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Journal of Bacteriology, November 2001, p. 6413-6421, Vol. 183, No. 21
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.21.6413-6421.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ⁷⁰ Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

Simon L. Dove and Ann Hochschild^*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received 3 May 2001/Accepted 6 August 2001

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

A number of transcriptional regulators mediate their effects through direct contact with the ⁷⁰ subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ⁷⁰ that harbors conserved region 4. This region of  contains a putative helix-turn-helix DNA-binding motif that contacts the 35 element of ⁷⁰-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti- factor Rsd and the ⁷⁰ subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ⁷⁰ and also that amino acid substitution R596H, within region 4 of ⁷⁰, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between  and two other regulators shown previously to contact region 4 of ⁷⁰. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the ⁷⁰ subunit of RNAP from this organism. We found that amino acid substitution R600H in ⁷⁰ from P. aeruginosa, corresponding to the R596H substitution in E. coli ⁷⁰, specifically weakens the interaction between AlgQ and ⁷⁰. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ⁷⁰ and probably regulate gene expression through this contact.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Sigma factors are subunits of bacterial RNA polymerase (RNAP) that direct the holoenzymes that contain them to promoters of a specific class (20). In Escherichia coli there are seven different species of  factors, and ⁷⁰ is the principal  factor (27). The presence of different types of  factors within a cell with distinct DNA sequence binding specificities provides a mechanism for coordinate regulation of genes that are controlled by promoters of the same class. Competition between different  factors for the available RNAP core enzyme in part determines which genes are transcribed within a cell at any given time (27). This competition can be influenced by anti- factors, which are regulatory proteins that bind  factors and often prevent their association with the RNAP core enzyme (19, 26). Anti- factors ultimately inhibit transcription from the class of promoters recognized by the  factors that they sequester.

The ⁷⁰ subunit of RNAP participates in a number of protein-protein interactions, including interactions with other subunits of the polymerase complex (43, 52) and interactions with transcriptional regulators (18, 23). The regulators that interact with ⁷⁰ often contact a region of  that contains a putative helix-turn-helix DNA-binding motif responsible for contacting the 35 elements of ⁷⁰-dependent promoters (18, 23, 37). This DNA-binding region of  is conserved in members of the ⁷⁰ family of proteins and is called region 4 (36).

Recently, Jishage and Ishihama identified a protein in E. coli that was preferentially made by cells during the stationary phase of growth and was associated with the ⁷⁰ subunit of RNAP in stationary-phase extracts (28). The protein was named Rsd (which stands for regulator of sigma D) since it was found to associate specifically with ⁷⁰ (but not with several alternative  factors) and was shown to be capable of inhibiting ⁷⁰-dependent transcription from certain promoters in vitro (28). The binding site for Rsd on ⁷⁰ was mapped to a C-terminal tryptic fragment encompassing conserved region 4 (28). On the basis of these observations and because the synthesis of Rsd coincides with the general shutdown in ⁷⁰-dependent transcription that occurs as cells enter the stationary phase of growth, Jishage and Ishihama suggested that Rsd might be an anti- factor (28). Subsequent work has shown that consistent with this idea, Rsd may facilitate the replacement of ⁷⁰ by the stationary-phase-specific  factor ³⁸ in functional RNAP holoenzyme complexes as cells go from the exponential phase to the stationary phase of growth (29).

The sequence of putative anti- factor Rsd is similar to the sequence of a regulator of alginate production in Pseudomonas aeruginosa called AlgQ (or AlgR2) (28). Alginate is an important virulence factor that imparts the characteristic mucoid phenotype to P. aeruginosa isolated from the lungs of cystic fibrosis patients (17). P. aeruginosa isolates from other sources are typically nonmucoid and do not exhibit activated expression of genes involved in alginate production (10). However, the production of alginate is believed to promote survival of P. aeruginosa in the special environment of the lungs of cystic fibrosis patients, contributing to resistance to both immune responses and antibiotics (10). AlgQ was originally identified as a positive transcriptional regulator of the key alginate biosynthetic gene algD (8, 32), which is expressed at high levels in mucoid cells. The regulation of the algD gene is complex and involves two different  factors; transcription initiates from two superimposed promoters, one of which is recognized by RNAP containing ^E (AlgU/AlgT) and the other of which is recognized by RNAP containing ⁵⁴ (RpoN) (3, 9, 11, 21, 38, 49, 59). Furthermore, at least one DNA-binding protein, AlgR (AlgR1), is known to bind to specific sites upstream of the algD promoter and activate transcription (31, 41, 42). The mechanism by which AlgQ positively regulates transcription of the algD gene is not known.

