Effect of Integration Host Factor on RNA II Synthesis in Replication of Plasmid Containingorip15A

PLASMID

40, 150 –157 (1998) PL981361

ARTICLE NO.

Effect of Integration Host Factor on RNA II Synthesis in Replication of Plasmid Containing orip15A Elz˙bieta Hiszczyn´ska-Sawicka and Jo´zef Kur Department of Microbiology, Technical University of Gdan´sk, ul. G. Narutowicza 11/12, 80-952 Gdan´sk, Poland Received March 5, 1998; revised April 30, 1998 The synthesis rates of the replication control RNAs of plasmid orip15A, RNA I, an inhibitor of replication, and RNA II, the primer, have been determined using lacZ fusion plasmids, hybridization assay, and reverse transcription polymerase chain reaction (RT-PCR) in Escherichia coli integration host factor-positive (IHF1) and -negative (IHF2) strains containing pACYC184 plasmid (orip15A). In the absence of IHF (E. coli IHF2), expression of the lacZ gene from the PRNAII promoter increased by a factor of 4 compared with the E. coli wild type (IHF1). Also, the increase in expression was more pronounced when the IHF protein was mutated in the ihfB gene than in the ihfA gene. For the PRNAII promoter of oripMB1 (pBR322), no significant differences were found in expression of the lacZ gene in he E. coli strains examined. The level of b-galactosidase expression from the PRNAI promoter of orip15A shows that the absence of functional IHF in the transformed strains has no effect on expression of the lacZ gene. The synthesis RNA II:RNA I ratio obtained in hybridization assays was 2.4 for E. coli IHF1 and 4.4 for E. coli IHF2. Densitometric analysis of RT-PCR products indicates that the relative levels of RNA I in E. coli IHF1 and IHF2, are equal, but the relative level of RNA II in E. coli IHF2 is about four times higher than in E. coli IHF1. These results indicate that the IHF protein inhibits transcription from the PRNAII promoter of orip15A plasmid. © 1998 Academic Press

Key Words: integration host factor; ihf site; histone-like protein; pBR322; pACYC184; orip15A; RNA II primer; RNA I; reverse transcription polymerase chain reaction; b-galactosidase.

The integration host factor (IHF)1 of Escherichia coli is a histone-like, heterodimeric DNA-binding protein consisting of the ihfA/ himA and the ihfB/hip gene products. The IHF protein participates in several regulatory processes, including transcription modulation, phage packaging, plasmid replication and transfer, and bacterial phase variation (see review by Friedman, 1988). It is known from published data that both ihfA and ihfB mutants fail to maintain plasmid pSC101 (Gamas et al., 1986) and a truncated form of plasmid R6K (only with g ori) (Filutowicz and Appelt, 1988). IHF can either repress or stimulate transcription (Goosen and van de Putte, 1995). For example, the IHF protein presents both activities at the ilvGMEDA; it represses an upstream promoter, pG1, and activates a downstream promoter, pG2. For 1 Abbreviations used: IHF, integration host factor; wt, wild type; SDS, sodium dodecyl sulfate; RT, reverse transcription; PCR, polymerase chain reaction.

0147-619X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

several operons of E. coli, IHF has been shown to inhibit transcription in vivo and in vitro. In some cases IHF seems to act as a classical repressor, as one or more IHF binding sites have been found to overlap with the 210 and/or 235 region of the promoter (Griffo et al., 1989; Kur et al., 1989a,b; Tsui et al., 1991; Pagel et al., 1992; Ditto et al., 1994). IHF can also inhibit transcription indirectly through modulation of a regulator protein (Goosen and van de Putte, 1995). We have previously demonstrated that the E. coli IHF binds to the single site (ihf) of the DNA fragment containing the ori of plasmid p15A (Hiszczyn´ska-Sawicka and Kur, 1995). Our footprinting experiments on IHF protection of that ihf site show three distinct areas of protection. One of these coincides with the 210 region of the promoter for synthesis of a primer precursor transcript (RNA II). One ihf binding site was also found in the oripMB1 region se-

