APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.5.2183–2190.2001

May 2001, p. 2183–2190 Vol. 67, No. 5

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Organization and Transcriptional Analysis of a Six-Gene Clusteraround the rplK-rplA Operon of Corynebacterium glutamicum

Encoding the Ribosomal Proteins L11 and L1CARLOS BARREIRO,1 EVA GONZALEZ-LAVADO,2 AND JUAN F. MARTIN1,2*

Instituto de Biotecnologia (INBIOTEC), Parque Cientifico de Leon, 24006 Leon,1 and Facultad de CienciasBiologicas y Ambientales, Area de Microbiologia, Universidad de Leon, 24071 Leon,2 Spain

Received 5 December 2000/Accepted 22 February 2001

A cluster of six genes, tRNATrp-secE-nusG-rplK-rplA-pkwR, was cloned and sequenced from a Corynebacteriumglutamicum cosmid library and shown to be contiguous in the C. glutamicum genome. These genes encode atryptophanyl tRNA, the protein translocase component SecE, the antiterminator protein NusG, and theribosomal proteins L11 and L1 in addition to PkwR, a putative regulatory protein of the LacI-GalR family. S1nuclease mapping analysis revealed that nusG and rplK are expressed as separate transcriptional units andrplK and rplA are cotranscribed as a single mRNA. A 19-nucleotide inverted repeat that appears to correspondto a transcriptional terminator was located in the 3* region downstream from nusG. Northern analysis withdifferent probes confirmed the S1 mapping results and showed that the secE-rplA four-gene region gives riseto four transcripts. secE was transcribed as a 0.5-kb monocistronic mRNA, nusG formed two transcripts of 1.4and 1.0 kb from different initiation sites, and the two ribosomal protein genes rplK and rplA were cotranscribedas a single mRNA of 1.6 kb. A consensus L1 protein binding sequence was identified in the leader region of therplK-rplA transcript, suggesting that expression of the rplK-rplA cluster was regulated by autogenous regulationexerted by the L1 protein at the translation level. The promoters of the nusG and rplK-rplA genes weresubcloned in a novel corynebacterial promoter-probe vector and shown to confer strong expression of thereporter gene.

Ribosomal proteins of both gram-positive and gram-nega-tive bacteria are involved in the translational control of theexpression of genes for the initiation of physiological and mor-phological differentiation (29), although the molecular mech-anisms involved are poorly known. One of these mechanisms,relA control, involved in adaptation of the cells to amino acidstarvation, is mediated by the hyperphosphorylated guanosinetetraphosphate and pentaphosphate [(p)ppGpp]. These com-pounds are formed from GTP and the pyrophosphoryl groupof ATP in a reaction mediated by the RelA factor that isassociated with ribosomal proteins. The RelA protein becomesactive when uncharged tRNA accumulates due to the lack ofthe corresponding amino acids, and ribosomes are unable towork (11).

In Escherichia coli, a functional ribosomal protein, L11, en-coded by the rplK gene, is required for the activation of theRelA factor (10). Similarly, a functional rplK gene product isrequired for (p)ppGpp biosynthesis in Bacillus subtilis (41) andStreptomyces coelicolor (30, 32).

Corynebacterium glutamicum and Brevibacterium lactofer-mentum, renamed Corynebacterium lactofermentum (2), arewidely used for industrial production of amino acids (22, 37). Alarge number of genes involved in primary metabolism havebeen cloned from corynebacteria (9, 24) and have been used toimprove the production of amino acids (25).

Amino acid accumulation in corynebacteria follows a de-

crease in rRNA synthesis and growth (E. Gonzalez-Lavado, C.Barreiro, and J. F. Martin, unpublished data). Initial evidenceindicates that the growth rate of corynebacteria is inverselycorrelated with the cellular (p)ppGpp concentration (36). Theroles of ribosomal proteins and the rel mechanism in the switchfrom the growth phase to the amino acid production phase incorynebacteria are of great interest.

