JOURNAL OF BACTERIOLOGY, June 1997, p. 3797–3800 0021-9193/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 179, No. 11
Integration Host Factor Is Required for 1,2-Propanediol-Dependent Transcription of the cob/pdu Regulon in Salmonella typhimurium LT2 MICHELLE R. RONDON†
AND
JORGE C. ESCALANTE-SEMERENA*
Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706 Received 24 February 1997/Accepted 20 March 1997
We show that integration host factor (IHF) is required for the activation of transcription of the cobalamin biosynthetic (cob) and 1,2-propanediol (1,2-PDL) utilization (pdu) operons in Salmonella typhimurium LT2. A lack of IHF affected transcription of the cob/pdu regulon in at least two ways. First, the level of the regulatory protein PocR was decreased in ihfB (formerly himD) mutants, as judged by Western blot analysis with polyclonal antiserum raised against PocR. Second, even when PocR was available, in the absence of IHF, PocR was unable to activate transcription of cob/pdu in response to 1,2-PDL. This result suggested an additional role for IHF in PocR-dependent transcription activation. Consistent with these findings, ihfB mutants of this bacterium were unable to use 1,2-PDL as a carbon or energy source. presence or absence of 1,2-PDL as described elsewhere (16). cbi-24::MudI1734 is a fusion within the cob operon, and expression of this fusion measures cob expression. pdu-12:: MudI1734 is located within the pdu operon (12). ihfB (formerly known as himD [14, 22]) mutants were completely defective in 1,2-PDL-dependent transcription, whereas the ihfB1 control strains were able to induce cob/pdu transcription in response to 1,2-PDL. We measured a 45-fold reduction in the expression of cbi-24::MudI1734 in the ihfB mutant JE4000 (ca. 2 U of b-galactosidase activity) relative to that in the ihfB1 strain JE1734 (ca. 90 U of b-galactosidase activity). Similarly, expression of pdu-12::MudI1734 in the ihfB mutant strain JE4001 (ca. 1 U of b-galactosidase activity) was reduced 75-fold relative to the level measured in the ihfB1 strain JE2507 (ca. 75 U of b-galactosidase activity). ihfB mutants were also defective in the transcription of fusion cbi-24::MudI1734 (JE4001) under anaerobic growth conditions (data not shown). As a control, we examined the transcription of the cobA gene, which encodes the ATP:corrinoid adenosyltransferase required for cobalamin synthesis. The cobA gene is unlinked to the cob operon at 43.5 centisomes, and its transcription is not affected by factors known to regulate expression of the main cob operon (9, 16, 17, 19, 20). Transcription of a cobA343:: MudI1734 fusion (strain JE1096) was unaffected by the introduction of the ihfB mutation (strain JE4002) (data not shown). These results indicate that IHF was required for the activation of cob/pdu expression in response to 1,2-PDL. Consistent with these data, ihfB mutants were unable to grow with 1,2PDL as their carbon or energy source (Fig. 1). Growth of the ihfB mutant on 1,2-PDL was restored by providing ihfB in trans by using plasmid pHX3-8, which contained wild-type copies of the ihfA and ihfB genes of Escherichia coli (Fig. 1) (14). Location of potential IHF-binding sites within the cob/pdu control region. Using the MacTargsearch program, developed to identify putative IHF-binding sites (11), we located several potential IHF sites between pdu and cob. The locations of these sites in the cob/pdu control region are shown in Fig. 2 (6, 7, 15, 18); scores for similarity among these sites are also presented in Fig. 2. Although these have not been shown to be functional IHF-binding sites in vivo, they provided a starting point for investigating the role of IHF in this system. A recent analysis of the role of IHF in the regulation of
Transcription of the cobalamin biosynthetic (cob) and 1,2propanediol (1,2-PDL) utilization (pdu) operons in Salmonella typhimurium is induced in response to 1,2-PDL in the medium (5, 16). This regulation is mediated by PocR, a protein belonging to the AraC family of transcription factors (15, 18). Transcription of cob/pdu is also influenced by the cyclic AMP (cAMP) receptor protein-cAMP complex and by ArcA (1–3, 5, 8). Although the effects of these global regulatory proteins have not been shown directly, it is thought that the cAMP receptor protein-cAMP complex acts by inducing pocR transcription (1), while ArcA may increase pocR transcription under anoxic conditions and may activate cob transcription directly (1, 2). In vitro, 1,2-PDL had a strong positive effect on the binding of PocR to the cob promoter region (15). In vivo, 1,2-PDL may also play a role in the modulation of the level of PocR in the cell, since PocR–1,2-PDL may activate transcription from the upstream pduF promoters. These promoters (see Fig. 2) were