identification of the rph (rnase ph) gene of bacillus subtilis

Vol. 174, No. 14JOURNAL OF BACrERIOLOGY, JUlY 1992, p. 4727-47350021-9193/92/144727-09$02.00/0Copyright X 1992, American Society for Microbiology

Identification of the rph (RNase PH) Gene of Bacillus subtilis:Evidence for Suppression of Cold-Sensitive Mutations in

Escherichia coliMARK G. CRAVEN,1t DENNIS J. HENNER,2 DIANE ALESSI,1l ALAN T. SCHAUER,3

KAREN A. OST,4 MURRAY P. DEUTSCHER,4 AND DAVID I. FRIEDMAN"*Department ofMicrobiology and Immunology, University ofMichigan Medical School, Ann Arbor, Michigan

48109-06201; Department of Cell Genetics, Genentech Inc., South San Francisco, Califomia 940802;Department ofMicrobiology, University of Texas, Austin, Texas 78712-10953; and Department of

Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 060304

Received 14 February 1992/Accepted 10 May 1992

A shotgun cloning of Bacillus subtfis DNA into pBR322 yielded a 2-kb fragment that suppresses thecold-sensitive defect of the nusAl4O(Cs) Escherichia coli mutant. The responsible gene encodes an open readingframe that is >50%o identical at the amino acid level to the E. coli rph gene, which was formerly called orlE.This B. subtiuis gene is located at 2510 adjacent to the gerM gene on the B. subtiuis genetic map. It has beennamed rph because, like its E. colh analog, it encodes a phosphate-dependent exoribonuclease activity, RNasePH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses thecold-sensitive phenotype of other unrelated cold-sensitive mutants ofE. coil, but not the temperature-sensitivephenotype of three temperature-sensitive mutants, including the nusAII(Ts) mutant, that were tested.

The evolutionary divergence of Bacillus subtilis and Esch-erichia coli, easily observed in their different life styles, hasbeen verified at the molecular level (49). There are, however,a number of examples of functionally equivalent E. coli andB. subtilis gene products that are related by greater than 40%identity at the amino acid level (1, 7, 21, 45). Although someof these products are not essential for cell viability, all areinvolved in important physiological processes.The NusA transcription elongation factor, an essential E.

coli protein (31, 44), was first identified because it is aparticipant in the N transcription antitermination system ofcoliphage X (18, 41). Western blots (immunoblots) (18a)suggested that B. subtilis expresses a similar protein. Aselection designed to isolate the B. subtilis analog of the E.coli nusA gene uncovered a previously unidentified gene ofB. subtilis whose deduced protein sequence exhibits greaterthan 50% identity with an open reading frame, orfE, found inE. coli (40). More recently, it has been shown that orfEencodes RNase PH (37), a phosphate-dependent exoribonu-clease that removes nucleotides 3' to the CCA end of tRNA(13, 15) and adds nucleotides to the ends of RNA moleculesby using nucleoside diphosphates as substrates (36). TheorfE gene was renamed rph to reflect this activity (37).Although E. coli carrying a deletion and an insertion disrupt-ing rph grows normally (39), the rph gene product is requiredfor growth of an E. coli mutant strain missing a collection ofnuclease activities, RNase I, RNase II, RNase BN, RNaseD, and RNase T (37a).The rph gene is the first gene in a bicistronic operon

located at min 81 of the E. coli chromosome (40a). Thesecond gene, pyrE, encodes the enzyme phosphoribosyltransferase, one of six enzymes involved in the de novo

* Corresponding author.t Present address: Roche Institute of Molecular Biology, Nutley,

NJ 07110-1199.t Present address: Parke-Davis Pharmaceutical Division, Ann

Arbor, MI 48105.

synthesis of UTP (32). Expression ofpyrE is regulated by aUTP-mediated attenuation mechanism located in the inter-cistronic region between rph (orfE) andpyrE (4, 9, 27).We report the identification of the rph analog from B.

subtilis and show the similarity between its deduced aminoacid sequence and those of the E. coli and the Salmonellatyphimurium genes. Functional analysis demonstrates thatthe rph gene of B. subtilis encodes an activity analogous tothat of the rph-encoded RNase PH found in E. coli. Althoughthere is significant identity between the amino acid se-quences of these rph genes, suggesting selective pressure formaintenance of the protein, insertional inactivation of the B.subtilis rph gene, as shown for the E. coli analog (39), doesnot appear to affect bacterial viability.

MATERIALS AND METHODS

Strains. The bacteria used in these studies are listed inTable 1.Media. E. coli strains were grown in Luria-Bertani (LB)

broth supplemented, where appropriate, with antibiotics atthe following concentrations (micrograms per milliliter): 30,kanamycin and ampicillin; 15, tetracycline; and 12.5, chlor-amphenicol. B. subtilis strains were grown in tryptone bloodagar base or minimal glucose medium. Anaerobically cul-tured bacteria were grown in a Coy chamber (5).

Reagents. Restriction endonucleases, T4 DNA ligase, andphosphorylated MluI linkers were purchased from NewEngland Biolabs. Bacterial alkaline phosphatase was pur-chased from Bethesda Research Laboratories.