A relA (also similar to spoT) gene of C. glutamicum encodinga bifunctional enzyme with (p)ppGpp synthetase and (p)p-pGpp-degrading activity was cloned (45). However, the role ofthe L11 ribosomal protein in the synthesis of (p)ppGpp and inthe switch from the growth phase to the amino acid accumu-lation stage in corynebacteria remains unknown. Ribosomalprotein engineering is receiving increasing attention as a toolto modify growth-related control mechanisms (31). It was,therefore, of great interest to clone the gene encoding L11 andother ribosomal proteins to elucidate its role in the mechanismof rel control in C. glutamicum. We report the cloning, orga-nization, and transcriptional analysis of a six-gene region ofcorynebacteria that contains the genes for the L11 and L1ribosomal proteins.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. All bacterial strains andplasmids used in this work are listed in Table 1. E. coli was grown in LuriaBertani broth (38) at 37°C. C. glutamicum ATCC 13032 and B. lactofermentumR31, renamed C. lactofermentum (2), a high-efficiency host strain for plasmidtransformation (25, 40), were grown in trypticase-soy broth (TSB) at 30°C. E. colitransformants were selected in the presence of ampicillin (100 mg/ml), and C.glutamicum and C. lactofermentum transformants were selected in media withkanamycin (30 mg/ml).

* Corresponding author. Mailing address: Instituto de Biotecnologıa(INBIOTEC), Parque Cientıfico de Leon, Avda. del Real, no. 1, 24006Leon, Spain. Phone: (34 987) 210308. Fax: (34 987) 210388. E-mail:degjmm@unileon.es.

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DNA isolation and manipulation. E. coli plasmid DNA was obtained by alka-line lysis as described by Birnboim and Doly (3). Total C. glutamicum DNA wasprepared as described by Martın and Gil (25), and C. lactofermentum and C.glutamicum plasmidic DNA were prepared by the method of Kieser (17, 25).

DNA manipulations were performed as described by Sambrook et al. (38).DNA fragments were isolated from agarose gels using the Geneclean II kit (Bio101).

E. coli cells were transformed as described by Hanahan (12), whereas coryne-bacteria were transformed by electroporation (8, 25).

Southern hybridizations. DNA fragments were vacuum blotted from 0.8%agarose gels to nylon membranes (42). Labeling of the DNA probes was donewith digoxigenin (Boehringer Mannheim, Mannheim, Germany) according tothe manufacturer’s instructions.

DNA sequencing. The nucleotide sequence of the cloned region was deter-mined in both strands by the dideoxynucleotide chain termination method usingan automatic DNA sequencer (ALF; Pharmacia). The nucleotide and deducedamino acid sequences were compared with those in the EMBL and GenBankdatabases by using the Clustal W program (43).

RNA extraction. Total RNA from corynebacteria was extracted by a methodbased on that of Eikmanns et al. (9) except that the cell pellet, obtained aftercentrifugation, was frozen with liquid nitrogen and kept at 270°C before RNAextraction. The RNA concentration was determined spectrophotometrically bydetermining the absorbance at 260 nm.

S1 nuclease protection assays. Low-resolution S1 mapping was performed asdescribed by Sambrook et al. (38) using 200 mg of total C. glutamicum RNA and100 to 150 ng of probe DNA. Treatment with S1 nuclease was made for 30 min(longer times were found to be less suitable). tRNA from Saccharomyces cerevi-siae (type X-SA; Sigma) was used as a negative control for the hybridizations andthe complete probe, or only the homologous part of the probe, was used as apositive control.

Northern hybridization. Denaturing RNA electrophoresis was performed in0.9% agarose gels in MOPS (morpholine propane sulfonic acid) buffer (20 mMMOPS, 5 mM sodium acetate, 1 mM EDTA [pH 7.0]) with 17% (vol/vol)formaldehyde. RNA (30 mg) was dissolved in denaturing buffer (50% form-amide, 20% formaldehyde, 20% MOPS [53]) with 10% DYE (38) and 1%ethidium bromide. DNA probes were labeled with 32P by nick translation (Pro-mega).

Hybridizations were performed at 42°C with 50% formamide; membranewashings were carried out as described by Sambrook et al. (38) except that thesecond washing was done at 42°C. The labeled bands were detected by autora-diography with exposition times of 72 and 96 h.

