Vol. 174, No. 14

JOURNAL OF BACrERIOLOGY, JUlY 1992, p. 4727-4735

0021-9193/92/144727-09$02.00/0 Copyright 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 coli MARK 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 of Michigan 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 the cold-sensitive defect of the nusAl4O(Cs) Escherichia coli mutant. The responsible gene encodes an open reading frame 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 been named rph because, like its E. colh analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E. coil, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusAII (Ts) mutant, that were tested.

The evolutionary divergence of Bacillus subtilis and Escherichia coli, easily observed in their different life styles, has been verified at the molecular level (49). There are, however, a number of examples of functionally equivalent E. coli and B. subtilis gene products that are related by greater than 40% identity at the amino acid level (1, 7, 21, 45). Although some of these products are not essential for cell viability, all are involved in important physiological processes. The NusA transcription elongation factor, an essential E. coli protein (31, 44), was first identified because it is a participant in the N transcription antitermination system of coliphage X (18, 41). Western blots (immunoblots) (18a) suggested that B. subtilis expresses a similar protein. A selection designed to isolate the B. subtilis analog of the E. coli nusA gene uncovered a previously unidentified gene of B. subtilis whose deduced protein sequence exhibits greater than 50% identity with an open reading frame, orfE, found in E. coli (40). More recently, it has been shown that orfE encodes RNase PH (37), a phosphate-dependent exoribonuclease that removes nucleotides 3' to the CCA end of tRNA (13, 15) and adds nucleotides to the ends of RNA molecules by using nucleoside diphosphates as substrates (36). The orfE gene was renamed rph to reflect this activity (37). Although E. coli carrying a deletion and an insertion disrupting rph grows normally (39), the rph gene product is required for growth of an E. coli mutant strain missing a collection of nuclease activities, RNase I, RNase II, RNase BN, RNase D, 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). The second gene, pyrE, encodes the enzyme phosphoribosyl transferase, one of six enzymes involved in the de novo *

synthesis of UTP (32). Expression of pyrE is regulated by a UTP-mediated attenuation mechanism located in the intercistronic region between rph (orfE) and pyrE (4, 9, 27). We report the identification of the rph analog from B. subtilis and show the similarity between its deduced amino acid sequence and those of the E. coli and the Salmonella typhimurium genes. Functional analysis demonstrates that the rph gene of B. subtilis encodes an activity analogous to that of the rph-encoded RNase PH found in E. coli. Although there is significant identity between the amino acid sequences of these rph genes, suggesting selective pressure for maintenance of the protein, insertional inactivation of the B. subtilis rph gene, as shown for the E. coli analog (39), does not appear to affect bacterial viability. MATERIALS AND METHODS

Strains. The bacteria used in these studies are listed in Table 1. Media. E. coli strains were grown in Luria-Bertani (LB) broth supplemented, where appropriate, with antibiotics at the following concentrations (micrograms per milliliter): 30, kanamycin and ampicillin; 15, tetracycline; and 12.5, chloramphenicol. B. subtilis strains were grown in tryptone blood agar base or minimal glucose medium. Anaerobically cultured bacteria were grown in a Coy chamber (5). Reagents. Restriction endonucleases, T4 DNA ligase, and phosphorylated MluI linkers were purchased from New England Biolabs. Bacterial alkaline phosphatase was purchased from Bethesda Research Laboratories. Transduction. PBS-1 was used for B. subtilis essentially according to the method outlined by Hoch et al. (25). P1 transduction was employed for E. coli (29). EOP. The growth of conditionally defective mutants at nonpermissive temperatures was quantitatively measured by using the efficiency of plating (EOP) method. Bacteria containing test plasmids were cultivated overnight at their permissive temperature in LB broth supplemented with the

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.