Transduction. PBS-1 was used for B. subtilis essentiallyaccording to the method outlined by Hoch et al. (25). P1transduction was employed for E. coli (29).EOP. The growth of conditionally defective mutants at

nonpermissive temperatures was quantitatively measured byusing the efficiency of plating (EOP) method. Bacteria con-taining test plasmids were cultivated overnight at theirpermissive temperature in LB broth supplemented with the

4727

4728 CRAVEN ET AL.

TABLE 1. Bacterial strains

Species and Relevant genotype and/or Sourcestrain parent strain

E. coliK37 Wild type These laboratoriesaK95 nusAl These laboratoriesaK1914 K37 nusAIO(Cs) These laboratorieseK3909 K37 nusAll(Ts) M. GottesmanK4206 K37 nusAIO(Cs) pmc-1 This workD80 Uncharacterized; cold sensitive J. IngrahamIQ346 ssyA3(Cs) K. ItoCG431 nusB136(Cs) C. GeorgopoulosRR100 poL4(Ts) P. RockwellK3732 K1914 + pBB1 This workK4713 rph::CamTpoL4(Ts) This workK4714 rph::Cam' This workK4716 K37 rph::Camr This workNT675 ftsZ84(Ts) N. TrunNT701 dnaA204(Ts) N. Trun18-11 ma mb md mt RNase BNb These laboratories

B. subtilis1168 trpL2 J. HochQB936 ald-i aroG932 leuA8 trpC2 BG-SC(#lAY)a University of Michigan.b Relevant phenotype.c University of Connecticut.

was constructed by placing the SphI fragment containing the3' portion of rphF, from pUE281 into SphI-digested pBB2.pHEB5 was constructed by replacing the SphI fragmentcontaining the 3' portion of rphEc in pUE281 with the SphIfragment from pBB1 containing the 3' portion of rphBS.pFBO2, a plasmid that expresses a p-galactosidase (13-Gal)-RphEC fusion protein, was constructed by using pUR288(42), which has a polycloning site inserted into the 3' end oflacZ. Because appropriate restriction sites were not avail-able to clone rphEc directly into pUR288, a plasmid interme-diate, pUE80, was constructed by removing the 872-bp MspIfragment containing the complete rphE gene plus 13 addi-tional nucleotides upstream of the AUG from pUE281,ligating it into the AccI site in pUC8, and screening byrestriction digests for the desired orientation. The BamHI-HindIII fragment of pUE80 was cloned into pUR288. Theresulting plasmid, pFBO2, contains the complete rphEc cod-ing sequence plus seven additional codons upstream of thefirst AUG sequence fused in frame to the 3' end of the lacZgene.The following plasmids were used in B. subtilis. pJHrph

was created by cloning the EcoRI fragment from pBB1 intothe integrative plasmid, pJH101 (16). pJHrphD5 was createdby cloning a 250-bp HincII-BglII fragment from the B.subtilis rph gene into pJH101.

Transformations. The transformation procedure used forB. subtilis was essentially the method of Anagnostopoulos

appropriate antibiotics. The overnight culture was dilutedinto the same medium and grown to 2 x 108 cells per ml atthe permissive temperature. Dilutions of the bacteria werespread on LB plates, and the plates were incubated atpermissive and nonpermissive temperatures. The EOP iscalculated by dividing the number of colonies observed atthe nonpermissive temperature by the number observed atthe permissive temperature.

Plasmids. The following plasmids contained B. subtilisDNA (Fig. 1). pBB1 was selected from a library created byshotgun cloning B. subtilis DNA into the EcoRI site ofpBR322. pBB2 is a derivative of pBB1 with an SphI-createddeletion covering 612 bp of insert and 566 bp of vector DNA.pUBi is a pUC18 derivative with a 1-kb HpaI-EcoRI frag-ment from pBB1 containing the complete B. subtilis rph(rphBs) gene cloned between its SmaI and EcoRI sites in theopposite orientation with respect to Plac pUB2 is a pUC19derivative with an EcoRI-BamHI fragment from pBB1 con-taining the complete rphBs gene cloned between its BamHIand EcoRI sites in the same orientation as P1aC.The following plasmids contained rphE, (Fig. 2). pGA2,

which has the rph-pyrE operon on an 8-kb insert (2), was agift from James Friesen. pBE1 is a pBR322 derivative withthe 1,080-bp Clal-BamHI fragment from pGA2 and containsthe E. coli gene plus a portion of the upstream pyrE genes.pUE281 is a pUC18 derivative with the EcoRI-BamHIfragment from pBEl oriented so that rphEC can be tran-scribed from Plac, pUE185 has the FnuDII fragment (MluIlinked) of pBR328 carrying Camr cloned into the MluI site ofpUE281, resulting in a disrupted rph gene. pRPH1, whichhas been previously described under the name pORFE-1(37), is a derivative of pHC79 that contains a wild-type copyof rphEc (24).The following plasmids contained hybrid rph genes (Fig.

3). A shared SphI restriction site was used to constructhybrid rph genes that maintained the proper reading frame atthe junction of the B. subtilis and E. coli mph genes. pHBEl

rph B. s.

pBB1

pBB2

E-1264

E-1264

H

H

H H-244

lH H

-244

+190

I IE S

S+190

.rph B. s.P:4 -

pUB1

pUB2

E S *B+190 -244

rph B. s.