We were interested in testing the idea that like Rsd, AlgQ interacts with region 4 of the ⁷⁰ subunit of RNAP. In this study we tested this idea explicitly by using a bacterial two-hybrid system. We found that both Rsd and AlgQ can interact with a ⁷⁰ moiety encompassing region 4 in vivo, and we identified an amino acid substitution in region 4 that specifically weakens the interaction of Rsd and AlgQ with the  moiety.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids and strains. E. coli XL1-blue (Stratagene) was used as the recipient strain for all plasmid constructions. E. coli KS1 harbors on its chromosome the lac promoter derivative placO_R2-62 driving expression of a linked lacZ reporter gene and has been described previously (14). E. coli SF1 harbors an F' episome containing the lac promoter derivative plac O_R2-55/Cons 35 driving expression of a linked lacZ reporter gene and has also been described previously (13).

Plasmids pACcI, pACcI, pBR, pBR-³⁸, pBR-⁷⁰, and pBR-⁷⁰ (R596H) have all been described previously (13, 14). Plasmid pACcI-Rsd encodes cI (residues 1 to 236) fused to Rsd (residues 1 to 158) via three alanine residues. pACcI-Rsd was made by cloning the appropriate NotI-BamHI-digested PCR product into NotI-BstYI-digested pACcI32 (24); expression of the cI-rsd fusion gene was therefore under the control of the lacUV5 promoter. Plasmid pACcI-AlgQ encodes cI (residues 1 to 236) fused to AlgQ (residues 1 to 160) via three alanine residues, and it was made in a manner similar to the manner in which pACcI-Rsd was made. Expression of the cI-algQ fusion gene on pACcI-AlgQ is also under the control of the lacUV5 promoter. Plasmid pACcI-AsiA encodes cI (residues 1 to 236) fused to AsiA from bacteriophage T4 (residues 1 to 90) via three alanine residues, and it was made in a manner similar to the manner in which pACcI-Rsd was made. Plasmid pAC-35cI-AsiA contains the cI-asiA fusion gene from plasmid pACcI-AsiA under the control of a lacUV5 promoter variant in which the 35 element of the promoter has been deleted. Plasmid pAC-35cI-AsiA, therefore, expresses less of the cI-AsiA fusion protein than plasmid pACcI-AsiA expresses under identical conditions. pAC-35cI-AsiA was made by cloning the appropriate HindIII-BstYI fragment from pACcI-AsiA into plasmid pA3B2 (57) cut with both HindIII and BstYI. Plasmid pBR-⁷⁰PA encodes residues 1 to 248 of the  subunit of E. coli RNAP fused to residues 532 to 617 of the ⁷⁰ subunit of P. aeruginosa RNAP. The hybrid -⁷⁰PA gene was made by performing PCR and was cloned into HindIII-BamHI-digested pBR. Expression of the chimeric gene on pBR-⁷⁰PA is, therefore, under the control of tandem lpp and lacUV5 promoters. pBR-⁷⁰PA (R600H) is a derivative of pBR-⁷⁰PA in which the R600H substitution in the  moiety of the chimera was introduced by PCR.

The lac promoter derivative placCOP-93+O_L2-62 was made by the PCR and contains two  operators; a near-consensus  operator (TACCACCGGCGGTGATA) and O_L2 (CAACACCGCCAGAGATA) are centered 93 and 62 bp, respectively, upstream of the transcriptional start site of the lac core promoter. Plasmid pFW11-COP-93+O_L2-62 was constructed by cloning an EcoRI-HindIII-cut PCR product containing placCOP-93+O_L2-62 into pFW11 (56) cut with EcoRI and HindIII. Plasmid pFW11-COP-93+O_L2-62 was then transformed into strain CSH100, and the promoter-lacZ fusion was recombined onto an F' episome and mated into strain FW102 (56) to create reporter strain F'93+62. The PCR-amplified regions of all plasmids were sequenced to confirm that no errors had been introduced as a result of the PCR process.