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TABLE 1. Bacterial strains Species and strain

E. coli K37 K95 K1914 K3909 K4206 D80 IQ346 CG431 RR100 K3732 K4713 K4714 K4716 NT675 NT701 18-11

Relevant genotype and/or parent strain

Source

Uncharacterized; cold sensitive ssyA3(Cs) nusB136(Cs) poL4(Ts) K1914 + pBB1 rph::CamTpoL4(Ts) rph::Cam' K37 rph::Camr ftsZ84(Ts) dnaA204(Ts) ma mb md mt RNase BNb

These laboratoriesa These laboratoriesa These laboratoriese M. Gottesman This work J. Ingraham K. Ito C. Georgopoulos P. Rockwell This work This work This work This work N. Trun N. Trun These laboratories

1168

trpL2

J. Hoch

QB936

ald-i aroG932 leuA8 trpC2

BG-SC(#lAY)

Wild type

nusAl K37 nusAIO(Cs) K37 nusAll(Ts) K37 nusAIO(Cs) pmc-1

B. subtilis

University of Michigan. b Relevant phenotype. c University of Connecticut.

a

appropriate antibiotics. The overnight culture was diluted into the same medium and grown to 2 x 108 cells per ml at the permissive temperature. Dilutions of the bacteria were spread on LB plates, and the plates were incubated at permissive and nonpermissive temperatures. The EOP is calculated by dividing the number of colonies observed at the nonpermissive temperature by the number observed at the permissive temperature. Plasmids. The following plasmids contained B. subtilis DNA (Fig. 1). pBB1 was selected from a library created by shotgun cloning B. subtilis DNA into the EcoRI site of pBR322. pBB2 is a derivative of pBB1 with an SphI-created deletion covering 612 bp of insert and 566 bp of vector DNA. pUBi is a pUC18 derivative with a 1-kb HpaI-EcoRI fragment from pBB1 containing the complete B. subtilis rph (rphBs) gene cloned between its SmaI and EcoRI sites in the opposite orientation with respect to Plac pUB2 is a pUC19 derivative with an EcoRI-BamHI fragment from pBB1 containing the complete rphBs gene cloned between its BamHI and 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 a gift from James Friesen. pBE1 is a pBR322 derivative with the 1,080-bp Clal-BamHI fragment from pGA2 and contains the E. coli gene plus a portion of the upstream pyrE genes. pUE281 is a pUC18 derivative with the EcoRI-BamHI fragment from pBEl oriented so that rphEC can be transcribed from Plac, pUE185 has the FnuDII fragment (MluI linked) of pBR328 carrying Camr cloned into the MluI site of pUE281, resulting in a disrupted rph gene. pRPH1, which has been previously described under the name pORFE-1 (37), is a derivative of pHC79 that contains a wild-type copy of rphEc (24). The following plasmids contained hybrid rph genes (Fig. 3). A shared SphI restriction site was used to construct hybrid rph genes that maintained the proper reading frame at the junction of the B. subtilis and E. coli mph genes. pHBEl

was constructed by placing the SphI fragment containing the 3' portion of rphF, from pUE281 into SphI-digested pBB2. pHEB5 was constructed by replacing the SphI fragment containing the 3' portion of rphEc in pUE281 with the SphI fragment 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 of lacZ. Because appropriate restriction sites were not available to clone rphEc directly into pUR288, a plasmid intermediate, pUE80, was constructed by removing the 872-bp MspI fragment containing the complete rphE gene plus 13 additional nucleotides upstream of the AUG from pUE281, ligating it into the AccI site in pUC8, and screening by restriction digests for the desired orientation. The BamHIHindIII fragment of pUE80 was cloned into pUR288. The resulting plasmid, pFBO2, contains the complete rphEc coding sequence plus seven additional codons upstream of the first AUG sequence fused in frame to the 3' end of the lacZ gene. The following plasmids were used in B. subtilis. pJHrph was created by cloning the EcoRI fragment from pBB1 into the integrative plasmid, pJH101 (16). pJHrphD5 was created by cloning a 250-bp HincII-BglII fragment from the B. subtilis rph gene into pJH101. Transformations. The transformation procedure used for B. subtilis was essentially the method of Anagnostopoulos rph B. s.

pBB1 E -1264

H

E -1264

H

pBB2

.rph P:4

I E

H H -244

+190

H H -244

+190

l

B. s.

I

S

S

-

pUB1 E

S

*B -244

+190

rph B. s.

pUB2

1

II

B

*

-244

S

I E

+190

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

rph GENE OF BACILLUS SUBTILIS

VOL. 174, 1992

pyrE E. c.

rph E. c.

-P.

P1

pGA2

PO.