postulated to transcribe pocR (6). Thus, 1,2-PDL may affect both the activity and the level of PocR in the cell. We demonstrate herein that the global regulatory protein integration host factor (IHF) is required for transcription of the cob/pdu regulon. IHF is a sequence-specific DNA-binding protein which participates in many cellular processes, including site-specific recombination, DNA replication, and regulation of transcription (10). Data showing that IHF is required at multiple steps during the induction of cob/pdu transcription in response to 1,2-PDL are presented. IHF mutants are unable to transcribe the cob/pdu regulon in response to 1,2-PDL. Expression of cbi and pdu operon fusions was measured as a function of IHF in the cell. b-Galactosidase activity was measured in strains JE1734 (metE205 ara-9 cbi-24:: MudI134), JE4000 (metE205 ara-9 cbi-24::MudI1734 ihfB:: Cm), JE2507 (metE205 ara-9 pdu-12::MudI1734), and JE4001 (metE205 ara-9 pdu-12::MudI1734 ihfB::Cm) grown in the * Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, 1550 Linden Dr., Madison, WI 53706-1567. Phone: (608) 262-7379. Fax: (608) 262-9865. E-mail:
[email protected]. † Present address: Department of Plant Pathology, University of Wisconsin—Madison, Madison, WI 53706. 3797
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FIG. 1. Growth on 1,2-PDL requires IHF. Cells were grown aerobically in NCE medium containing methionine (0.5 mM), MgSO4 (0.1 mM), 1,2-PDL (50 mM), and cyanocobalamin (75 nM). Ampicillin was added to 20 mg/ml for the maintenance of pHX3-8. Growth was monitored by measuring the A650. Symbols: open circles, TR6583 (ihfB1); open squares, JE3999 (ihfB:Cm); closed circles, JE4003 (ihfB1)/pHX3-8; closed squares, JE4004 (ihfB::Cm)/pHX3-8. All strains were derivatives of strain TR6583 (metE205 ara-9). Plasmid pHX3-8 carries bla1, whose product (b-lactamase) provides resistance to ampicillin (14).
transcription compared 20 putative IHF-binding sites in E. coli operons where binding was confirmed by DNase I footprinting and/or gel retardation assay (10). For these sites, binding affinity was less correlated to the similarity score obtained by
J. BACTERIOL.
MacTargsearch, with the average score being only 46.8. Since many of the operons tested had altered expression in an ihf mutant background (10), this finding suggested that the lowerscoring sites in the cob/pdu control region may be physiologically relevant. ihfB mutants produce detectable levels of PocR protein. To determine the effect of IHF on PocR levels in vivo, we measured the levels of PocR in strains containing an ihfB, pduF, or pocR insertion mutation by Western blot analysis and compared those levels to the level in the wild-type strain. Cells were grown aerobically on NCE medium (21) containing succinate (30 mM), pyruvate (2 mM), MgSO4 (1 mM), methionine (0.5 mM), and 1,2-PDL (25 mM). These conditions were found to maximize the level of PocR in the cell. One-milliliter samples were collected at an A650 of 0.5 and resuspended in 0.1 ml of loading buffer (4) per 0.5 A650 unit. Thus, equal amounts of cells of each strain were used as a source of protein and were loaded onto the gel. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13) and then transferred to an Immobilon-P membrane (Millipore, Bedford, Mass.) by using a Mini Trans-Blot apparatus at 100 V for 1 h according to the manufacturer’s instructions (Bio-Rad, Richmond, Calif.). Western blots were performed as outlined in the ECL kit manual (Amersham, Arlington Heights, Ill.), with a few modifications. Membranes were blocked at 4°C overnight, and Tris-buffered saline with 0.05% Tween 20 and 5% dry milk was used for all incubations. Rabbit polyclonal antibodies directed against PocR were prepared by the Animal Care Facility of the Medical School at the University of Wisconsin— Madison and were used at a 30,000-fold final dilution. The secondary antibody was horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Pierce, Rockford, Ill.) and was diluted 1:20,000. X-ray film (XAR-50; Kodak) was used to detect the signal. As shown in Fig. 3, the level of PocR in strains containing an
FIG. 2. cob/pdu region and locations of putative IHF-binding sites. (A) This schematic diagram is not to scale. Lines with arrows indicate the direction and origin of the inferred transcripts. The small boxes indicate the locations of the putative IHF-binding sites. The region upstream of the cob promoter is enlarged below the main diagram to show the relative locations of the PocR-binding sites (hatched boxes) and the putative IHF-binding site within that region. (B) The MacTargsearch program provides a similarity score relating the putative IHF-binding site to the consensus sequence, with the average score being 60 and the lowest score being 46.2 (11).