Promoter-probe plasmids. A new promoter-probe plasmid, pULCE0, wasconstructed from the vector pUL880M (23, 26). pUL880M was linearized withBglII. The protruding BglII ends were partially filled with dGTP and dATP usingthe Klenow fragment of the DNA polymerase. A polylinker containing therestriction sites BstXI and ClaI (sites that were absent in the rest of the vector)was synthesized and linked to the linearized pUL880M vector. The appropriateorientation of the polylinker was confirmed by PCR using the following primers:Oligo 1 (59-TCGCCAGAGCTCTGGATCGATA-39), identical to that used toconstruct the polylinker, and Oligo 2 (59-GGGAGCGGCGATACCGTAAA-39),internal to the kanamycin resistance gene of pUL880M. An amplification bandof 804 bp was obtained that corresponded to the insertion of the polylinker in thecorrect orientation (Fig. 1). Finally, the region of the polylinker was sequencedin both strands (Fig. 1), and the functionality of the polylinker was confirmed by

hydrolysis with restriction endonucleases. This vector (unlike pUL880M) alloweddirected cloning of promoter sequences with several different cohesive ends.

Subcloning of the promoters. The promoter region upstream of rplK, control-ling expression of rplK-rplA, was amplified by PCR as a 225-bp fragment con-taining its own translation start codon and was subcloned in pULCE0. Thispromoter region was named PrplKA. The construction with the promoter PrplKA

was introduced by transformation into C. lactofermentum R31 (a high-efficiencyhost strain for initial transformation), reisolated, and then transformed into C.glutamicum. Direct transformation of C. glutamicum with the ligation mixturewas inefficient, but transformation with plasmid isolated from C. lactofermentumR31 was quite efficient.

FIG. 1. Construction of the promoter-probe vector pULCE0. Thisvector was constructed by inserting a polylinker (shaded nucleotidesequence) with unique restriction sites (absent from other places in thevector) in the BglII site upstream from the promoterless kanamycinresistance marker. One of the BglII sites was removed (X) by appro-priate selection of oligonucleotides (arrowhead). Reverse-type lettersindicate BglII ends partially filled to avoid vector religation. KmR,kanamycin-resistance gene; HygR, hygromycin resistance gene; ApR,ampicillin resistance gene. ori E. coli is the colE1 plasmid replicationorigin, and ori B. lactofermentum corresponds to the replication originof plasmid pBL1 of B. lactofermentum (synonymous with C. lactofer-mentum). Ttrp, transcriptional terminator of the C. lactofermentumtryptophan operon (to avoid readthrough expression).

TABLE 1. Strains and plasmids used in this work

Strain or plasmid Characteristics Source or reference

E. coli DH5a F2 recA1 endA1 gyrA96 thi-1 hsdR17 (rk2mk

1) sup44 relA1 l2

(w80dlacZDM15) D(lacZYA-argF) U16913

E. coli WK6 mutS F9 lacIqZDM15 proAB D(lac-roAB) galE strA mutS215::Tn10 18C. glutamicum ATCC 13032 Wild type; Nalr ATCCa

B. lactofermentum R31 MeLysr Aecr Nalr 39Plasmids

pBluescript SK(1) or KS(1) E. coli vector; Apr lacZ ori f1 1M13K07 M13-derived phage; Kmr; used as helper phage for ssDNAb isolation 44, 27pUL880M E. coli-Corynebacterium vector; Apr Hygr; neo gene without promoter

used for promoter analysis21

pULCE0 pUL880M with new cloning sites This work

a ATCC, American Type Culture Collection.b ssDNA, single-stranded DNA.

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Similarly, the promoter region upstream of nusG (named PnusG) was sub-cloned as a 567-bp fragment with its own translation start codon in the promoter-probe vector pULCE0 and was introduced first into C. lactofermentum and theninto C. glutamicum.

Ten transformants containing the plasmid with the PrplKA promoter (namedpULCErplKA) and another 10 containing the plasmid with the PnusG promoter(named pULCEnusG) were selected and used to test the promoter strength bygrowing them in plates of TSB medium with increasing concentrations of kana-mycin (from 100 to 1,200 mg/ml).

