Gene Product Identification and Promoter Analysis ofhigLocus of Plasmid Rts1

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 679–684 (1996)ARTICLE NO. 1229

Gene Product Identification and Promoter Analysisof hig Locus of Plasmid Rts1

Qing Bao Tian, Tetsuya Hayashi, Takahiro Murata, and Yoshiro Terawaki1

Department of Bacteriology, Shinshu University School of Medicine, Matsumoto 390, Japan

Received June 26, 1996

The hig (host inhibition of growth) genes of plasmid Rts1 belong to the plasmid-encoded proteic killergene family. Compared with other proteic killer genes described so far, hig is unique in that the toxic part(higB) exists upstream of the antidote gene (higA). Here we describe results of the promoter analysis ofhig genes together with identification of the proteic gene products of higA and higB. Two promoters wereidentified in the hig locus; a stronger one, named Phig, is located upstream of higB and a weaker one,PhigA, is upstream of higA within the higB coding region. The Phig activity was negatively regulated byHigA and this regulation was augmented by HigB, whereas PhigA was not subjected to such a regulation.q 1996 Academic Press, Inc.

Proteic killer genes have been identified and characterized from several plasmids (1). Allof them consist of two genes encoding small polypeptides, a toxin and an antidote. Physiologicalfunction of these genes is thought to maintain the population of plasmid-containing cells byselective killing of the plasmid-free cells. The mechanisms are now explained as follows; ina plasmid-containing cell, the toxin is being kept inactive by forming a complex with theantidote. Upon loss of the plasmid, the less stable antidote is degraded by some host proteasesmore rapidly than the stable toxin, leaving the active toxin. Activation of the toxin results inthe host cell filamentation and cessation of the viable plasmid-lacking cell production.

A general structure of the proteic killer genes has been proposed (1). According to the model,the antidote and toxin genes exist in this order and compose an operon. The transcriptions ofthe operons are negatively regulated by antidote alone or by the complex of antidote and toxin.This regulation is considered as an important mechanism to protect the host cell from theaction of the toxin.

We have recently identified on the Rts1 genome a genetic locus hig (host inhibition ofgrowth) (2) that related to the temperature sensitive host cell growth (3). The hig consistedof two genes (higB and higA), encoding short polypeptides of 92 and 104 amino acid residues,respectively. From the functional analysis of the cloned genes, higB was found to encode akiller function and higA to suppress the higB function both in cis and in trans (2). These datasuggest that the hig genes belong to the proteic killer gene family.

A most characteristic feature of hig is in its unique gene arrangement where the toxin geneexists upstream of the antidote gene, in a reversed order compared with all other killer genes.This suggested that the expression of the hig genes might be controlled by some uniquemechanism(s) different from other systems. In this study, therefore, we analyzed the promoteractivity of the hig genes. Identification of the proteic gene products of higA and higB was alsodescribed.

1 Corresponding author. Fax: /81 (263) 340176. E-mail: [email protected].

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

679

AID BBRC 5188 / 6906$$$761 07-29-96 09:45:47 bbrca AP: BBRC

Vol. 225, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. (A) Gene arrangement of the hig locus and fragments used in this study. The numbers indicate the startor end position of each fragment. pTQB plasmids containing each fragment are shown on the right side. (B) TheDNA sequences used in the promoter assay for Phig (a) and PhigA (b), respectively. Two potential promoter regionspredicted by computer analysis and putative SD sequences are underlined. The arrows in (a) indicate two invertedrepeats found in the Phig region.

MATERIALS AND METHODSBacterial strains, plasmids, and cultural conditions. E. coli HB101 (4) (leuB6 supE44 thi-1 hsdS20 recA13 ara-14

proAB lacY1 galK2 rpsL20 xyl-5 mtl-1) was used as host for transformation. E. coli SE4006 (5) (araD relA thi rpsLrecA56 Dlac169 rsl:Tn10) was used for b-galactosidase assay. E. coli BL21 (6) [ompT [lon] hsdSB (r0B, m0