4 wz

l

s

cC -83

B

rph E. c.

pBEl

P1

J)

E C;

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S

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pUE281 E C

Im

C S

B

S

P Camrr

pUE185

P.

l,TA

E C

and Spizizen (3), and that for E. coli was essentially the method of Cohen et al. (10). '~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Preparation of anti-RphF, antibody. The B-Gal-RphEc fusion protein was isolated by gel fractionation from cellular extracts of a bacteria with the fusion-expressing plasmid pFBO2. The mashed gel slice, mixed with complete Freund's adjuvant, was used for primary and secondary immunization of New Zealand White rabbits. Serum isolated 2 weeks after the secondary immunization was employed in the experiments. Protein techniques. Western blots were run essentially as described previously (6). Proteins labeled in the steady state with [35S]methionine were analyzed by two-dimensional gel electrophoresis as previously described (35). Enzyme assays. RNase PH and polynucleotide phosphorylase were assayed as described previously (14, 15, 37). DNA sequencing. Restriction enzyme-generated fragments from the insert in pBB1 were cloned into M13 vectors and sequenced by the dideoxy method (8). Construction of the rph::Camr chromosomal insertion. The rph::Camr insertion was crossed from pUE185 onto the E. coli chromosome in apoLA(Ts) pyrE' strain (N7622) according to a previously described method (23). P1 transduction was employed to move rph::Camr to strain K37. Nucleotide sequence accession number. The sequence of the rphBS gene and surrounding sequences has been deposited in GenBank under accession no. M85163. RESULTS

B

M

4729

FIG. 2. Plasmid with rphE, inserts. Signals and conventions are as listed in the legend to Fig. 1. Locations of the full coding regions of rphE, andpyrE are indicated by thick arrows; partial regions are indicated by thick lines. P1 is the identified promoter for the rph-pyrE operon. Positions of relevant restriction enzyme sites are shown: B, BamHI; C, ClaI; E, EcoRI; M, MluI. The ClaI site is located 83 bp upstream of the first AUG in the rph coding sequence. This map is not drawn to scale. pBE1 is derived from pBR322, while pUE185, pUE281, and pUE291 are pUC derivatives. The position of the Camr insert in the rph gene of pUE185 is indicated.

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 screened for nusA-complementing activity by using a bacterial strain (K1914) that has the nusAlO(Cs) allele (44). This mutant allele is composed of two nucleotide changes in nusA that cause a cold-sensitive phenotype (12, 26); bacteria carrying nusAlO(Cs) fail to grow at or below 32°C. Ampicillin-resistant transformants of K1914 were selected at 30°C. One transformant, designated K3732, obtained with this complementation selection was chosen for further study. We demonstrated in two ways that the plasmid in K3732, named pBB1, confers cold resistance. First, derivatives of K3732 Hybrid rph B.sIE.c.

D, l1

pHBE1 Eco RI

Hpa I

ATG

Sph I

-1264

-244

+1

+190

Bam HI Sph I

Hybrid rph E.c.lB.s. P1_4

pHEB5 Eco RI

Cla I -83

ATG +1

Sph I

Eco RI

Sph I

+190

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 symbols correspond to B. subtilis sequences, and white portions correspond to E. coli sequences. Relevant restriction enzyme sites, as well as the distances in base pairs from the first AUG in the coding sequence, are listed below each map. The map is not drawn to scale.

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CRAVEN ET AL.