Nucleotide sequence accession number. The nucleotide sequence of the4.42-kb fragment containing the tRNATrp-secE-nusG-rplK-rplA-pkwR cluster wasdeposited in the EMBL database under accession number AJ300822.

RESULTS

Cloning the DNA region encoding L11 and L1 ribosomalproteins. Since the rplA and rplK genes (encoding the L1 andL11 proteins, respectively) are linked to the secE and nusGgenes in some gram-positive bacteria, a search for this genecluster in the cosmid library of C. glutamicum was made usingtwo probes, A and B. Probe A corresponded to an 800-bpBglII-SalI fragment containing the secE gene of Streptomyceslividans (J. Blanco and J. F. Martın, unpublished data), andprobe B was a 1,500-bp KpnI fragment containing the S. livi-dans nusG gene.

The results of Southern hybridization with total DNA of C.glutamicum digested with SalI showed a single 6.6-kb SalI bandthat hybridized with both probes. Similarly, a 5.5-kb BamHIfragment hybridized with both probes, suggesting that the secEand nusG genes are linked and probably maintain the sameorganization as in other gram-positive bacteria.

The C. glutamicum cosmid library was then hybridized withan 800-bp KpnI fragment containing the secE of S. lividans, and10 positive cosmids were selected and reconfirmed by a secondhybridization with the secE and nusG probes (see above). Cos-mid pCG1 was selected and used for further studies. pCG1DNA was digested with BamHI, and the hybridizing 5.5-kbband was subcloned in pBluescript SK(1) to form the newplasmid pB1.

The cloned DNA fragment comprises six genes, includingthe rplA-rplK cluster. Cosmid pCG1 was mapped with restric-tion endonucleases. A PstI-BamHI region of 4.42 kb was en-tirely sequenced in both strands, showing a G1C content of53.48%. Computer analysis of the sequence taking into ac-count the codon usage of corynebacteria (23) revealed fiveopen reading frames (ORFs) in the sequenced DNA fragment(Fig. 2A).

Upstream of the first ORF, we found a gene encoding atRNATrp (73 bp). Comparison of the nucleotide sequences andthe deduced amino acid sequences of the five ORFs with thesequence of proteins in the Swiss-Prot data bank indicates thatthe first, ORF1, encodes a protein of 111 amino acids thatshows homology with the membrane protein SecE of differentgram-positive and gram-negative bacteria (36.1% identity withSecE of Mycobacterium tuberculosis). ORF1 was separatedfrom the tRNATrp gene by an intergenic region of 230 bp. Asequence of 60 bp present in the upstream region of ORF1 isidentical to that of a promoter region that was randomlycloned as a transcription initiation region (34). Downstreamfrom ORF1 and in the same orientation we found ORF2,which encodes a protein of 318 amino acids and a deduced

molecular mass of 34.7 kDa that shows very high homologywith the antiterminator protein NusG (48.9% identity withNusG from M. tuberculosis). Downstream from nusG, ORF3and ORF4 encode proteins of 145 and 236 amino acids thatshow extensive homology with the ribosomal proteins L11 andL1, respectively (85.4% identity with the L11 protein of M.tuberculosis and 71.5% identity with the L1 protein of M. tu-berculosis). These two genes have been named rplK and rplA,respectively.

ORF5 is located at the end of the sequenced fragment andin the opposite orientation to ORF4. It corresponds to a trun-cated gene (918 bp) encoding a protein fragment (lacking theN-terminal end) with significant homology (63.3% identity atthe nucleotide level) with the pkwR gene of Thermomonosporacurvata (14). PkwR is a putative regulatory protein that is amember of the LacI-GalR family of regulatory proteins.

These results clearly indicate that the region cloned in cos-mid pCG1 contains the desired genes, rplK and rplA, for theribosomal proteins L11 and L1. In some gram-positive bacte-ria, the genes rplJ and rplL, encoding other ribosomal proteins,map downstream of rplA. To study the possibility that a DNArearrangement might have occurred during cosmid packaging,the entire region of cosmid pCG1 and the total DNA of C.glutamicum were digested in parallel and hybridized with a1,820-bp HindIII-BamHI probe (Fig. 3) that contains both thecomplete rplA gene and the sequenced ORF5.