B; an E. coli Bstrain)] was used for expressing gst-higB fusion. E. coli BL21(DE3) (7) [ompT (lon) hsdS gal(lcIts857 ind1 Sam7 nin5lacUV5-T7 gene 1)] was used for expressing hig locus. Plasmids pTQB30 and pTQB31 are pACYC184 (8) recombinantplasmids containing higA and higBA fragments, respectively, in the BamHI and HindIII sites. In those plasmids theexpression of hig genes are under the control of their own promoters. Plasmid pFZY1 (9) is a miniF derived transcriptionallacZ fusion cloning vector and was used for constructing transcriptional lacZ fusions. Plasmids in which lacZ is transcriptedfrom Phig and PhigA were constructed by inserting Phig (Fig.1A, a) and PhigA (Fig.1A, b) fragments into BamHI andHindIII sites of pFZY1, resulting plasmids pTQB40 and pTQB41, respectively. Plasmid pSU2718GI (B-H) is a plasmidderived from pTI185 (10) in which the polylinker was replaced by a synthesized linker containing BamHI and HindIIIsites and transcriptional stop codons in all three reading frames between the polylinker and lacZ. Plasmids pTQB50 andpTQB51 are plasmids containing higA and higBA fragments without their own promoters in BamHI and HindIII sites ofpSU2718GI(B-H), respectively. In those plasmids the expression of hig genes are under the control of Plac. PlasmidpGEX4T-1 (Pharmacia) is a GST fusion vector. Plasmid pTQB70 was constructed by inserting higBA fragment (lackingPhig and the initiation codon of higB) into BamHI and SalI sites of pGEX4T-1. Plasmid pTQB60 and pTQB61 are pDP1(NEN products) recombinants with higA and higBA fragments. In those plasmids the expression of hig genes are under

680



the control of T7 promoter. Luria-Bertani medium (Difco) was used for transformation, Penassay broth (Difco) for selectionof transformants, 2YT (11) for isolation of plasmid DNA, and B broth (11) for the b-galactosidase assay. Screening ofclones for b-galactosidase assay was done on B agar (11) containing 50 mg/ml of X-gal (5-bromo-4-chloro-3-indolyl-b-galac-topyranoside). The following antibiotics were included in the media when needed: ampicillin (100mg/ml), chloram-phenicol (10 mg/ml), kanamycin (30 mg/ml), and tetracycline (10 mg/ml). Cells were cultured at 377C and growth wasmonitored by turbidity at 600 nm.

DNA manipulation. Plasmid DNA isolation, restriction enzyme digestion, ligation and transformation were performedusing standard methods (12). DNA fragments derived from restriction enzyme digestion were purified from agarosegels using a Prep-A-Gene DNA purification kit (Bio-Rad Laboratories, Richmond, Calif.). All the DNA fragmentsused in this study were generated by PCR (polymerase chain reaction), using a DNA Thermal Cycler (model IJ2000;Perkin Elmer Cetus, Norwalk, Conn.) and a GeneAmp PCR reagent kit with Ampli-Taq DNA polymerase (PerkinElmer). Oligonucleotides used as primers were designed to contain appropriate restriction sites at the 5* ends forsubcloning and were synthesized using a DNA synthesizer (model 391; Perkin Elmer). The 1.0 kb SacI-HindIIIfragment (Fig. 1A) cloned in pUC18 was used as the template. Nucleotide sequences of the cloned fragments wereconfirmed by the dideoxy chain termination method using an automated DNA sequencer (model 370A; Perkin Elmer).b-Galactosidase assay. The b-galactosidase activities of cultures of cells harboring plasmids concerned were assayed

by a modification of the method of Miller (11) as described by Easton and Rownd (13).Overproduction of HigBA and GST-HigB fusion proteins. E. coli strains harboring plasmids for the overproduction

of HigBA proteins and GST (glutathione S-transferase)-HigB fusion protein were cultured to mid-log phase, theninduced by IPTG (isopropyl-b-D-thiogalactopyranoside) at a final concentration of 1 mM. After induction for threehours, cells were collected by centrifugation, and solubilized with SDS (sodium dodecyl sulfate) loading buffer (1 mlper ml culture per OD600). Three microliter of each sample was then subjected to SDS-PAGE (polyacrylamide gelelectrophoresis) analysis using a tricine buffer system (14) and a 16.5% gel. The gel was stained with coomassiebrilliant blue R-250.

RESULTS AND DISCUSSION

Identification of hig gene products. To confirm that hig is a member of plasmid-encodedproteic killer gene family, it is important to identify the proteic gene products of higA andhigB. For this purpose, two DNA fragments containing higA alone or higBA (Fig. 1A) wereinserted into the downstream of T7 promoter of pDP1 expression vector. The resulting plasmids,pTQB60 and pTQB61, were transformed into an E. coli strain BL21(DE3). After inductionwith IPTG, total cellular proteins were analyzed by SDS-PAGE using tricine buffer system.Strains carrying plasmids pTQB60 or pTQB61 overproduced a 12 kDa polypeptide which wasnot expressed in cells harboring pDP1 (Fig. 2, lanes 1 to 3), indicating the 12 kDa polypeptidewas the higA gene product. In this experiment, we could not detect higB product though apossibility that the product comigrated with HigA protein was not excluded.