cured of pBB1 are cold sensitive. Second, derivatives of the cold-sensitive parental strain K1914 transformed with pBB1 are cold resistant. pBB1 DNA was shown by restriction enzyme analysis to have a 2-kb insert and by Southern analysis to hybridize to B. subtilis DNA and not toE. coli DNA (data not shown). A deletion derivative was constructed by treating pBB1 with SphI, which cleaves at a site in the insert and at a site in the plasmid. The resulting plasmid, pBB2, has a 612-bp deletion of insert DNA and fails to complement the cold-sensitive defect of K1914. Complementation of other nusA mutations. We next determined whether pBB1 complementation of the nusAIO(Cs) mutation reflects expression of a product with NusA-like activity that complements the cold-sensitive defect or an activity unrelated to NusA that indirectly suppresses the cold-sensitive defect. If the former were the case, we would expect that pBB1 would complement the mutant phenotypes of other nusA mutations. However, if the latter were the case, we might expect pBB1 not to complement these mutant 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 N transcription antitermination system of bacteriophage X. This is evidenced by a failure of X growth in a nusAl mutant at 42°C (19). E. coli with the nusAll(Ts) mutation are not viable at 42°C (31). Neither of these mutant phenotypes is suppressed by pBB1 (data not shown). Since both of these mutant alleles are recessive, the failure to observe complementation suggests that pBB1 does not express a NusA-like activity. Thus, the cold resistance conferred by pBB1 to the nusAlO mutant is likely to result from some type of suppression. Analysis of B. subtilis DNA in pBB1. The sequence of the 2-kb insert in pBB1 was determined, and an analysis of the sequence revealed a 735-bp open reading frame located at one end of the insert. A computer-based comparison revealed no identities between this DNA sequence or its derived protein sequence with either the E. coli nusA DNA or protein sequences. A GenBank search failed to identify significant homologies with any DNA sequences. However, a search of protein sequences uncovered an open reading frame of 238 codons in E. coli called orfE (rph) with significant 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 amino acid at position 173 of the E. coli rph-encoded protein sequence) shows identical amino acids at 132 of 238 positions (56% identity) (Fig. 4). Moreover, 30 of the differing amino acids are conservative changes. This B. subtilis open reading frame will be referred to as rphBS, while the analogous sequence from E. coli will be called rphEr. Salmonella typhimurium, a close relative of E. coli, also has a bicistronic pyrE operon. The sequence of the 3' end of the promoterproximal gene has been determined, is extremely close to that of rphEc (33), and thus will be referred to as rphs5. The portion of the S. typhimurium DNA sequence available for comparison begins at the codon corresponding to amino acid 130 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 these positions differ in the alignment of rphE,,, with lphB., three of the nine encoded by the rphs, gene match with the amino acids at the corresponding positions in rph.1, i.e., at least in

10 B. E.

subtilis coli

S.

typhimurium

20

30

40

50

MRHDGRGHDELRPITFDLDFISHPEGSVLITAGNTKVICNASVEDRVPPF

B.s. similarity to E.c. or S.t.

B. subtilis E. coli S. typhimurium

B.s. similarity to E.c. or S.t.

B. E.

subtilis coli

S.

typhimurium

B.s. similarity to E.c. or S.t.

B. E. S.

subtilis coli typhimurium

B.s. similarity to E.c. or S.t.

B.

subtilis

100 80 90 60 70 LRGGGKGWITAEYSMLPRATNGRTIRESSKGKISGRTMEIQRLIGRALRA LKGQGQGWITAEYGMLPRSTHTRNAREAAKGKQGGRTMEIQRLIARALRA

L-G-G-GWITAEY-MLPR-T--R-*RE--KGK--GRTMEIQRLI-RALRA 150 140 120 130 110 VVDLEKLGERTIWIDCDVIQADGGTRTASITGAFLAMAIAIGKLIKAGTI L GKL AVDLKALGEFTITLDCDVLQADAWTRTASITGACVA A TGACVAI

AI L

GKL

*VDL- -LGE -T I--DCDV*QAD *-TRTAS ITGA- -A-A-A* -KL* --G- *

200 170 180 190 160 KTNPITDFLAAISVGIDKEQGILLDLNYEEDSSAEVDMNVIMTGSGRFVE YVEDSAAETDMNVVMTEDGRIIE KTNPMKGMVAAVSVGIVNGE EYVEDSAAETDMNVVMTEDGRIIE KTNPMKGMVAAVSVGIVNGE KTNP---- *AA*SVGI-----I--DL-Y-EDS-AE-DMNV*MT--GR-*E

240 230 210 220 LQGTGEEATFSREDLNGLLGLAEKGIQELIDKQKEVLGDSLPELK

E. coli

S.

typhimurium

B.s similarity to E.c. or S.t.