IIB *

1 IS E

-244 +190

FIG. 1. Plasmids with rphB inserts. The flanking vector arms arerepresented as thin lines, while inserts are represented by open bars.Locations of the coding regions of rphB, are indicated by a thickarrow or, in the case of pBB2, which contains only a portion of thecoding sequence, a thick line. The location of the lac promoterupstream of the insert in the pUC derivative is labeled, and thedirection of transcription is indicated by the short arrow. Positionsof relevant restriction enzyme sites are also indicated: B, BamHI; E,EcoRI; H, HpaI; S, SphI. The SmaI site lost in the cloning isindicated by an asterisk. The numbers below each drawing give theposition, in base pairs, relative to the first AUG in the codingsequence (not drawn to scale). pBB1 and pBB2 are pBR322 deriv-atives, while pUB1 and pUB2 are pUC18 derivatives.

J. BAc-rERIOL.

rph GENE OF BACILLUS SUBTILIS 4729

rph E. c. pyrE E. c.P1-P. PO.4wz '~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Cl

c s-83

B

rph E. c.P1

J) IE C; S B

rph E. c.

C ImE C S B S

CamrrP

l,T P.

AE C M B

FIG. 2. Plasmid with rphE, inserts. Signals and conventions are

as listed in the legend to Fig. 1. Locations of the full coding regionsof rphE, andpyrE are indicated by thick arrows; partial regions areindicated by thick lines. P1 is the identified promoter for therph-pyrE operon. Positions of relevant restriction enzyme sites areshown: B, BamHI; C, ClaI; E, EcoRI; M, MluI. The ClaI site islocated 83 bp upstream of the first AUG in the rph coding sequence.This map is not drawn to scale. pBE1 is derived from pBR322, whilepUE185, pUE281, and pUE291 are pUC derivatives. The position ofthe Camr insert in the rph gene of pUE185 is indicated.

and Spizizen (3), and that for E. coli was essentially themethod of Cohen et al. (10).

Preparation of anti-RphF, antibody. The B-Gal-RphEc fu-sion protein was isolated by gel fractionation from cellularextracts of a bacteria with the fusion-expressing plasmidpFBO2. The mashed gel slice, mixed with completeFreund's adjuvant, was used for primary and secondaryimmunization of New Zealand White rabbits. Serum isolated2 weeks after the secondary immunization was employed inthe experiments.

Protein techniques. Western blots were run essentially asdescribed previously (6). Proteins labeled in the steady statewith [35S]methionine were analyzed by two-dimensional gelelectrophoresis as previously described (35).Enzyme assays. RNase PH and polynucleotide phosphory-

lase were assayed as described previously (14, 15, 37).DNA sequencing. Restriction enzyme-generated fragments

from the insert in pBB1 were cloned into M13 vectors andsequenced by the dideoxy method (8).

Construction of the rph::Camr chromosomal insertion. Therph::Camr insertion was crossed from pUE185 onto the E.coli chromosome in apoLA(Ts) pyrE' strain (N7622) accord-ing to a previously described method (23). P1 transductionwas employed to move rph::Camr to strain K37.

Nucleotide sequence accession number. The sequence ofthe rphBS gene and surrounding sequences has been depos-ited in GenBank under accession no. M85163.

RESULTS

A B. subtilis gene that complements the E. coli nusAIO(Cs)mutation. A library, formed by cloning EcoRI-digested B.subtilis DNA into the EcoRI site of pBR322, was screenedfor nusA-complementing activity by using a bacterial strain(K1914) that has the nusAlO(Cs) allele (44). This mutantallele is composed of two nucleotide changes in nusA thatcause a cold-sensitive phenotype (12, 26); bacteria carryingnusAlO(Cs) fail to grow at or below 32°C. Ampicillin-resis-tant transformants of K1914 were selected at 30°C. Onetransformant, designated K3732, obtained with this comple-mentation selection was chosen for further study. We dem-onstrated in two ways that the plasmid in K3732, namedpBB1, confers cold resistance. First, derivatives of K3732

Hybrid rph B.sIE.c.

D,pHBE1

Eco RI

-1264Hpa I

-244

ATG Sph I+1 +190

l1

Bam HI Sph I

Hybrid rph E.c.lB.s.P1_4

pHEB5Eco RI Cla I ATG Sph I

-83 +1 +190Eco RI Sph I

FIG. 3. Plasmids with hybrid B. subtilis-E. coli rph genes. Signals and conventions are as listed for Fig. 1 and 2. Black portions of symbolscorrespond to B. subtilis sequences, and white portions correspond to E. coli sequences. Relevant restriction enzyme sites, as well as thedistances in base pairs from the first AUG in the coding sequence, are listed below each map. The map is not drawn to scale.

pGA2

pBEl

pUE281

pUE185

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4730 CRAVEN ET AL.