The results (Fig. 3) showed unequivocally that ORF5(pkwR) is adjacent to rplA both in the chromosome and in thecosmid. In M. tuberculosis, the region downstream of rplA doesnot contain rplJ and rplL (5).

Transcriptional analysis of the secE-rplA region. Knowledgeof the expression of the ribosomal protein genes is extremelyimportant to understand the downshift in growth rate andprotein synthesis that leads to amino acid accumulation. Low-resolution S1 mapping with the “heterologous tail” probe pro-cedure (33) was used to locate the transcription initiation re-gions that were studied in detail. The modified heterologoustail probe method allows us to distinguish in a simple formRNA-DNA hybrids from reannealed DNA-DNA formed bybase pairing of the two strands of the probe.

Protection analysis of the nusG-rplK intergenic region wasdone with probe PvuII (1,721 bp) and, in the case of therplK-rplA intergenic region, with the 855-bp PvuII fragment asthe probe (Fig. 4).

Three positive protection bands for the nusG-rplK regionwere found (Fig. 4A); the largest one clearly corresponds tothe reannealed probe with the tail (1.72 kb); the second, witha size of 950 bp, corresponds to the protected band of nusGmRNA; and the third corresponds to the protected mRNA ofrplK (300 nucleotides [nt]). The lack of a protection bandcorresponding to the complete ApaI-SpeI fragment (without atail) indicated that there was not a bicistronic transcript forboth the nusG and rplK genes. The intergenic region betweennusG and rplK-rplA contains a long (19-nt) inverted repeatforming a very stable stem-and-loop structure and a shortpoly(U) tail (Fig. 2B) that may work as a transcriptional ter-minator. The presence of this terminator structure is in agree-ment with the formation of separate transcripts from nusG andrplK-rplA. The 300-nt protected band corresponding to rplKindicates that the transcription start point of rplK is located

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FIG. 2. (a) Restriction map and locations of the six genes found in the sequenced 4.42-kb DNA fragment (see the text). The wavy arrowscorrespond to the transcripts of the genes. The shaded bars show the S1 protection probes, and the solid bars represent the probes used in Northernanalysis (see Fig. 4 and 5). (B) Stem-and-loop structure formed in the intergenic region between nusG and rplK-rplA corresponding to an imperfectinverted repeat of 19 nt. (C) Alignment of the L1-binding sequence in the 59 untranslated region of the rplK-rplA transcript with those of S.coelicolor (35) and S. griseus (20).

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about 180 nt upstream of the ATG translation initiation codon.In summary, all of the evidence indicates that there is a pro-moter region between nusG and rplK and that the two genesare expressed as separate transcripts.

Similarly, low-resolution S1 mapping of the rplK-rplA regionshowed two positive hybridizing bands (Fig. 4B) that corre-sponded exactly to the reannealed probe (with its tail) and toan RNA protection band of 725 nt that coincides with theexpected size of a bicistronic transcript of both the rplK andrplA genes, i.e., both rplK and rplA are transcribed as a singlemRNA (see Discussion).

Northern analysis of the secE-rplA region shows that it istranscribed as four separate mRNAs. Northern analysis of theentire region confirmed the results obtained by low-resolutionS1 mapping and provided evidence that the secE gene is ex-pressed as a separate unit, unlike what occurs in E. coli. Theresults of Northern analysis with a secE probe (Fig. 5A)showed that secE forms a transcript of 0.5 kb that is clearlysmaller than any of the other transcripts formed from thegenes in the region. This mRNA size is in good agreement withthe size of the secE gene (333 nt).

Interestingly, hybridizations with the nusG probe revealedtwo transcripts of 1.4 and 1.0 kb (Fig. 5B), neither of whichcoincides with other transcripts in the region. This result indi-cated that there are two overlapping mRNAs for the nusGgene, probably differing in the transcription start point, be-cause low-resolution S1 mapping indicates that there is a single39 end.

Northern hybridizations of the RNA with an rplK-rplA probe

showed a single transcript of 1.6 kb (Fig. 5C). When the hy-bridization was repeated with individual probes internal toeither rplA or rplK, the same 1.6-kb band appeared, thus con-firming that both rplK and rplA are transcribed as a singlebicistronic mRNA, in agreement with the conclusions of the S1nuclease protection studies.