Experimental procedures. Cells were grown in LB supplemented with kanamycin (50 µg/ml), chloramphenicol (25 µg/ml), carbenicillin (50 µg/ml), and isopropyl--D-thiogalactoside (IPTG) at the concentration indicated. Cells were permeabilized with sodium dodecyl sulfate-CHCl₃ and assayed for -galactosidase activity essentially as described previously (39). Assays were performed at least three times in duplicate on separate occasions, and representative data sets are shown below. The values are averages based on one experiment; duplicate measurements differed by less than 10%.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

cI-Rsd activates transcription from a test promoter in the presence of a chimeric -subunit harboring region 4 of E. coli 70. We sought to detect an interaction between Rsd and region 4 of ⁷⁰ in vivo by using a bacterial two-hybrid system that we had recently developed (12, 14). This bacterial two-hybrid system is based on the finding that any sufficiently strong interaction between a protein bound upstream of a suitable test promoter and a component of RNAP can activate transcription in E. coli (12, 14). Thus, two proteins that interact with one another can mediate transcriptional activation in E. coli provided that one protein is fused to a DNA-binding protein and the other is fused to a component of RNAP (12, 14).

Our strategy for detecting an interaction between Rsd and region 4 of ⁷⁰ involved the use of two chimeric proteins, one comprising Rsd fused to the repressor of bacteriophage  (cI) and the other comprising a modified form of the -subunit of RNAP in which the C-terminal domain (CTD) of  has been replaced by a C-terminal fragment of ⁷⁰. We reasoned that RNAP containing the resulting -⁷⁰ chimera would display a target for Rsd that could be contacted by a DNA-bound cI-Rsd dimer (Fig. 1A). Having fused the entire Rsd protein (residues 1 to 158) to the C terminus of cI, we placed the gene encoding this chimeric protein on a plasmid vector downstream of the IPTG-inducible lacUV5 promoter, thus creating plasmid pACcI-Rsd. We used plasmid pBR-⁷⁰ as a source of the -⁷⁰ chimera. This plasmid encodes a chimera in which residues 528 to 613 of E. coli ⁷⁰ are fused to residues 1 to 248 of the -subunit of RNAP (13). We introduced plasmids pACcI-Rsd and pBR-⁷⁰ into E. coli KS1 (14), which harbors on its chromosome the lac promoter derivative placO_R2-62 (bearing a single  operator centered 62 bp upstream of the transcriptional start site) linked to a lacZ reporter gene. We then tested the ability of the cI-Rsd chimera to activate transcription from the placO_R2-62 test promoter in the presence of the -⁷⁰ chimera. Figure 1B shows that cI-Rsd activated transcription from the test promoter up to ~24-fold in cells containing the -⁷⁰ chimera compared to control cells containing only wild-type . An additional control revealed that cI (lacking the fused Rsd moiety) did not activate transcription from the test promoter in the presence of the -⁷⁰ chimera (Fig. 1B). We also found that cI-Rsd did not activate transcription from the test promoter in the presence of an -³⁸ chimera (comprising residues 1 to 248 of  fused to residues 243 to 330 of ³⁸) encoded by plasmid pBR-³⁸ (13; data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Transcriptional activation by cI-Rsd in the presence of the -70 chimera. (A) Replacement of the RNAP -CTD by a C-terminal fragment of E. coli 70 (residues 528 to 613) permits interaction with the Rsd moiety of a cI-Rsd chimera bound to DNA. The diagram depicts the test promoter placOR2-62, which bears the  operator OR2 centered 62 bp upstream from the transcriptional start site of the lac core promoter. In reporter strain KS1 the placOR2-62 test promoter is located on the chromosome and drives expression of a linked lacZ gene. The N-terminal domain of  is designated NTD. (B) Effect of cI-Rsd on transcription in vivo from placOR2-62 in the presence of the -70 or -70(R596H) chimera. KS1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity.