cured of pBB1 are cold sensitive. Second, derivatives of thecold-sensitive parental strain K1914 transformed with pBB1are cold resistant.pBB1 DNA was shown by restriction enzyme analysis to

have a 2-kb insert and by Southern analysis to hybridize toB. subtilis DNA and not toE. coli DNA (data not shown). Adeletion derivative was constructed by treating pBB1 withSphI, which cleaves at a site in the insert and at a site in theplasmid. The resulting plasmid, pBB2, has a 612-bp deletionof insert DNA and fails to complement the cold-sensitivedefect of K1914.Complementation of other nusA mutations. We next deter-

mined whether pBB1 complementation of the nusAIO(Cs)mutation reflects expression of a product with NusA-likeactivity that complements the cold-sensitive defect or anactivity unrelated to NusA that indirectly suppresses thecold-sensitive defect. If the former were the case, we wouldexpect that pBB1 would complement the mutant phenotypesof other nusA mutations. However, if the latter were thecase, we might expect pBB1 not to complement thesemutant phenotypes.Two other nusA mutations were employed to test the

complementing activity of pBB1, nusAl and nusAll(Ts). E.coli carrying the nusAl mutation fails to support the Ntranscription antitermination system of bacteriophage X.This is evidenced by a failure of X growth in a nusAl mutantat 42°C (19). E. coli with the nusAll(Ts) mutation are notviable at 42°C (31). Neither of these mutant phenotypes issuppressed by pBB1 (data not shown). Since both of thesemutant alleles are recessive, the failure to observe comple-mentation suggests that pBB1 does not express a NusA-likeactivity. Thus, the cold resistance conferred by pBB1 to thenusAlO mutant is likely to result from some type of suppres-sion.

Analysis of B. subtilis DNA in pBB1. The sequence of the2-kb insert in pBB1 was determined, and an analysis of thesequence revealed a 735-bp open reading frame located atone end of the insert. A computer-based comparison re-vealed no identities between this DNA sequence or itsderived protein sequence with either the E. coli nusA DNAor protein sequences. A GenBank search failed to identifysignificant homologies with any DNA sequences. However,a search of protein sequences uncovered an open readingframe of 238 codons in E. coli called orfE (rph) withsignificant identity to the open reading frame in the insert.Alignment of the amino acid sequences of the E. coli and B.subtilis open reading frames (allowing a gap of one aminoacid at position 173 of the E. coli rph-encoded proteinsequence) shows identical amino acids at 132 of 238 posi-tions (56% identity) (Fig. 4). Moreover, 30 of the differingamino acids are conservative changes. This B. subtilis openreading frame will be referred to as rphBS, while the analo-gous sequence from E. coli will be called rphEr. Salmonellatyphimurium, a close relative of E. coli, also has a bicistronicpyrE operon. The sequence of the 3' end of the promoter-proximal gene has been determined, is extremely close tothat of rphEc (33), and thus will be referred to as rphs5. Theportion of the S. typhimurium DNA sequence available forcomparison begins at the codon corresponding to amino acid130 of rphE, and in that region, encoding 109 amino acids,there are 9 amino acid differences between rphE,; and rphs,.Interestingly, while eight of the nine amino acids at thesepositions differ in the alignment of rphE,,, with lphB., three ofthe nine encoded by the rphs, gene match with the aminoacids at the corresponding positions in rph.1, i.e., at least in

B. subtilisE. coliS. typhimuriumB.s. similarityto E.c. or S.t.




B. subtilisE. coliS. typhimuriumB.s similarityto E.c. or S.t.

10 20 30 40 50MRHDGRGHDELRPITFDLDFISHPEGSVLITAGNTKVICNASVEDRVPPF

60 70 80 90 100LRGGGKGWITAEYSMLPRATNGRTIRESSKGKISGRTMEIQRLIGRALRALKGQGQGWITAEYGMLPRSTHTRNAREAAKGKQGGRTMEIQRLIARALRA

L-G-G-GWITAEY-MLPR-T--R-*RE--KGK--GRTMEIQRLI-RALRA

110 120 130 140 150VVDLEKLGERTIWIDCDVIQADGGTRTASITGAFLAMAIAIGKLIKAGTIAVDLKALGEFTITLDCDVLQADAWTRTASITGACVA A L GKL

TGACVAI AI L GKL

*VDL--LGE-TI--DCDV*QAD*-TRTASITGA--A-A-A*-KL*--G-*

160 170 180 190 200KTNPITDFLAAISVGIDKEQGILLDLNYEEDSSAEVDMNVIMTGSGRFVEKTNPMKGMVAAVSVGIVNGE YVEDSAAETDMNVVMTEDGRIIEKTNPMKGMVAAVSVGIVNGE EYVEDSAAETDMNVVMTEDGRIIE

KTNP----*AA*SVGI-----I--DL-Y-EDS-AE-DMNV*MT--GR-*E

210 220 230 240LQGTGEEATFSREDLNGLLGLAEKGIQELIDKQKEVLGDSLPELK

FIG. 4. Comparison of translated rph sequences. Amino acidsare given in the single-letter code. The Rph sequences from B.subtilis (complete), E. coli (complete), and S. typhimurium (partial)are compared. Dashed lines indicate gaps placed to optimize thealignment. The bottom sequence indicates the similarities betweenthe B. subtilis sequence and either or both of the E. coli and S.typhinurium sequences. Letters indicate amino acids found incommon, asterisks indicate conservative differences, and dashesindicate nonconservative differences. Differences between rphEcand rphs, are indicated by boxes. The rphEc sequence used in thiscomparison was obtained from SWISSPROT. The carboxy terminusof rphst was described previously (33).