Upstream of the rplK-rplA cluster in the leader region ofrplK mRNA, we have found a putative L1 binding sequencethat shows strong conservation (Fig. 2C) with the reported L1binding sequence in the rplK-rplA transcript of Streptomycesgriseus (20) and S. coelicolor (35).

Promoter studies. The promoter region upstream of secEwas isolated previously (34) and was not studied further. Thetwo other promoter regions upstream of nusG and rplK-rplAwere analyzed by subcloning them in a new promoter-probevector, pULCE0 (Fig. 1), to quantify their transcription initi-ation abilities. The subcloned promoter fragments containedthe 235 and 210 regions and their ribosome binding sites.

All transformants expressing the kanamycin resistance markerfrom the PrplKA promoter were able to grow in the presence ofup to 975 mg of kanamycin/ml, whereas the control untrans-formed strain was sensitive to 20 mg/ml. Similarly, the level ofresistance to kanamycin of the transformants with the PnusG

promoter was 800 mg/ml.Figure 6 compares the efficiencies of PrplKA and PnusG with

those of other endogenous promoters of the C. lactofermentumplasmid pBL1 (4). The efficiency of PrplKA was clearly higherthan those of the other known corynebacterial promoters withthe same marker and vector, indicating that there is a strongexpression of the rplK-rplA operon in C. glutamicum.

DISCUSSION

The ribosomal protein L11 is involved in control of theexpression of genes regulated by the rel mechanism (16, 30,45). The L11 protein is required for ribosome-dependent ac-cumulation of (p)ppGpp (32). We have cloned this gene (rplK)and found that it is associated with rplA (encoding the ribo-somal protein L1) in a cluster with the secE, nusG, and pkwRgenes, in addition to a tRNATrp gene located in the distalregion (with respect to rplK-rplA) of the cluster.

There are notable differences in the conservation of thegenes in this region in different bacteria. The rplK gene hasbeen cloned from E. coli (6) and other gram-negative bacteria,and more recently from gram-positive microorganisms, includ-ing B. subtilis (19), M. tuberculosis (5), Streptomyces virginiae(15), and S. coelicolor (35). In all these organisms, as in C.glutamicum, the gene encoding L11 is clustered with the secE,nusG, and rplA genes.

A gene homologous to the pkwR gene of the actinomycete T.curvata (14) was located downstream from rplA and in oppositeorientation to it in C. glutamicum. In M. tuberculosis (5), theregion downstream of rplA contains five small ORFs in oppo-site orientation to that of rplA (4), and none of these ORFscorrespond to the pkwR gene, whereas in B. subtilis (19), S.virginiae (15), S. coelicolor (http://www.sanger.ac.uk/Projects/S_coelicolor/), and other bacteria, such as E. coli and Thermo-toga maritima (28), rplA is followed by the genes rplJ and rplL,encoding the ribosomal proteins L10 and L7/L12.

These results suggest that there are two types of organiza-

FIG. 3. Southern hybridization showing that ORF5 (pkwR) is ad-jacent to rplA both in the cosmid pCG1 (lanes 1, 3, 5, 7, 9, and 11) andin the chromosome (lanes 2, 4, 6, 8, 10, and 12). Cosmid DNA and totalDNA were digested with BamHI plus SpeI (lanes 1 and 2), BamHI plusSacI (lanes 3 and 4), BamHI plus EcoRI (lanes 5 and 6), BamHI plusBstXI (lanes 7 and 8), BamHI plus ApaI (lanes 9 and 10), and BamHI(lanes 11 and 12).

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FIG. 4. Low-resolution S1 mapping experiments. The probes used in each protection experiment are shown below the cloned DNA fragment.Note that each probe contains a homologous region (solid) and a heterologous region (shaded) belonging to lacZ and lacI. (A) Protected bandsin the nusG-rplK region. Lanes: 1, probe A; 2, homologous part of probe A; 3, negative control with probe A and tRNA; 4, S1 protection assay(protected bands are indicated by arrows); 5, molecular size marker. (B) Lanes: 1, probe B; 2, homologous part of probe B; 3, negative controlwith probe and tRNA; 4, S1 protection assay (60-min digestion with nuclease S1); 5, S1 protection assay (30-min digestion with nuclease S1) (theprotected band is indicated by the arrow); 6, size marker. Sizes in nucleotides are indicated at the right. Commercial tRNA from S. cerevisiae wasused as a negative control instead of C. glutamicum RNA.