VOL. 179, 1997
NOTES
FIG. 3. ihfB mutants contain a level of PocR similar to that observed in pduF mutants. A Western blot of total cell extract probed with anti-PocR antiserum is shown. Equal amounts of total cells were loaded; thus, the levels of PocR in different lanes can be directly compared. PocR was detected as described in the text. Lane 1, TR6583 (pduF1 pocR1 ihfB1); lane 2, JE3999 (pduF1 pocR1 ihfB:: Cm); lane 3, JE1925 (pduF501::Tn10d(Tc) pocR1 ihfB1); lane 4, JE1935 (pduF1 pocR106::Tn10d(Tc) ihfB1). All strains were derivatives of strain TR6583 (metE205 ara-9).
ihfB or a pduF mutation was decreased relative to the amount of PocR in the wild-type strain (Fig. 3, lanes 2 and 3, respectively), suggesting that both these mutations interfere with maximal PocR production in the presence of 1,2-PDL. As expected, no PocR was detected in the strain containing the pocR insertion (Fig. 3, lane 4). The important point is that the amount of PocR in the ihfB strain was equal to or greater than that found in the pduF strain, suggesting that IHF-deficient mutants, which produce PocR protein at a level comparable to that seen in the pduF mutant, still require IHF to transcribe cob/pdu. These results imply that IHF was involved at more than one level in the 1,2-PDL-dependent activation of cob and pdu expression and was not required simply for PocR production. ihfB mutants have a more severe effect on cob/pdu transcription than pduF mutants. Since ihfB mutants were shown to have levels of PocR protein equal to or higher than those of pduF mutants, one might expect that the effects of the two mutations on cob/pdu transcription would be similar; this was not the case. We compared the effects of both mutations on cob and pdu expression and found that the two mutations had very different effects. Results obtained with fusion pdu-12:: MudI1734 are shown in Table 1. Similar results were obtained with strains carrying the cbi-24::MudI1734 fusion (data not shown). Cells were grown for these assays under the same growth conditions that were used for the detection of PocR levels; thus, the results from the two experiments can be compared. In spite of the observed variability, the effect of the ihfB mutation was clearly similar to the effect of the pocR mutation, not to that of a pduF mutation. The ihfB mutation completely TABLE 1. Comparison of the effects of an ihfB mutation and a pduF mutation on pdu transcriptiona b-Galactosidase activityc b
Strain
Relevant genotype
Without 1,2-PDL
With 1,2PDL
JE2507
pdu-12::MudI1734
1
124 (675)
JE2510
pdu-12::MudI1734 pduF501::Tn10d(Tc)
1
44 (621)
JE2511
pdu-12::MudI1734 pocR106::Tn10d(Tc)
1
1
JE4001
pdu-12::MudI1734 ihfB::Cm
1
1
a
The results are the averages of two experiments. Activity measurements were performed in duplicate in each experiment. b All strains were derivatives of TR6583 (metE205 ara-9). Cells were grown as described in the text. c b-Galactosidase activity is expressed in nanomole of o-nitrophenyl-b-D-galactosidase per minute per A650 unit.