the carboxy terminus, rphB5 more closely corresponds torphst.

rphBS encodes an RNase PH-like enzyme. The similarity ofthe rphBS open reading frame with that of rphEC led us to testwhether pBB1 expresses an RNase PH-like activity. Previ-ous experiments have established that RNase-deficient E.coli 18-11 can be used as the background to assess RNasePH expression from plasmids (37). The substrate for theenzyme assay is tRNA-CCA-[ H]Cn. A measure of thespecificity of RNase PH activity is obtained by comparing itsnucleolytic action on the tRNA substrate to that on[3H]poly(A); RNase PH, unlike polynucleotide phosphory-lase, shows relatively low activity on the poly(A) substrate(15).As shown in Fig. 5, extracts from the derivatives of strain

18-11 harboring pRPH1 and pBB1 have higher levels of thenuclease activity that processes the tRNA substrate than doextracts from the control derivative harboring the pHC79parent vector. The relatively lower activity against thepoly(A) substrate demonstrates the specificity of these reac-tions. Interestingly, the B. subtilis extracts are more activefor the tRNA substrate than are the E. coli extracts. BecausepRPH1, with rphEC, expressed lower levels of RNase PHactivity than was expressed by pBB1 with rphBS, we testedtwo other plasmid constructs with rphEC to determinewhether they express levels of the E. coli RNase PHcomparable to those expressed by pBB1. A pBR322 deriva-tive, pBE1, contains a 1-kb ClaI-BamHI insert with rphEr

J. BACrERIOL.

-QGT*E---FS-E*L--LL*LA--Gl--**--QK-*L--


1 0

8

tscu

0

EN

c

0

CU_co

6

4

0-0) v

0. 0.I trXL ff

co m m0. 0. P

Plasmid

_.

I D0. X.

w

0.

FIG. 5. Relative levels of RNase PH activity. Enzyme activities expressed from the listed plasmids in strain 18-11 were measured asindicated in the Materials and Methods, and all values were normalized to that found for the control plasmid, pHC79. Enzyme activities weremeasured at 37°C by determining the Pi-dependent specific activity with a tRNA-CCA-[3H]C, substrate for RNase PH (dark bars) or a[3H]poly(A) substrate for PNPase (light bars). Strains with pUC19 and pUE281 were not tested for activity with the latter substrate.

that includes the identified promoter for the rph-pyrE operonas well as part of the downstream pyrE gene. A pUC18derivative, pUE281, containing the rphEc gene, was con-structed with the insert oriented so that the rph gene istranscribed from the lacZ promoter (Fig. 2). Transformantsof E. coli 18-11 derivatives with these plasmids were assayedfor RNase PH activity. As shown in Fig. 5, pBEl fails toexpress elevated enzyme activity, while pUE281 expressesonly a low level of activity, 50% of the increase expressedfrom pRPHl.E. coli rph and the nusA4O(Cs) mutation. We next deter-

mined whether rphEr could also confer cold resistance to thenusAlO(Cs) bacterium, i.e., allow growth at 30°C. Althoughwe were unable to obtain a plasmid that expressed levels ofthe E. coli RNase PH activity comparable to the levels of B.subtilis RNase PH activity expressed by pBB1, we consid-ered it worthwhile to test the different plasmids with rphEcfor suppression of the nusAlO(Cs) phenotype because it wasconceivable that the suppressing activity might not correlatewith RNase PH activity.

Table 2 presents results of a quantitative assessment of theeffect of rph plasmid constructs on the growth of K1914derivatives at low temperatures. Because pUC plasmids arenot stably maintained in the presence of the nusAlO(Cs)mutation, we used K4206, a derivative of K1914 with anadditional mutation, pmc-1, that allows maintenance ofpUCplasmids in nusAlO(Cs) mutants without affecting the cold-sensitive phenotype. Thepmc-1 mutation located at min 2 onthe E. coli chromosome is otherwise uncharacterized (11).Both plasmids with an intact rphBS gene permit growth of

the K4206 nusAlO(Cs) strain at 30°C. All plasmids withrphE,, including pRPH1, fail to exhibit a similar phenotype.

These studies also revealed that pUC derivatives carryingrphBS can be maintained by the nusAlO(Cs) strain in theabsence of thepmc-1 mutation at all temperatures, althoughpUC derivatives with rphEC, like pUC parent plasmids,could not be maintained in this host under identical condi-tions.

Identification of the rph protein. Western blots were em-ployed to measure expression of the Rph protein indepen-dently of enzymatic activity. Antibody was raised to therphE, gene product by using a ,B-Gal-Rph fusion protein asthe immunogen (see Materials and Methods). As shown inFig. 6, pBE1 (lane 4) fails to express detectable levels of Rph

TABLE 2. Effect of plasmids containing rph genes onnusAlO(Cs) cold sensitivity

EOPI (colonies at 30'C/colonies at40°C)

Plasmid GeneK1914 K4206

(nusA1O) (nusAlOpmc-1)

pUC18 None __b <0.001pBE1 rph c <0.001 <0.001pUE281 rphE_b <0.001pBB1 rphBs 1.0 0.9pBB2 A?phBs <0.001 <0.001pUB2 rphBs 1.0 0.9pHC79 None NDC <0.001pRPH1 rphEc ND <0.001

a EOP was determined as described in Materials and Methods.b Stable transformants could not be obtained.c ND, not done.