To identify the higB gene product, a DNA fragment containing higBA structural genes (butnot containing the initiation codon for higB) was synthesized by PCR and inserted into theBamHI/HindIII sites being located at the 3* end of GST gene on the pGEX4T-1 plasmid. Thisrecombinant plasmid, named pTQB70, was transformed into an E. coli strain, BL21. Afterinduction with IPTG, the total cellular proteins were analyzed by SDS-PAGE. The cells withpTQB70 overproduced a 38 kDa protein, in addition to a 12 kDa HigA protein which wasnot detected in the cells harboring pGEX vector plasmid (Fig. 2, lane 4 and 5). The size ofthe induced protein was in a good agreement with a calculated molecular mass of GST-HigBfusion protein (37.7 kDa). It should be mentioned here that the GST-HigB fusion protein wasalso highly toxic to E. coli cells and its toxic activity was neutralized by HigA protein asobserved for a native HigB protein (data not shown). This finding not only supported the ideathat the polypeptide of 92 amino acid residues encoded by the higB is responsible for its killerfunction but also implies a potential use of this type of fusion protein for the biochemicalanalyses of hig gene products in the future.

In conclusion, the observations described above, together with our previous finding that asingle base substitution in the initiation codon of higB led to the complete loss of its toxicactivity (2), confirmed that the hig of Rts1 belongs to the proteic killer gene family.

Presence of two promoters in hig. A computer analysis of the nucleotide sequence predicted

681



FIG. 2. Expression of Hig proteins and GST-HigB fusion protein. Plasmids used in this experiment are pDP1 (lane1), pTQB60 (lane 2), pTQB61 (lane 3), pGEX4T-1 (lane 4) and pTQB70 (lane 5). Host strains are BL21(DE3) forlanes 1-3 and BL21 for lanes 4 and 5. After induction with IPTG, total cellular proteins were separated on a 16.5percent SDS-polyacrylamide gel using a tricine buffer system. Protein bands of GST-HigB, GST and HigA areindicated.

two potential promoter sequences in hig, one is located upstream of higB (named Phig ) andthe other is upstream of higA within the higB coding region (PhigA ) (Fig. 1A). In order todetermine the promoter activities, two DNA fragments containing each sequence were synthe-sized by PCR and inserted upstream of a promoterless lacZ gene in a pFZY1 plasmid. Resultingplasmids pTQB40 and pTQB41 were transformed into an E. coli strain SE4006. The trans-formants with either plasmid formed blue-colored colonies on the plates containing X-gal afterincubated at 377C for 16 hours. This indicated that both fragments had promoter activities inthe transcriptional fusion. To compare strength of the promoter activities, the b-galactosidaseassay was carried out. Phig showed a strong activity with 1500 Miller units (an average ofthree independent experiments), whereas PhigA displayed a much weaker activity with 103Miller units, but a 2.5 fold excess of the level of a vector plasmid alone (Fig. 3). The existenceof the second promoter was unique to hig, which might reflect its unique gene arrangementof the toxin and antidote genes.

Negative regulation of Phig by HigA and its augmentation by HigB. To know whether thepromoters were regulated by any of hig gene products as found in other killer gene systems,we constructed pTQB30 and pTQB31 plasmids which were pACYC184-recombinant plasmids

FIG. 3. b-galactosidase activity of E. coli strain SE4006 harboring plasmids pFZY1, pTQB40 or pTQB41. Theaverage values (Miller units) of three independent experiments are presented.

682



TABLE 1Repression of Phig by Hig Proteins

b-Galactosidase activityCoresident Hig protein(s)plasmids encoded Miller units (%)

pACYC184 None 952 100.0pTQB30 HigA 438 46.0pTQB31 HigB and HigA 85 8.9

Note. Mid-log phase culture of E. coli SE4006 containing the Phig-lacZ fusion plasmid pTQB40 and the coresident plasmid indicated wereassayed for b-galactosidase activities as described under Materials andMethods. Specific activities are expressed in Miller units and representthe mean values of three independent experiments for each strain.

carrying higA or higBA genes, respectively. In these plasmids, hig genes were under the controlof their own promoters. After introducing the plasmids into the cell containing pTQB40, effectsof HigA alone or along with HigB on Phig were determined by measuring the level of b-galactosidase activities. The presence of pTQB30 and pTQB31 reduced the enzymatic activitiesof the Phig-lacZ gene of pTQB40 to 43% and 8.9% of the control, respectively (Table 1).This indicated that HigA repressed Phig weakly, and the repression by HigA was markedlyenhanced in the presence of HigB. On the contrary, PhigA was affected by neither HigA norHigA with HigB (data not shown).