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eliminated transcription of the pdu and the cob operon fusions. These results suggest that IHF had a role in the activation of cob/pdu transcription in addition to affecting the level of PocR in the cell. Conclusion. This work shows that in S. typhimurium LT2, ihfB mutants are unable to use 1,2-PDL as a carbon or energy source. IHF was required to produce wild-type levels of PocR protein in response to 1,2-PDL and appears to play an additional role in the activation of cob/pdu expression. The effect of a lack of IHF cannot be explained solely by the decreased level of PocR in the cell, indicating that IHF is also required at another step in transcription activation. We have previously shown that PocR binds to two regions upstream of the cob promoter (15). The documented role of IHF in binding and bending DNA suggests that it might be needed to help form a structure at the cob and pdu promoters which may be required for PocR function. Preliminary data suggest that IHF may bind to the cob promoter region in vitro (data not shown). Another possibility is that IHF is required indirectly for the production of a different, as-yet-unidentified factor which is required for PocR-dependent transcription activation. Further experiments are needed to understand this additional requirement for IHF in the activation of cob/pdu transcription. This work was supported in part by Public Health Service grant GM40313 to J.C.E.-S., by graduate school project 960116, and by the College of Agricultural and Life Sciences. We thank S. Maloy for providing ihf mutant strains and plasmid pHX3-8, M. Filutowicz for the gift of purified IHF protein, and W. McClure for the MacTargsearch program. REFERENCES 1. Ailion, M., T. A. Bobik, and J. R. Roth. 1993. Two global regulatory systems (Crp and Arc) control the cobalamin/propanediol regulon of Salmonella typhimurium. J. Bacteriol. 175:7200–7208. 2. Andersson, D. I. 1992. Involvement of the Arc system in redox regulation of the Cob operon in Salmonella typhimurium. Mol. Microbiol. 6:1491–1494. 3. Andersson, D. I., and J. R. Roth. 1989. Redox regulation of the genes for cobinamide biosynthesis in Salmonella typhimurium. J. Bacteriol. 171:6734–6739. 4. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current protocols in molecular biology, vol. 1. Greene Publishing Associates and Wiley-Interscience, New York, N.Y. 5. Bobik, T. A., M. Ailion, and J. R. Roth. 1992. A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation. J. Bacteriol. 174:2253–2266. 6. Chen, P., M. Ailion, T. Bobik, G. Stormo, and J. Roth. 1995. Five promoters integrate control of the cob/pdu regulon in Salmonella typhimurium. J. Bacteriol. 177:5401–5410. 7. Chen, P., D. I. Andersson, and J. R. Roth. 1994. The control region of the pdu/cob regulon in Salmonella typhimurium. J. Bacteriol. 176:5474–5482. 8. Escalante-Semerena, J. C., and J. R. Roth. 1987. Regulation of cobalamin biosynthetic operons in Salmonella typhimurium. J. Bacteriol. 169:2251–2258. 9. Escalante-Semerena, J. C., S.-J. Suh, and J. R. Roth. 1990. cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium. J. Bacteriol. 172:273–280. 10. Freundlich, M., N. Ramani, E. Mathew, A. Sirko, and P. Tsui. 1992. The role of integration host factor in gene expression in Escherichia coli. Mol. Microbiol. 6:2557–2563. 11. Goodrich, J. A., M. L. Schwartz, and W. R. McClure. 1990. Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 18:4993–5000. 12. Jeter, R. M. 1990. Cobalamin-dependent 1,2-propanediol utilization by Salmonella typhimurium. J. Gen. Microbiol. 136:887–896. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. 14. O’Brien, K., G. Deno, P. Ostrovsky de Spicer, J. F. Gardner, and S. R. Maloy. 1992. Integration host factor facilitates repression of the put operon in Salmonella typhimurium. Gene 118:13–19. 15. Rondon, M. R., and J. C. Escalante-Semerena. 1996. In vitro analysis of the interactions between the PocR regulatory protein and the promoter region of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2. J. Bacteriol. 178:2196–2203. 16. Rondon, M. R., and J. C. Escalante-Semerena. 1992. The poc locus is re-
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quired for 1,2-propanediol-dependent transcription of the cobalamin biosynthetic (cob) and propanediol utilization (pdu) genes of Salmonella typhimurium. J. Bacteriol. 174:2267–2272. 17. Rondon, M. R., R. Kazmierczak, and J. C. Escalante-Semerena. 1995. Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2. J. Bacteriol. 177:5434–5439. 18. Roth, J. R., J. G. Lawrence, M. Rubenfield, S. Kieffer-Higgins, and G. M. Church. 1993. Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J. Bacteriol. 175:3303–3316.
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