Substitution R596H in the  moiety of the -⁷⁰ chimera weakens the interaction between  and Rsd Our ability to link the protein-protein interaction between Rsd and its target on ⁷⁰ to transcriptional activation provided us with a useful genetic tool for dissecting this interaction. We were particularly interested in identifying mutant forms of ⁷⁰ that were specifically defective in the ability to interact with Rsd. We therefore introduced a variety of amino acid substitutions into the  moiety of the -⁷⁰ chimera and tested the effects of these substitutions on the ability of the cI-Rsd fusion protein to mediate transcriptional activation from the test promoter. Figure 1B shows that substitution R596H in the  moiety of the -⁷⁰ chimera strongly reduced the magnitude of cI-Rsd-dependent activation (to a factor of ~5). Substitution R596A in the  moiety of the -⁷⁰ chimera had a nearly identical effect on the magnitude of the activation (data not shown). In contrast, substitutions L573A, E591A, E591Q, H600A, and H600R did not decrease the magnitude of cI-Rsd-dependent activation (data not shown).

Substitution R596H in the  moiety of the -⁷⁰ chimera does not compromise the ability of a superactivating variant of cI to stimulate transcription from an appropriate test promoter. To determine whether the R596H substitution affects the interaction of ⁷⁰ with Rsd specifically, we tested its effect on the ability of another protein to interact with the  moiety of the chimera. We showed recently that the cI protein (a transcriptional activator as well as a repressor) can interact specifically with the  moiety of the -⁷⁰ chimera and stabilize its binding to a promoter 35 element (13). The in vivo assay which we designed to detect the interaction between cI and region 4 of ⁷⁰ is shown in Fig. 2A. In this experimental setup a DNA-bound cI dimer activates transcription from test promoter placO_R2-55/Cons-35 by stabilizing the binding of the  moiety of the -⁷⁰ chimera to the ectopic 35 element present upstream of the core promoter elements (13) (Fig. 2A). Transcriptional activation from this test promoter is dependent not only on the protein-protein interaction between cI and the tethered  moiety but also on the protein-DNA interaction between the tethered  moiety and the ectopic 35 element (13).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Transcriptional activation by cI superactivator in the presence of the -70 chimera. (A) cISa109 stabilizes the binding of region 4 of 70 to an ectopic 35 element. The diagram depicts the test promoter placOR2-55/Cons-35, which bears an ectopic 35 element and the  operator OR2 centered 45.5 and 55 bp, respectively, upstream from the transcriptional start site of the lac core promoter. In reporter strain SF1 the placOR2-55/Cons-35 test promoter is located on an F' episome and drives expression of a linked lacZ gene. (B) Effect of substitution R596H in the  moiety of the -70 chimera on the ability of cISa109 to activate transcription in vivo from placOR2-55/Cons-35. SF1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity.

We tested whether the R596H substitution in the -⁷⁰ chimera has an effect on the ability of a superactiviating variant of cI to activate transcription from test promoter placO_R2-55/Cons-35 (Fig. 2A). Reporter strain SF1 carries promoter placO_R2-55/Cons-35 fused to the lacZ gene in single copy on an F' episome (13). We assayed the ability of cI superactivator 109 (cISa109) (5, 13) to activate transcription from this reporter in the presence of the -⁷⁰ chimera with or without the R596H substitution in the  moiety. cISa109 activated transcription a maximum of ~5.5-fold from the test promoter in the presence of the -⁷⁰ chimera and a maximum of ~7.5-fold in the presence of the chimera harboring the R596H substitution (Fig. 2B). (Although the R596H substitution has previously been shown to inhibit the ability of wild-type cI to activate transcription from P_RM [35], we have observed that this substitution does not inhibit the ability of cISa109 to activate transcription from P_RM [see below].) We concluded that substitution R596H in the  moiety of the -⁷⁰ chimera does not result in a general defect in the ability of the  moiety to interact with other proteins.

We also tested the effect of the R596A substitution in the  moiety of the -⁷⁰ chimera on the ability of cISa109 to activate transcription from the same test promoter. Unlike the R596H substitution, the R596A substitution significantly reduced the magnitude of the activation by cISa109 (data not shown). Using another assay, however, we were able to determine that this reduction in activation was not due to a general defect caused by the R596A substitution (see below).

cI-AlgQ activates transcription from a test promoter in the presence of a chimeric -subunit harboring region 4 of P. aeruginosa ⁷⁰. Jishage and Ishihama (28) noted that Rsd exhibits 31% identity with the alginate regulatory protein AlgQ (also known as AlgR2) from P. aeruginosa. AlgQ was originally identified as a positive regulator of alginate production in P. aeruginosa (8, 32) but has subsequently been shown to regulate several other gene products in this organism (see below).