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

FIG. 4. Comparison of translated rph sequences. Amino acids are 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 the alignment. The bottom sequence indicates the similarities between the B. subtilis sequence and either or both of the E. coli and S. typhinurium sequences. Letters indicate amino acids found in common, asterisks indicate conservative differences, and dashes indicate nonconservative differences. Differences between rphEc and rphs, are indicated by boxes. The rphEc sequence used in this comparison was obtained from SWISSPROT. The carboxy terminus of rphst was described previously (33).

the carboxy terminus, rphB5 more closely corresponds to

rphst.

rphBS encodes an RNase PH-like enzyme. The similarity of the rphBS open reading frame with that of rphEC led us to test whether pBB1 expresses an RNase PH-like activity. Previous experiments have established that RNase-deficient E. coli 18-11 can be used as the background to assess RNase PH expression from plasmids (37). The substrate for the enzyme assay is tRNA-CCA-[ H]Cn. A measure of the specificity of RNase PH activity is obtained by comparing its nucleolytic action on the tRNA substrate to that on [3H]poly(A); RNase PH, unlike polynucleotide phosphorylase, 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 the nuclease activity that processes the tRNA substrate than do extracts from the control derivative harboring the pHC79 parent vector. The relatively lower activity against the poly(A) substrate demonstrates the specificity of these reactions. Interestingly, the B. subtilis extracts are more active for the tRNA substrate than are the E. coli extracts. Because pRPH1, with rphEC, expressed lower levels of RNase PH activity than was expressed by pBB1 with rphBS, we tested two other plasmid constructs with rphEC to determine whether they express levels of the E. coli RNase PH comparable to those expressed by pBB1. A pBR322 derivative, pBE1, contains a 1-kb ClaI-BamHI insert with rphEr

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10

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FIG. 5. Relative levels of RNase PH activity. Enzyme activities expressed from the listed plasmids in strain 18-11 were measured as indicated in the Materials and Methods, and all values were normalized to that found for the control plasmid, pHC79. Enzyme activities were measured 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 operon as well as part of the downstream pyrE gene. A pUC18 derivative, pUE281, containing the rphEc gene, was constructed with the insert oriented so that the rph gene is transcribed from the lacZ promoter (Fig. 2). Transformants of E. coli 18-11 derivatives with these plasmids were assayed for RNase PH activity. As shown in Fig. 5, pBEl fails to express elevated enzyme activity, while pUE281 expresses only a low level of activity, 50% of the increase expressed from pRPHl. E. coli rph and the nusA4O(Cs) mutation. We next determined whether rphEr could also confer cold resistance to the nusAlO(Cs) bacterium, i.e., allow growth at 30°C. Although we were unable to obtain a plasmid that expressed levels of the E. coli RNase PH activity comparable to the levels of B. subtilis RNase PH activity expressed by pBB1, we considered it worthwhile to test the different plasmids with rphEc for suppression of the nusAlO(Cs) phenotype because it was conceivable that the suppressing activity might not correlate with RNase PH activity. Table 2 presents results of a quantitative assessment of the effect of rph plasmid constructs on the growth of K1914 derivatives at low temperatures. Because pUC plasmids are not stably maintained in the presence of the nusAlO(Cs) mutation, we used K4206, a derivative of K1914 with an additional mutation, pmc-1, that allows maintenance of pUC plasmids in nusAlO(Cs) mutants without affecting the coldsensitive phenotype. Thepmc-1 mutation located at min 2 on the 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 with rphE,, including pRPH1, fail to exhibit a similar phenotype.

These studies also revealed that pUC derivatives carrying

rphBS can be maintained by the nusAlO(Cs) strain in the

absence of thepmc-1 mutation at all temperatures, although pUC derivatives with rphEC, like pUC parent plasmids, could not be maintained in this host under identical conditions. Identification of the rph protein. Western blots were employed to measure expression of the Rph protein independently of enzymatic activity. Antibody was raised to the rphE, gene product by using a ,B-Gal-Rph fusion protein as the immunogen (see Materials and Methods). As shown in Fig. 6, pBE1 (lane 4) fails to express detectable levels of Rph TABLE 2. Effect of plasmids containing rph genes on

nusAlO(Cs) cold sensitivity

Plasmid

Gene

EOPI (colonies at 30'C/colonies at 40°C) K1914

K4206

(nusA1O)

(nusAlOpmc-1)

Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli.

A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia...
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