................. i....................

t--------------------

.................I....................

..................... ..................... ......................................

.................... ..................... .................... -------------------

........................................................................................................

VOL. 174, 1992

a

4732 CRAVEN ET AL.

O> - min'-o V- V- 0ui C

mco W Wa mw

su

QL Cf 0f 0CL Q Cf

I I180,000oo116,000-84,000-

58,000-

48,50

36,500

26,600

1 2 3 4 5 6 7

FIG. 6. Immunoelectroblot identifying Rph protein. Extracts ofproteins from derivatives of strain K4206 carrying the plasmidslisted above the lanes were prepared and analyzed by immunoelec-troblot by using the procedures indicated in Materials and Methods.Equal amounts of protein extract (ca. 50 pg) were loaded. Thepositions of molecular weight standards are shown on the left, andthe positions of the band unique to some strains with plasmids withrph genes (i.e., the Rph protein) are indicated by the arrow to theright. The circle on the left indicates the position of the [-Gal-Rphfusion protein expressed from plasmid pFBO2 (lane 1) that was usedfor immunization.

protein, while pUE281 (lane 7) expresses detectable levels ofa protein migrating in the range expected for the inferredmolecular mass of Rph, 25 kDa. Note that the bacterial hostdid not express sufficient Rph protein to yield a band on theimmunoblot.We could detect no evidence from Western blots that

pBB1 or pUB2, a pUC derivative containing the clonedrphBS gene positioned to be read from PIac, expresses pro-

teins that cross-react with this antiserum (lanes 2 and 3).This failure to identify the rphBs protein probably reflects a

failure of the immune serum to react with the B. subtilisprotein, since pBB1 with the cloned rphB. expresses highlevels of its gene product as measured by RNase PH activity(see above) and by two-dimensional electrophoresis thatshowed high levels of a protein with the mobility expectedfor the product of the rphBS open reading frame (data notshown).Hybrid rph genes. To provide hybrid proteins to assess

whether specific domains might be implicated in rpSBS activ-ity, we constructed plasmids with hybrid genes containingportions of rph., and rphE. The first, pHBE1, a pBR322derivative, has B. subtilis DNA that encodes the first 63amino acids with the associated upstream region of rphB5fused in-frame to the portion of rphE<, that encodes aminoacids 64 through 238. The second, pHEB5, a pUC18 deriv-ative, has the DNA that encodes the first 63 amino acids withthe associated 81 bp of the upstream region of rphEr fused

J. BACrERIOL.

in-frame to the portion of rphBS encoding amino acids 64through 245. The insert is oriented so that the hybrid genecan be transcribed from the lac promoter in pUC. The insertcontaining the hybrid gene in pHBEl could not be clonedinto a pUC vector.We were unable to obtain stable transformants of the

nusAIO(Cs) strain K1914 with either pHBEl or pHEB5.However, stable transformants were obtained with E. coliK4206, which, as discussed above, carries in addition tonusAlO(Cs), the pmc-1 mutation. Tests for growth at lowtemperatures showed that neither pHEB5 nor pHBEl re-lieve the cold-sensitive defect of K4206 (data not shown).We note that K4206 containing pHEB5 grows poorly even athigher, permissive temperatures (42°C). This failure to sup-press the cold-sensitive mutant phenotype correlates withRNase PH activity; pHEB5 does not express RNase PHactivity, and pHBEl expresses only a low level of RNase PHactivity (Fig. 5).

Expression of Rph proteins from the plasmids with hybridrph genes was assessed by Western blots by using theantiserum raised against the P-Gal-RphE,, fusion protein(Fig. 6). The immune serum failed to react with any proteinin extracts from the K4206 derivative with pHEB5 (lane 5).This was not surprising, since this hybrid rph gene wasprimarily derived from rphBS and the product of that genedoes not react with the antiserum. Protein extracts from thestrain with pHBEl produced significantly higher levels ofdetectable protein than those observed in extracts frompUE281, the pUC-derived plasmid with rphE, (lanes 6 and 7,respectively), suggesting that the regulatory region associ-ated with the B. subtilis gene supported high levels of rphexpression in E. coli. However, pHBEl expressed only lowlevels of RNase PH activity (Fig. 5). Examination of thesequences 5' to the rphBS open reading frame failed to revealany obvious E. coli promoter sequences, although the regionwas rich in AT base pairs (data not shown).

rph expression and bacterial physiology. The effect of rphB.on E. coli gene expression was examined by using two-dimensional gel electrophoresis. Proteins produced in the E.coli K-12 strain K37 in the presence of pUBi (pUC18 withrphBS) were compared with those produced by the samebacterium carrying the control vector pUC18. With theexception of the expression of rphB. and 3-lactamase by thebacterium with pUB1, we were unable to observe anysignificant differences in the quantity or quality of theproteins expressed by these two bacteria (data not shown).