Very little is known about how AlgQ mediates its effects on gene expression. In order to test the hypothesis that AlgQ, like Rsd, can interact with region 4 of ⁷⁰, we made a cI-algQ fusion gene analogous to the cI-rsd fusion gene. We also made a fusion gene encoding a protein analogous to the -⁷⁰ chimera that contained region 4 of ⁷⁰ from P. aeruginosa. We fused the entire AlgQ protein (residues 1 to 160) to the C terminus of cI. We placed the gene encoding this chimeric protein on a plasmid vector downstream of the IPTG-inducible lacUV5 promoter, creating plasmid pACcI-AlgQ. We then fused residues 532 to 617 of P. aeruginosa ⁷⁰ (equivalent to residues 528 to 613 of E. coli ⁷⁰) to residues 1 to 248 of . The resulting chimera was called -⁷⁰PA. The hybrid -⁷⁰PA gene encoding this chimera was placed on a plasmid vector downstream of tandemly arranged lpp and lacUV5 promoters, creating plasmid pBR-⁷⁰PA. We introduced plasmids pACcI-AlgQ and pBR-⁷⁰PA into E. coli KS1. We then tested the ability of the cI-AlgQ chimera to activate transcription from placO_R2-62 in the presence of the -⁷⁰PA chimera (Fig. 3A). Figure 3B shows that cI-AlgQ activated transcription from the test promoter a maximum of ~17-fold in cells containing the -⁷⁰PA chimera compared to control cells containing only wild-type . An additional control revealed that cI without the fused AlgQ moiety did not activate transcription from the test promoter in the presence of the -⁷⁰PA chimera (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Transcriptional activation by cI-AlgQ in the presence of the -70PA chimera. (A) Replacement of the RNAP -CTD by a C-terminal fragment of P. aeruginosa 70 (residues 532 to 617) permits interaction with the AlgQ moiety of a cI-AlgQ chimera bound to DNA. The diagram depicts the test promoter placOR2-62, which bears the  operator OR2 centered 62 bp upstream from the transcriptional start site of the lac core promoter. In reporter strain KS1 the placOR2-62 test promoter is located on the chromosome and drives expression of a linked lacZ gene. (B) Effect of cI-AlgQ on transcription in vivo from placOR2-62 in the presence of the -70PA or -70PA(R600H) chimera. KS1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity.

Substitution R600H in the  moiety of the -⁷⁰PA chimera weakens the interaction between  and AlgQ. The ⁷⁰ subunits from E. coli and P. aeruginosa are very similar to one another, exhibiting ~83% identity over the length of the  fragments (86 amino acids) that we used in our experiments (36). We wanted to test whether substitution R600H in ⁷⁰ from P. aeruginosa, which corresponds to the R596H substitution in E. coli ⁷⁰, had any effect on the ability of cI-AlgQ to activate transcription in the presence of the -⁷⁰PA chimera. To do this, we made a version of the -⁷⁰PA chimera harboring substitution R600H [-⁷⁰PA(R600H)] and assayed the ability of cI-AlgQ to activate transcription in KS1 cells expressing this chimera. Figure 3B shows that cI-AlgQ activated transcription from the reporter gene a maximum of ~5-fold in the presence of the -⁷⁰PA(R600H) chimera, compared to a maximum of ~17-fold with the chimera derived from the wild-type form of P. aeruginosa ⁷⁰.

Substitution R600H in the  moiety of the -⁷⁰PA chimera does not compromise the ability of a superactivating variant of cI to stimulate transcription from an appropriate test promoter. In order to assess whether the effect of the R600H substitution was specific for the interaction between ⁷⁰PA and AlgQ, we first tested whether cISa109 could interact with the  moiety of the -⁷⁰PA chimera. We found that cISa109 stimulated transcription from test promoter placO_R2-55/Cons-35 up to ~3.5-fold specifically in the presence of the -⁷⁰PA chimera (Fig. 4). Furthermore, introduction of the R600H substitution into the  moiety of the -⁷⁰PA chimera did not abrogate the stimulatory effect of cISa109 (instead it resulted in a modest increase in the observed activation), suggesting that the effect of the R600H substitution is specific for the cI-AlgQ chimera (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Transcriptional activation by cI superactivator in the presence of the -70PA chimera. (A) cISa109 stabilizes the binding of region 4 of 70 from P. aeruginosa to an ectopic 35 element. The diagram depicts the test promoter placOR2-55/Cons-35, which bears an ectopic 35 element and the  operator OR2 centered 45.5 and 55 bp, respectively, upstream from the transcriptional start site of the lac core promoter. In reporter strain SF1 the placOR2-55/Cons-35 test promoter is located on an F' episome and drives expression of a linked lacZ gene. (B) Effect of substitution R600H in the  moiety of the -70PA chimera on the ability of cISa109 to activate transcription in vivo from placOR2-55/Cons-35. SF1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity.