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IHF EFFECT ON RNA II SYNTHESIS

quence (pBR322 plasmid), but that site is weaker compared with the ihf of orip15A (Hiszczyn´ska-Sawicka and Kur, 1995). We also showed that the copy number of the orip15A plasmid increases about four times in the ihfAihfB double-mutant (65–70 copies per cell) and ihfB single-mutant (50 –56 copies per cell) cells and is almost the same in the ihfA mutant (17–18 copies per cell) and wild-type (14 –16 copies per cell) cells (Hiszczyn´ska-Sawicka and Kur, 1997). The results suggested that IhfB can form homodimers, which are functionally competent for regulation of the orip15A plasmid copy number. We also showed that the absence of IHF (using ihfAihfB double mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid. In this study, we show that there is an effect of IHF on the RNA II synthesis level of orip15A plasmid. The PRNA II promoter of orip15A appears to be specifically inhibited by IHF binding to the promoter region. IHF binding within the PRNA II promoter results in a decrease in PRNA II promoter activity and this binding has no effect on the PRNA I promoter of orip15A activity. MATERIALS AND METHODS Bacterial Strains and Plasmids Four isogenic E. coli strains were kindly provided by M. Chandler (Laboratory of Molecular Genetics and Microbiology, CNRS, Toulouse, France): E. coli 1061 (wt, IHF1), araD D(ara leu) galV galK hsdS rpsL D(lacI OP2YA) X74; E. coli 1069 (double mutant, IHF), 1061 D82(ihfA)::Tn10, D3(ihfB)::Cm; E. coli 1068 (single mutant, ihfA), 1061 D82(ihfA)::Tn10; E. coli 1067 (single mutant, ihfB), 1061 D3(ihfB)::Cm. The plasmids used in this study were pACYC184 (Chang and Cohen 1978), pBR322 (Bolivar et al., 1977), and pTL61T (Linn and Pierre, 1990). Construction of Transcription Fusions To test promoters for relative transcription activity, they were cloned upstream of a promoterless E. coli lacZ reporter gene of the mul-

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ticopy pTL61T vector plasmid. A 204-bp AluI– AluI fragment of the pACYC184 plasmid (from 1305 to 1509 bp of pACYC184 DNA) containing the PRNA II promoter of orip15A was cloned into the SmaI site of the pTL61T plasmid (pPJ1 construct). A 375-bp AluI–HaeIII fragment of pBR322 plasmid (from 3035 to 3410 bp of pBR322 DNA) containing the PRNA II promoter of oripMB1 was cloned into the SmaI site of plasmid pTL61T (pPJ2 construct). To test transcription of the PRNA I promoter of orip15A, a 903-bp PvuI–SspI fragment of the pACYC184 plasmid (from 515 to 1418 bp of pACYC184 DNA) was cloned into the SmaI site of the pTL61T plasmid (pPJ3 construct). DNA, RNA Preparation and Recombinant DNA Technology The plasmid DNA preparation kit (S.N.A.P. Miniprep Kit) was purchased from Invitrogen (USA). DNA fragments for cloning were purified from agarose and polyacrylamide gels using the DNA Gel Out Kit (A&A Biotechnology, Poland). Ligation and transformation were performed as described by Maniatis et al. (1982).

b-Galactosidase Assays b-Galactosidase assays were carried out according to Miller (1972). Assays were carried out at room temperature. b-Galactosidase activity in CHCl2 3 and sodium dodecyl sulfatepermeabilized cells was measured by monitoring the hydrolysis of o-nitrophenyl-b-D-galactopyranoside. Activities are expressed in terms of the OD600 of cell suspensions by Miller’s (1972) formula. Each culture was assayed in triplicate; results were confirmed by at least three independent experiments. In Vivo Labeling of RNA In vivo labeling of RNA was performed as described by Lin-Chao and Bremer (1986) with some modifications. Strains of E. coli IHF1 (MC1061 with pACYC184) and E. coli IHF2 (MC1069 with pACYC184) were grown overnight in LB medium; then 1 ml of the cultures were used to inoculate 100 ml of casamino acids