To confirm the repressor activity of HigA, a plasmid named pTQB50 was constructed byinserting higA downstream of lac promoter of pSU2718GI(B-H) plasmid. Using this plasmidthat supplied a varied amount of HigA by adding different concentrations of IPTG to theculture, the repressor activity of HigA on Phig was determined. As increasing the IPTGconcentration, the promoter activity of Phig was repressed more prominently (Table 2), con-firming the repressor activity of HigA on Phig. Interestingly, in this experiment, the suppressionof Phig by HigA alone was again incomplete even at the high concentration of IPTG. On theother hand, when pTQB51 in which the higBA fragment lacking Phig was inserted downstreamof lac promoter of pSU2718GI(B-H) was introduced into the cell containing pTQB40, thePhig activity of pTQB40 was repressed almost completely as observed with pTQB31, even

TABLE 2Repression of Phig by Increasing Amounts of HigA

b-Galactosidase activityCoresident Concentrationplasmids of IPTG (mM) Miller units (%)

pSU2718GI(B-H) 1.00 945 100.0pTQB50 0.00 860 91.0pTQB50 0.01 875 92.6pTQB50 0.10 769 81.4pTQB50 1.00 286 30.3

Note. The assay and presentation of the data are the same as inTable 1. The production of HigA protein from pTQB50 was inducedby different concentrations of IPTG, and its effect on Phig of the reporterplasmid pTQB40 was measured.

683



under the condition when Plac was suppressed by the presence of glucose (data not shown).This suggested that the repressor function of HigA could be greatly enhanced in the presenceof a trace amount of HigB.

The feature of the transcriptional regulation by Hig proteins is similar to that of the pemlocus of plasmid R100 among the proteic killer gene family (15, 16, 17), although the genearrangement of antidote and toxin genes was different and the presence of the second promoter(PhigA) was unique to our hig locus. Regarding the negative regulation of Phig, it is worthyto note the existence of two inverted repeats which completely cover the Phig sequence(Fig.1B). These might provide binding sites for the repressor.

From the promoter activities described above, a subtle control mechanism of the hig proteickiller gene system could be drawn. A weak but constitutive promoter activity of PhigA wouldsupply a constant level of HigA. It would neutralize HigB and, at the same time, act on Phigto repress the expression of higB gene. Once HigB was produced in the cell, even in a traceamount, it would enhance the HigA repressor activity and shut down the expression of higBitself, while the production of HigA could continue by PhigA activity. Although other regulatorymechanism(s) could be involved in the expression of hig genes, this multiple regulatory systemat the transcription level would efficiently prevent an undesirable drive of hig gene system inthe host cell carrying the plasmid Rts1.

ACKNOWLEDGMENTSThe authors thank Dr Akira Tabuchi for providing plasmids, Miss Kaori Sato for her technical assistance, and Dr.

Makoto Ohnishi and Yong Fang Li for valuable discussions. This work was supported by a Grant-in-aid for ScientificResearch from the Ministry of Education, Science, and Culture of Japan and by grants from the Yakult Foundation.

REFERENCES1. Jensen, R. B., and Gerdes, K. (1995) Mol. Microbiol. 17, 205–210.2. Tian, Q. B., Ohnishi, M., Tabuchi, A., and Terawaki, Y. (1996) Biochem. Biophys. Res. Commun. 220, 280–284.3. Terawaki, Y., Kakizawa, Y., Takayasu, H., and Yoshikawa, M. (1968) Nature (London) 219, 284–285.4. Boyer, H. W., and Roulland-dussoix, D. (1969) J. Mol. Biol. 41, 459–472.5. Friedman, S. A., and Austin, S. J. (1988) Plasmid 19, 103–112.6. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Meth. Enzymol. 185, 60–89.7. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113–130.8. Chang, A. C. Y., and Cohen, S. N. (1978) J. Bacteriol. 134, 1141–1156.9. Koop, A. H., Hartley, M. E., and Bourgeois, S. (1987) Gene 52, 245–256.

10. Tabuchi, A., Min, Y.-N., Womble, D. D., and Rownd, R. H. (1992) J. Bacteriol. 174, 7629–7634.11. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor, Laboratory Press, Cold Spring

Harbor, NY.12. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, NY.13. Easton, A. M., and Rownd, R. H. (1982) J. Bacteriol. 152, 829–839.14. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368–379.15. Tsuchimoto, S., and Ohtsubo, E. (1993) Mol. Gen. Genet. 237, 81–88.16. Tam, J. E., and Kline, B. C. (1989) J. Bacteriol. 171, 2353–2360.17. Davis, T. L., Helinski, D. R., and Roberts, R. C. (1992) Mol. Microbiol. 6, 1981–1994.

684


Documents

Gene Product Identification and Promoter Analysis ofhigLocus of Plasmid Rts1