rph effect on other temperature-dependent mutations. Wenext asked whether rphBS suppression of cold sensitivity wasallele specific. Three E. coli strains with unrelated cold-sensitive mutations were employed. CG431 carries thenusBl36(Cs) mutation (20). NusB, like NusA, has beenimplicated in transcription elongation (for a review, seereference 18). IQ346 contains the ssyA3(Cs) mutation, whichwas isolated as a suppressor of a secY thermal-sensitivemutation (46). The secY gene product is essential for trans-location of proteins across the cytoplasmic membrane, andthe ssyA3 mutation results in a slowing of the rate oftranslation (47). D80 has an uncharacterized mutation thatcauses cold sensitivity (25a). Cloned rphBS suppresses thecold-sensitive phenotype of both the nusB136(Cs) (CG431)and the ssyA3(Cs) (IQ346) strains, but it fails to suppress thecold-sensitive phenotype of the uncharacterized strain(D80). The suppression of cold sensitivity was measured byusing the EOP to compare growth of the mutant with that ofthe wild-type strain at the low, nonpermissive temperature.Plasmids pBB1 and pUB2 suppress cold sensitivity in CG341


TABLE 3. Effect of plasmids carrying rph genes on the coldsensitivity of cold-sensitive mutants

EOPaPlasmid Gene D80 (cold CG431 IQ346

sensitive) (nusB136) (ssyA3)

pUC18 None __b <0.001 apBE1 rphEc <0.001 <0.01 NDcpUE281 rphE _ _ d 0.1pBB1 rphBs <0.01 1.0 1.0pBB2 Arph], <0.001 <0.001 0.1pUB2 rphB, <0.001 1.0 1.1

a EOP was determined as described in Materials and Methods.b Stable transformants could not be obtained.C ND, not done.d Transformants were obtained with this plasmid, and they grew in liquid

culture with 30 pg of ampicillin per ml. However, when they were diluted andplated on tryptone broth plates containing ampicillin at the permissivetemperature, no colonies grew.

and IQ346, as evidenced by EOPs of 1.0 (Table 3). Controlsthat failed to suppress the cold sensitivity of the test bacte-rium include the cloning vector pBR322, pBB2 (a derivativeof pBB1 that is deleted for the 3' end of rph), and a numberof vectors with the cloned rphE, gene. Note that the EOP ofthe IQ346 controls was significantly higher than those of theother controls. None of the plasmids with rphB, permitsurvival of D80, the strain with the uncharacterized cold-sensitive mutation, at low temperatures. Moreover, pUC18,pUE281, and pUE185 were not maintained in D80 even athigher, permissive temperatures.To explore the possible effects of rphBS on thermal-

sensitive mutations, we examined the survival at high tem-peratures of E. coli derivatives NT675 and NT701 carryingtheftsZ84(Ts) or dnaA204(Ts) mutation, respectively. Thesetwo mutants were selected for study because their thermal-sensitive phenotype is tight. The failure of these mutantbacteria to survive at high temperatures was not influencedby the presence of pBB1 (data not shown).Mapping rphB3. The position of rphBS was located on the

B. subtilis chromosome by using an integrative plasmid,pJH101 (16). The EcoRI fragment from pBB1 was clonedinto pHJ101, creating pJHrphB5. B. subtilis 1168 was trans-formed with pJHrphB8, and integrative recombinants wereselected as chloramphenicol-resistant derivatives. Prelimi-nary mapping studies employing mating placed the Camrmarker near aroG and leuA at position 2500 on the B. subtilischromosome. A more-accurate mapping was obtained bytransduction. Phage PBS-1 was grown in one of theseintegrative recombinants, I168::pJHrphB., and the lysatewas used to transduce strain QB936 with independent selec-tions for Camr, Leu+, or Aro+. An analysis of recombinantssuggested the following order of markers: aroG-Camr-leuA8.This locates rph at about the 2520 position on the B. subtilischromosome. A subsequent comparison of surrounding se-quences with newly available B. subtilis sequences revealedthat immediately upstream of the rph gene in our clone is anoverlap with gerM located at 251° (43).

Testing the essentiality of rph products. To assess possiblephysiological roles for Rph in B. subtilis, we constructed a B.subtilis strain with a disrupted rphBS gene by integrating aplasmid with an internal fragment of rphBS into the chromo-some. This plasmid, pJHrphDS, was constructed by placinga 249-bp HincII-BglIH fragment of rph (codons 102 to 185)into pJH101. Because this fragment contains an internal

fragment of rph, integration of pJHrphdDS by homologousrecombination into the chromosome disrupts the chromo-somal gene, creating two truncated copies of rph; one copyencodes a protein missing the first 101 amino acids, and thesecond encodes a protein lacking the last 61 amino acids.Strain 1168 carrying the integrated pJHrphDS showed noobvious growth defect on plates at 40°C and appeared tosporulate normally, as judged by pigment formation on theplates (data not shown).The role of the rphE, gene product in bacterial growth was

examined by using a derivative of our standard laboratorystrain K37, K4716, that has a Camr cassette inserted in thechromosomal rph gene (see Materials and Methods). Growthwas qualitatively assessed by comparing colony formationby K4716 with that by K37 under a variety of conditions.There were no observable differences between K4716 andK37 in colony formation on rich (LB) or minimal (M9-glucose-uracil) medium grown aerobically at 30, 32, or42.5°C or anaerobically at 37°C (data not shown). Theseresults are consistent with previous studies (39), demonstrat-ing that disruption of rphEc has no effect on growth in normallaboratory media other than imposing a pyrimidine require-ment.