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mineral medium. Cultures were grown 6 h at 37°C; then an equal number of cells (2 3 1012) were withdrawn and centrifuged. The pellet was resuspended in 1.5 ml of the supernatant; 10 mCi of [3H]uridine was added. After incubation for 5 min the cells were centrifuged and RNA was isolated using the S.N.A.P. Total RNA Isolation Kit. A 0.1-ml sample of this isolated RNA from an E. coli IHF1 (MC1061 with pACYC184) strain contained about 19,000 acid-precipitable cpm and that from an E. coli IHF2 (MC1069 with pACYC184) strain, about 64,000 total cpm.

filters were removed to new bottles with 10 ml of hybridization buffer containing isolated labeled RNA. After further overnight incubation at 42°C with low-speed shaking, the filters were washed twice with 50 ml of 42°C 23 SSC/0.1% SDS buffer for about 5 min and twice with 50 ml 0.13 SSC/0.1% SDS buffer for about 15 min. After drying at room temperature, squares of nitrocellulose membrane filter corresponding to M13mp18RII, M13mp19RI, or M13mp18 DNA were cut out and placed into scintillation vials with scintillation fluid, and then radioactivity was counted.

DNA Preparations for Hybridization

RT-PCR

Two derivatives of M13mp18 and M13mp19 were constructed by inserting the EcoRI–XbaI fragment containing the replication origin of pACYC184 (from 1 to 1424 bp of pACYC184 DNA) into the EcoRI–XbaI site of the replicative form of phage M13mp18 or M13mp19. The phage whose plus strand is homologous to the replication primer, RNA II, was named M13mp18RII; the phage with the other orientation, whose plus strand is homologous to the plasmid replication inhibitor, RNA I, was named M13mp19RI. Supercoiled M13 replicative form DNA was prepared using S.N.A.P. Miniprep Kit. Single-stranded M13 phage DNA was prepared as described by Messing (1983).

Strains E. coli IHF1 (MC1061 with pACYC184) and E. coli IHF2 (MC1069 with pACYC184) were each grown in LB medium 6 h at 37°C and then equal numbers of cells (2 3 1011) were withdrawn and centrifuged. The cellular RNA was isolated using the S.N.A.P. Total RNA Isolation Kit. cDNA synthesis is limited by stable secondary structures. This problem occurs in the cases of RNA II and RNA I, and may be circumvented by increasing reaction temperature and using thermostable reverse transcriptase, e.g., Tth DNA polymerase. For full activity of Tth polymerase an optimal reaction temperature of 70°C and Mn21 ions are required. The synthetic oligonucleotides that anneal to RNA II, 59-GAAGTCATGCGCCGGTTAAGGCT (RT-RNA II primer), and RNA I, 59-GAAAAAACCGCCTTGCAGGGCGGT (RT-RNA I primer), were used as primers for cDNA synthesis. Oligonucleotides RT-RNA I, and RT-RNA II were used in the PCR for the RNA II generating a 135-bp amplicon. Oligonucleotides RNA I, 59-TTTGGTGACTGCGCTCCTCCAAGC, and RTRNA I were used in the PCR for the RNA I generating a 110-bp amplicon. RT and PCR were carried out using a Personal Thermocycler (Biometra, Germany). The RT mixture (20 ml) contained 10 mM Tris–HCl (pH 8.3); 90 mM KCl; 0.2 mM each of the deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP); 1 mM MnCl2; 20 pM RTRNA II or RT-RNA I primer; 5 units of Tth DNA polymerase; and 1

Binding of Single-Stranded DNA to Nitrocellulose Filters and Hybridization Assay Samples (20 ml) of M13mp18RII, M13mp19RI, or M13mp18 DNA corresponding to 5 mg of single-stranded DNA were diluted with 100 ml of the 203 SSC and added to wells in a slot-blot apparatus (Bio-Rad) with nitrocellulose membrane filters. The wells were washed with 100 ml of 203 SSC, and the filters were placed on a sheet of Whatman 3 paper and baked for 2 h at 60°C in a vacuum oven. The filters loaded with different single-stranded phage DNAs (M13mp18 as background control, M13mp18RII, or M13mp19RI) were placed in hybridization bottles containing 10 ml of hybridization buffer. After 6 h incubation at 42°C (prehybridization to reduce background), the