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tion of the rplK-rplA genes in actinomycetes: (i) the arrange-ment of rplK-rplA-pkwR that occurs in C. glutamicum and (ii)that known in species of Streptomyces (16, 20). The pkwR geneencodes a putative regulatory protein, a member of the LacI-GalR family of regulatory proteins, but its exact role is un-known (14). It is likely that the larger cluster of ribosomalproteins in Streptomyces species may have been formed byrecruiting genes from other gram-positive or gram-negativebacteria.

Our results with transcriptional analysis indicate that rplKand rplA are transcribed as a single bicistronic mRNA, whereassecE and nusG form separate transcripts. Similar results havebeen reported in E. coli (6), S. virginiae (15), and S. griseus (20).The rplK-rplA promoter region of C. glutamicum is preceded bya putative transcriptional terminator (a long stem-and-loopstructure), further supporting the conclusion that nusG formsa transcript separate from that of rplA-rplK.

The simultaneous transcription of rplK-rplA suggests that theformation of the ribosomal proteins L11 and L1 is coordinatedas expected. In E. coli there is, in addition to transcriptionalregulation, an elegant mechanism of autoregulation by the L1protein at the translational level (7). The protein L1 binds to abox in the mRNA upstream of the rplK translation initiationsite and prevents translation of the mRNA by the ribosomes.In this way, an excess of the ribosomal protein L1 prevents thewasteful synthesis of additional L1 and L11 proteins. Similarly,in the leader region of rplK-rplA we have found a putative L1binding sequence that is similar to the L1 binding sequence ofS. griseus (20) and S. coelicolor (35), suggesting that the rplK-rplA expression is also autoregulated in C. glutamicum.

Promoter analysis confirmed that nusG and rplK-rplA arepreceded by separate promoters. In addition, a promotercloned by random search of transcription-initiating activity byPatek et al. (34) fully coincided with a fragment of the nucle-otide sequence upstream of secE. The promoter region up-stream of rplK-rplA is very efficient for transcription initiation,since it confers resistance to up to 975 mg of kanamycin/mlwhen coupled to the Tn5 kanamycin resistance gene, higherthan previously reported promoters from corynebacteria avail-able to us (24, 25). The nusG promoter is slightly less efficientin transcription initiation. The regulation of these promoters inresponse to nutrient starvation, osmotic stress, and the relcontrol is the subject of further research. These strong pro-moters may be used for efficient gene expression in corynebac-teria.

FIG. 5. Northern analysis of transcripts of the cloned region with probes corresponding to secE, nusG, rplK-rplA, rplA, or rplK. The probes usedare solid in Fig. 2A and were labeled by nick translation. Note that rplK and rplA are expressed as a single transcript of 1.6 kb.

FIG. 6. Levels of resistance to kanamycin (in solid TSB plates)conferred by expressing the kanamycin resistance gene of pULCE0from the rplK-rplA promoter and from the nusG promoter in compar-ison with expression from the promoters obtained from pBL1 of C.lactofermentum (4).

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ADDENDUM IN PROOF

A related article on a defined deletion within the rplK genewill appear in Microbiology (L. Wehmeier, O. Brockmann-Gretza, A. Pisabarro, A. Tauch, A. Puhler, J. F. Martın, and J.Kalinowski, Microbiology, in press).

ACKNOWLEDGMENTS

This research was supported by a grant from the European Union(BIO4-CT96-0145). Carlos Barreiro received a fellowship from theMinistry of Science and Technology (Spain), and Eva Gonzalez-Lavado was granted a fellowship by the Basque Government (Vitoria,Spain). We thank H. Sahm for providing the initial cosmid library, A.Rodrıguez-Garcıa for his scientific support, and M. Mediavilla, B.Martın, J. Merino, R. Barrientos, and M. Corrales for excellent tech-nical assistance.

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