increase in the expression of pyrR-lacZ. These results indicate that the leader ORF is actually translated in the cell and the translation affects the expression of the downstream gene. Interestingly, the effect of the translation of the leader ORF was not observed in the genetic background of a pyrR deletion mutant, indicating that the effect involves the action of PyrR. Addition of uracil to the culture medium resulted in a decrease in the expression of pyrR-lacZ in all the strains tested. We also tested the effect of the translation of the leader ORF in the strain with the PyrRbinding loop sequence deleted, but no effect of leader ORF translation was observed in this strain, as expected (Fig. 4C). We also tested the effect of overexpression of the leader ORF in trans by introducing the expression plasmid (pCRC807). Introduction of the pCRC807 resulted in a 25-fold increase in the level of mRNA for the leader ORF (Fig. 4E). We detected the FLAGtagged protein for the leader ORF by Western blotting (Fig. 4D), confirming its translation in C. glutamicum. However, overexpression of the leader ORF did not affect the expression of pyrRlacZ in either the wild-type or the pyrR deletion mutant background (Fig. 4F). These results indicate that the translation of the leader ORF affects the expression of the downstream gene in cis and that the protein product of the leader ORF does not affect the expression of pyrR-lacZ, at least under the conditions used.


Effects of rho deletion on the expression of pyrR-lacZ. The results described above indicate the existence of a uracil-controlled regulator other than PyrR. We assumed that regulation of the pyrimidine de novo biosynthesis genes in C. glutamicum is also controlled at the transcriptional termination or elongation step. We deleted rho, encoding the transcription termination factor Rho, to test whether it is involved in the regulation of pyrimidine de novo biosynthesis genes (Fig. 5). Deletion of rho resulted in an increase in pyrR-lacZ expression of about 2-fold. However, the repression effect of uracil was still observed. We next tested the effect of a double deletion of rho and pyrR on the expression of pyrR-lacZ. Deletion of both rho and pyrR resulted in further increased expression of pyrR-lacZ compared to the single deletion of pyrR or rho (Fig. 2D and 5A). Interestingly, the uracil-mediated downregulation of pyrR-lacZ was not observed in the double deletion mutant. In the absence of the PyrR-binding loop, the addition of uracil showed no effect on the pyrR-lacZ expression in either the rho single mutant or the rho pyrR double deletion mutant, as expected (Fig. 5B). To verify the role of Rho in the uracil response, we examined the mRNA levels of the pyrimidine de novo biosynthesis genes (Fig. 6A). In all the genes tested, the mRNA levels were upregulated 2- to 4-fold by deletion of rho. The mRNA levels of these genes, besides pyrF, in the wild-type cells were downregulated by 40% in response to uracil supplementation (Fig. 6B). The uracil effect on pyrR, pyrB, and pyrC was slightly reduced by deletion of the rho gene: addition of uracil resulted in a 20% decrease in the mRNA levels. The effect of rho deletion was not clear for carA, carB, and pyrF mRNA. Although it has not been reported that Rho is involved in the regulation of pyrimidine de novo biosynthesis genes in E. coli or B. subtilis, our results indicate that Rho is involved in the downregulation of pyrR, pyrB, and pyrC in response to the addition of uracil in C. glutamicum. DISCUSSION

In this study, we investigated the regulation of the pyrimidine de novo biosynthesis genes in C. glutamicum. The PyrR protein has


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FIG 6 Effects of rho deletion on the expression of pyr mRNAs. (A) Total RNAs prepared from wild-type or rho gene deletion cells (⌬rho) grown in nutrient-rich A medium with or without 1 mM uracil supplementation were subjected to qRT-PCR analysis using primers specific for the pyr genes. mRNA levels are presented relative to the value obtained for the wild-type cells grown without uracil supplementation. The values are the means from three independent experiments, and standard deviations are indicated. (B) Ratio of the pyr mRNA expression in cells with uracil supplementation to that in cells without uracil supplementation.

67% identity and 82% similarity to M. smegmatis PyrR, which is involved in the regulation of the pyrR operon through binding to the mRNA (8). In addition, a sequence which is similar to the PyrR-binding loop is present upstream of the pyrR gene (Fig. 1). We observed that expression of the pyrR-lacZ translational fusion is upregulated by deletion of pyrR (Fig. 2D). We also observed that deletion of the PyrR-binding loop sequence results in a higher level of pyrR-lacZ expression and eliminates the effect of pyrR deletion (Fig. 2E). These results indicate that PyrR negatively regulates the pyrR-lacZ expression through the PyrR-binding loop sequence. Binding of PyrR to the binding loop sequence probably stabilizes the Shine-Dalgarno (SD)–anti-SD base pair, as has been suggested for M. smegmatis (8). The effect of deletion of the PyrR-