IHF EFFECT ON RNA II SYNTHESIS

mg of total RNA. Reactions were incubated at 94°C for 2min, then at 60°C for 2 min (annealing of RT primer) and at 70°C for 20 min for the RT step. Following the RT 30 ml of PCR mixture was added, containing 10 mM Tris–HCl (pH 8.3), 0.1 mM KCl, 0.75 mM EGTA, 0.05% Tween-20, 5% glycerol, 0.2 mM each of the dNTPs, 2.5 mM MgCl2, and 20 pM RT-RNA I or RNA I primer. The general incubation mixture (50 ml) was then amplified as follows: 30 s at 93°C, 30 s at 60°C, and 30 s at 72°C for 35 cycles. Aliquots of 10 ml were analyzed by electrophoresis on 6% polyacrylamide gel and stained with ethidium bromide for densitometric analysis. The calculation of optical density was performed with the ScanPack software (Biometra, Germany). RESULTS Effect of IHF on PRNA II and PRNA I Promoter Expression in Vivo Using lacZ Fusion Plasmids The regulation of PRNA II and PRNA I promoters was studied in vivo using lacZ fusion plasmids. To examine the effect of IHF on PRNA II and PRNA I expression, different lacZ constructs of the pTL61T (Linn and Pierre 1990) plasmid, containing oripMB1, were used. Plasmid pPJ1 contains the AluI 204-bp restriction fragment of pACYC184 plasmid (orip15A) harboring the PRNA II promoter. Plasmid pPJ2 contains the AluI–HaeIII 375-bp restriction fragment of pBR322 plasmid (oripMB1) harboring the PRNA II promoter. Plasmid pPJ3 contains the PvuI–SspI 903-bp restriction fragment of pACYC184 plasmid (orip15A) harboring the PRNA I promoter. Plasmids were transformed into the wild-type E. coli 1061 (IHF1), and into isogenic derivatives affected in the IHF double mutant, single mutant ihfA, or single mutant ihfB. We first investigated the pTL61T, pPJ1, pPJ2, and pPJ3 plasmid content in the abovementioned E. coli strains using the densitometry method according to Hiszczyn´ska-Sawicka and Kur (1997). The relative plasmid content per cell (copy number per cell) in double and single IHF mutants was similar to that in the wt strain (results not shown) independent of the plasmid

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and cultivation phase (15 copies). These results confirm our earlier observations that the absence of IHF had no effect on the copy number of the plasmid containing oripMB1 (Hiszczyn´ska-Sawicka and Kur, 1997). The cloned fragments, containing partial sequence of the orip15A, into the pTL61T, had no effect on the copy number of the pPJ1-3 plasmids. In the absence of IHF (E. coli ihfAihfB double mutant), expression of the lacZ gene of the pPJ1 plasmid (PRNA II promoter of orip15A) increased by a factor of 4 compared with the E. coli wt (IHF1) (Table 1). IHF protein is involved in the PRNA II promoter activity of orip15A probably as a modulator of transcription, decreasing the level of RNA II synthesis. When measured in single IHF mutants, the factor of increase was 1.15 in ihfA and 1.54 in ihfB single mutant. Thus, expression is more pronounced when the IHF protein is mutated in the ihfB than in the ihfA gene. This confirms our earlier data that homodimer IhfBIhfB of the IHF protein is more active in vivo than the homodimer IhfAIhfA (Zablewska and Kur, 1995; Hiszczyn´ska-Sawicka and Kur, 1997). There are no significant differences in lacZ expression among four isogenic E. coli strains (wt, ihfAihfB double mutant, ihfA and ihfB single mutants) containing pPJ2 plasmid (PRNA II promoter of oripMB1). We have also estimated the level of b-galactosidase expression from the PRNA I promoter of orip15A (plasmid pPJ3). The results show that the absence of functional IHF in the transformed strains has no effect on the expression of the lacZ gene (Table 1). The transformants show almost identical b-galactosidase activities under the growth conditions examined. IHF protein does not seem to be involved in the PRNA I promoter activity of orip15A. Effect of IHF on PRNA II and PRNA I Promoter Transcription in Vivo Using Hybridization Assay The amounts of labeled plasmid RNA I and RNA II were also determined by filter hybridization to specific probes, constructed from the single-stranded DNA of phage M13, carrying