DISCUSSION

We have identified a gene from B. subtilis, rphB5, whoseexpression from a plasmid suppresses a number of cold-sensitive mutations of E. coli. The striking homology at theamino acid level between rphBS and rphEc, as well as the 3'end of what appears to be the rph gene of S. typhimurium,suggests selective pressure for a common function. Previousstudies have indicated that rphEr encodes RNase PH, aphosphate-dependent exoribonuclease that cleaves the 3'ends of tRNA precursor (15, 37). The studies presented heredemonstrate that rphB. encodes a similar, if not identical,activity.Most of the studies reported in this paper were initiated

and completed before the function of the E. coli rph geneproduct was identified. The underlying presumption wasthat, once this function was identified, the raison d'etre forthe suppression of cold-sensitive mutant phenotypes wouldbecome apparent. Such is not the case. We still are unable toprovide a definitive explanation for the suppression of coldsensitivity provided by rphBs.Remarkably, the plasmid-based rphBS gene has biological

activities in E. coli not observed with a similarly plasmid-based phE,. Plasmids containing rphBS, identified becauseof suppression of the cold sensitivity of the nusAIO(Cs)mutation, also suppress the cold-sensitive phenotype ofmutations in other genes, as well as permitting maintenanceof pUC plasmids in the nusAIO(Cs) mutant. Plasmids con-taining rphEr do not exhibit any of these phenotypes. Thisapparent dissimilarity in function might reflect a qualitativedifference in enzymatic activities between the two RNases.This is despite the fact that our assays indicate a similarmode of action for the two rph gene products. Alternatively,a quantitative explanation is consistent with our data, i.e.,the higher levels of product expressed by the cloned rphBScause suppression of the cold-sensitive mutations. Consis-tent with this argument are measurements of rph expressionboth by Western and two-dimensional gel electrophoresisshowing significantly higher levels of expression from theplasmid with the cloned 5' region of rphB. than from theanalogous plasmid with the cloned 5' region of rphE.However, these high levels of hybrid protein are not re-

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4734 CRAVEN ET AL.

flected in either RNase PH activity or the suppression ofcold-sensitive phenotypes, suggesting that the hybrid proteinis nonfunctional; these results highlight the correlation be-tween RNase PH activity and suppression.The fact that rphBS suppresses a cold-sensitive but not a

thermal-sensitive nusA mutation, coupled with the observa-tions that rphB. suppresses other cold-sensitive and notthermal-sensitive mutations, suggests a specificity for sup-pression of cold-sensitive mutations. This could reflect adifference in the physiological character of the bacterium atlower temperatures, an argument that appears particularlyplausible in light of studies with both prokaryotes andeukaryotes which show that sets of genes with cold-sensitivemutations can be quite different from those with thermal-sensitive mutations (30). Accordingly, these physiologicaldifferences could result from differences between the re-quirements for growth at lower versus higher temperatures.Two examples of gene products that appear to be requiredonly at low temperatures are those of the yeast SACi gene,an apparent component of the cytoskeleton (34), and the E.coli nusB gene, a transcription elongation factor (20). More-over, in E. coli, cold-sensitive but not thermal-sensitivemutations affecting ribosome assembly could be isolated (22,48). According to this line of reasoning, the rphBS geneproduct might influence a physiological process active onlyat low temperatures, permitting the suppression of mutationsin a variety of genes at those temperatures.How might RNase PH activity fit into such a scheme? If

RNase PH influences the efficiency of tRNA (or other RNA)processing, it might indirectly influence the efficiency oftranslation. Translation efficiency is thought to be a centralfactor in mediating cell growth (28, 38).Both the E. coli and B. subtilis rph gene products appear

to be dispensable, with the exception of special conditions inE. coli in which a large number of other RNase activities arealso removed (28a). The fact that the derived rph proteinsequences are highly conserved suggests that these proteinsare physiologically significant. Could the rph products servean important functional role and not be essential? There isprecedence for postulating such functions in E. coli. Forexample, the small DNA-binding protein, IHE, influences anumber of processes in E. coli including recombination,replication, and expression of a number of genes, but it hasnot been shown to have an essential function (17). An rphproduct could thus serve as a modulator of a number ofimportant processes and not be essential for bacterial viabil-ity.Although we are unable to offer a definitive explanation

for the role of the rph gene product in suppressing a varietyof cold-sensitive mutations, this study shows that the B.subtilis rph gene product, like its E. coli analog, has RNasePH activity that processes 3' ends of tRNA precursors. It islikely that, as the role for RNase PH in cellular physiology ismore clearly delineated, the explanation for cold-sensitivesuppression will be determined.

ACKNOWLEDGMENTSWe thank Ruth VanBogelen, M. Elizabeth Hutton, and Frederick

Neidhardt for help in the two-dimensional electrophoretic analysisof proteins. Dan Yansura is gratefully acknowledged for providingthe plasmid bank from which pBB1 was isolated. We thank LathamClaflin for help in preparing the antibody, and we thank Angie Grossfor help in preparing the manuscript.Experiments at the University of Michigan were supported by

NIH grant AI1459-10, and those at the University of Connecticutwere supported by NIH grant GM16317.

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