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binding loop sequence is more prominent than that of a deletion of pyrR alone or of both pyrR and rho. Sequestration of the SD sequence may occur, to some extent, in the absence of PyrR. We also found that deletion of the PyrR-binding loop sequence results in the increased expression of pyrR-pyrB-lacZ (Fig. 3), suggesting that PyrR regulates the expression of aspartate carbamoyltransferase, encoded by the pyrB gene. The translation termination codon of pyrR lies adjacent to the translation initiation codon of the downstream gene, pyrB. Therefore, expression of pyrB might be translationally coupling with pyrR and is also under the control of PyrR. We found no PyrR-binding loop sequence other than in the upstream region of pyrR on the genome of C. glutamicum. Therefore, regulation of pyrD or pyrE may be different from that




of pyrR. Further studies, using pyrD-lacZ or pyrE-lacZ strains, are needed to elucidate the regulatory mechanism of these genes. In the 5= upstream region of pyrR, we found an open leading frame (Fig. 1B). The leader ORF is conserved in the C. glutamicum strains SCgG1 and SCgG2 but not in C. glutamicum ATCC 13032 or M. smegmatis. Disruption of the SD sequence and the initiation codon of the leader ORF resulted in the reduced expression of pyrR-lacZ (Fig. 4). Inversely, changing the initiation codon to ATG, which is expected to increase the translation of the leader ORF, resulted in the increased expression of pyrR-lacZ. These results indicate that the leader ORF is translated in the cell and this translation positively affects the expression of the downstream gene. Plasmid-based expression of the leader ORF did not increase the pyrR-lacZ expression (Fig. 4), indicating that the 78-aminoacid polypeptide derived from the leader ORF is not involved in the control of the pyr gene cluster. Currently, the role of this small protein remains unidentified. The effect of the translation of the leader ORF was not observed in the pyrR deletion mutant, suggesting that this effect is mediated through the action of PyrR. Since the translation of the leader ORF terminates in the PyrRbinding loop region, the ribosome translating the leader ORF may mask the anti-SD sequence, resulting in the revelation of the SD sequence of the pyrR gene, or the translating ribosome may simply inhibit the binding of PyrR to the PyrR-binding loop. The expression of pyrR-lacZ is downregulated in the presence of uracil in both the wild-type and pyrR deletion mutant backgrounds (Fig. 2D). However, the uracil-dependent downregulation of pyrR-lacZ was not observed in the absence of the PyrRbinding loop sequence (Fig. 2E). These results suggest that there is a uracil-responsive regulatory mechanism dependent on the PyrR-binding loop but independent of PyrR. It is reported that in Lactobacillus plantarum, the pyr genes are controlled by two PyrR proteins (23). PyrR1 responds to pyrimidine availability of the cell, and PyrR2 responds to inorganic carbon. However, there is no other PyrR family protein in C. glutamicum. Interestingly, we found that simultaneous deletion of rho and pyrR results in the loss of the uracil response of pyrR-lacZ (Fig. 5). We also observed that the expression levels of pyrR, pyrB, and pyrC mRNAs are downregulated in response to uracil. This downregulation was partially recovered by the rho gene deletion (Fig. 6). The remaining uracil effect is likely attributable to the PyrR-mediated regulation. Because deletion of both pyrR and rho is required to eliminate the downregulation by uracil, we conclude that the pyrimidine de novo biosynthesis gene cluster is regulated by the two mechanisms mediated by PyrR and Rho. Because the uracil effect is insignificant in the strain with a deletion of the PyrRbinding loop sequence, regulation by both PyrR and Rho requires the PyrR-binding loop region. It would be interesting to determine whether binding of PyrR to the PyrR-binding loop region inhibits the action of Rho. Regulation of pyrimidine de novo biosynthesis genes in E. coli and B. subtilis is known to be controlled by transcriptional attenuation, but it has not been reported that Rho is involved in the regulation in these bacteria. How does uracil affect the expression of pyrR, pyrB, and pyrC mRNAs through the action of Rho? It is possible that the leader region of pyrR senses the increased pyrimidine level by U-rich sequence like the pausing sequence of the E. coli pyrBI (2–4). Another possibility is that the leader region of pyrR directly binds to uracil or its derivative like an adenine riboswitch (24–26). Entry of Rho into the


pyrR leader region may be modulated by these mechanisms. However, we could not rule out the possibility that the effect of rho deletion on the expression of pyrR, pyrB, and pyrC is indirect. Further studies such as in vitro transcription assays with the purified Rho protein are required to reveal the mechanism of the uracil response in C. glutamicum. To date, most of the studies of the gene regulation in C. glutamicum have been focused on the transcriptional initiation step mediated by DNA-binding transcriptional regulators. Our study showed the presence of posttranscriptional regulation mediated by mRNA binding protein in this industrially important microorganism, and these examples should be expanded in the near future (27, 28). ACKNOWLEDGMENT This work was partially supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO).

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Regulation of de novo sphingolipid biosynthesis by the ORMDL proteins and sphingosine kinase-1.

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