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TABLE 1 In Vivo b-Galactosidase Expression of the PRNA II and PRNA I Promoter Constructs in Four Isogenic E. coli Strains: wt (IHF1), ihfAihfB Double Mutant (IHF2), ihfA and ihfB Single Mutants

b-Galactosidase (units)a

Increasing factor in relation to E. coli wt

orip15A orip15A orip15A orip15A

1300 5200 1500 2000

1.00 4.00 1.15 1.54

PRNA II PRNA II PRNA II PRNA II

oripMB1 oripMB1 oripMB1 oripMB1

410 300 350 280

1.00 0.73 0.85 0.68

PRNA I PRNA I PRNA I PRNA I

orip15A orip15A orip15A orip15A

700 710 680 730

1.00 1.01 0.97 1.04

Strain of E. coli

Plasmid

wt ihfAihfB double mutant ihfA single mutant ihfB single mutant

pPJ1 pPJ1 pPJ1 pPJ1

PRNA II PRNA II PRNA II PRNA II

wt ihfAihfB double mutant ihfA single mutant ihfB single mutant

pPJ2 pPJ2 pPJ2 pPJ2

wt ihfAihfB double mutant ihfA single mutant ihfB single mutant

pPJ3 pPJ3 pPJ3 pPJ3

a

Promoter

Data shown are averages of at least three experiments.

regions homologous to RNA I and RNA II, respectively. In the case of the E. coli IHF1 (MC1061 with pACYC184) strain, about 60 cpm hybridized to the probe for RNA II on the filter, corresponding to 0.32% of the total acidprecipitable radioactivity present, and 25 cpm (0.18% of the total) hybridized to the probe for RNA I (Fig. 1). With the E. coli IHF2 (MC1069 with pACYC184) strain about 110 cpm hybridized to the probe for RNA II on the filter, corresponding to 0.17% of the total acid-precipitable radioactivity present, and 25 cpm (0.04% of the total) hybridized to the probe for RNA I (Fig. 1). The synthesis RNA II:RNA I ratio obtained was 2.4 for E. coli IHF1 and 4.4 for E. coli IHF2. These results confirm the inhibitory effect of the IHF protein on transcription from the PRNA II promoter of the orip15A plasmid. The difference in efficiency of RNA labeling between E. coli IHF1 (19,000 cpm) and E. coli IHF2 (64,000 cpm) probably means that the absence of the IHF protein increases the synthesis of many other RNAs in bacterial cells. Effect of IHF on PRNA II and PRNA I Promoter Transcription in Vivo Using RT-PCR The total RNA from E. coli IHF1 (MC1061 with pACYC184) and E. coli IHF2 (MC1069

with pACYC184) cells was reverse transcribed into cDNA and then subjected to 35 cycles of PCR amplification, giving RNA II and RNA I products. The same volume of RT-PCR products was fractionated by gel electrophoresis and visualized with ethidium bromide (Fig. 2). The densitometric analysis shown in Fig. 2 indicates that the relative level of RNA I is equal in E. coli IHF1 and E. coli IHF2. However, the relative level of RNA II in E. coli IHF2 is about fourfold higher than that in E. coli IHF1. The number of cycles was so chosen that the products would be detectable on the electrophorogram, but the yield of products would not reach the plateau (usually three to five cycles less than the plateau value; results not shown). These results again confirm the inhibitory effect of the IHF protein on transcription from the PRNA II promoter of the orip15A plasmid. DISCUSSION In our previous report (Hiszczyn´ska-Sawicka and Kur, 1995) we showed that IHF binds to the single site of the orip15A region of pACYC184 plasmid that coincides with the 210 region of the PRNA II promoter for synthesis of a primer precursor transcript (RNA II). The affinity of

IHF EFFECT ON RNA II SYNTHESIS

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FIG. 1. Hybridization of RNA II and RNA I, pulse-labeled in vivo, to filters loaded with single-stranded DNA of phage M13mp18RII (RNA II columns) and M13mp19RI (RNA I columns). Data shown are averages of three experiments.

IHF to the orip15A ihf site is rather high, as it is very similar to that observed for the H9 ihf of l attP [the same relative IHF concentration resulted in a complete shift of the fragment (Hiszczyn´ska-Sawicka and Kur, 1995)]. The sequence of the ihf site of orip15A shows sequence similarity to all published IHF consensus sequences with two mismatches. The results of this study were not sufficient to assume that the presence of an IHF binding site in the primer promoter region might contribute significantly to the regulation of transcription and plasmid replication. However, the presence of an IHF

binding site that overlaps the PRNA II promoter led us to the hypothesis that IHF functions as a repressor of RNA II transcription. In the next paper (Hiszczyn´ska-Sawicka and Kur, 1997) we showed that the relative plasmid content per cell (copy number per cell) of the orip15A plasmid increases about four times in ihfAihfB double-mutant (65–70 copies) and ihfB single-mutant (50 –56 copies) cells as compared with wt cells (14 –16 copies per cell) in the stationary phase. In the ihfA mutant the relative plasmid content per cell (copy number per cell) was approximately the same as in the wt strain

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FIG. 2. Relative RNA I and RNA II content of E. coli IHF1 or IHF2 cells deduced from densitometric analysis of ethidium bromide-stained bands for RNA I and RNA II products after RT-PCR.

in each phase of growth (18 and 15 copies in the stationary phase, respectively). These results also confirmed earlier reports regarding the functionality of the IhfB homodimer (Zablewska and Kur, 1995; Zulianello et al., 1994). To confirm the above results we carried out the radioactive thymidine incorporation assay. The method allows direct evaluation of DNA synthesis and may be used in the study of plasmid DNA replication in IHF mutant strains. When the orip15A plasmid-harboring IHF double-mu-

tant bacteria grow exponentially during the first 4 h of the experiment, the synthesis of plasmid DNA (pulse-labeling) per bacterial mass remains at almost a constant level (increasing factor in relation to wt 5 1.58 at 4 h cultivation time). However, during the early stationary phase, the synthesis of plasmid DNA increases (increasing factor in relation to wt 5 3.8 at 6 h cultivation time; the calculated copy number was 80 compared with 21 in the wt strain) (Hiszczyn´ska-Sawicka and Kur, 1997).

IHF EFFECT ON RNA II SYNTHESIS

The purpose of the present work was to test whether the IHF affects the copy number of the p15A plasmid and the replication level of PRNA II or/and PRNA I promoter transcription regulation. We found that the PRNA II promoter is about four times more active in a host lacking IHF than in wild-type cells. It is possible that IHF directly regulates transcription initiation at PRNA II and interferes with the binding of RNA polymerase to the PRNA II promoter. We propose that IHF binding to the PRNA II promoter region partially blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure. As it is known from our last report (Hiszczn´ska-Sawicka, and Kur, 1997) DNA replication (copy number of plasmids per cell) is sensitive to an increased level of IHF protein in the stationary phase of cultivation. It seems that IHF protein controls the copy number of the orip15A derivative plasmids mainly in the stationary phase, where the IHF level is high, by partially blocking transcription from the PRNA II promoter. Our present results also suggest that the IhfB subunit of IHF can form functional homodimers in vivo with lower activity than the IhfAIhfB heterodimer. However, the effect on transcription is low (1.5-fold) and could be indirect. ACKNOWLEDGMENT This work was supported by the Chemical Faculty of the Technical University of Gdan´sk.

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