The E. coli protein Rsd can interact with region 4 of ⁷⁰ from P. aeruginosa, and the P. aeruginosa protein AlgQ can interact with region 4 of ⁷⁰ from E. coli. Given the high degree of similarity between the regions of ⁷⁰ from E. coli and P. aeruginosa used in our experiments, we thought that each regulator might be able to contact region 4 of ⁷⁰ from either E. coli or P. aeruginosa. We explicitly tested whether Rsd could interact with region 4 of ⁷⁰ from P. aeruginosa and also whether AlgQ could interact with region 4 of ⁷⁰ from E. coli. Figure 5A shows that the cI-Rsd chimera activated transcription from the test promoter in KS1 cells a maximum of ~52-fold in the presence of the -⁷⁰PA chimera, compared to ~24-fold in the presence of the E. coli -⁷⁰ chimera. We also found that introduction of substitution R600H into the -⁷⁰PA chimera reduced the ability of cI-Rsd to stimulate transcription from the test promoter to a factor of ~13 (Fig. 5A).

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Rsd can interact with region 4 of 70 from P. aeruginosa, and AlgQ can interact with region 4 of 70 from E. coli. (A) Effect of cI-Rsd on transcription in vivo from placOR2-62 in the presence of the -70PA, -70PA(R600H), or -70 chimera. KS1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity. (B) Effect of cI-AlgQ on transcription in vivo from placOR2-62 in the presence of the -70, -70(R596H), or -70PA chimera. KS1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity.

Figure 5B shows that the cI-AlgQ chimera activated transcription from the test promoter in KS1 cells a maximum of ~7-fold in the presence of the -⁷⁰ chimera, compared to ~15-fold in the presence of the P. aeruginosa -⁷⁰PA chimera. Introduction of substitution R596H into the  moiety of the E. coli -⁷⁰ chimera reduced the ability of cI-AlgQ to activate transcription from the test promoter to a factor of ~2 (Fig. 5B).

cI-AsiA activates transcription from a test promoter in the presence of either - chimera. We took advantage of another protein known to interact with region 4 of E. coli ⁷⁰ to test further the specificity of the effect of the R596H substitution on the interaction of ⁷⁰ with either Rsd or AlgQ. The AsiA protein encoded by bacteriophage T4 is an anti- factor that has been shown to interact specifically and very strongly with region 4 of E. coli ⁷⁰ (1, 7, 22, 40, 50, 51). AsiA is thought to work by interacting with ⁷⁰ that is free in solution rather than with ⁷⁰ that is already complexed with the RNAP core enzyme (22). The interaction between AsiA and E. coli ⁷⁰ inhibits the activity of RNAP holoenzyme (containing the AsiA-⁷⁰ complex) by preventing region 4 of  from contacting the 35 element of ⁷⁰-dependent promoters (7, 51).

We therefore sought to detect the interaction between AsiA and region 4 of ⁷⁰ by fusing AsiA to cI and measuring the ability of the resulting cI-AsiA chimera to activate transcription from placO_R2-62 in the presence of either the -⁷⁰ chimera or the -⁷⁰PA chimera. We fused the entire AsiA protein (residues 1 to 90) to the C terminus of cI. We placed the gene encoding this chimeric protein on a plasmid vector downstream of the IPTG-inducible lacUV5 promoter, creating plasmid pACcI-AsiA. Initial experiments demonstrated that the cI-AsiA chimera was extremely toxic to cells at the levels provided by the expression vector pACcI-AsiA (data not shown). For subsequent experiments we constructed a different expression vector, pAC-35cI-AsiA, in which the chimeric cI-asiA gene was under control of an IPTG-inducible variant of the lacUV5 promoter that lacked the normal 35 element and therefore directed the synthesis of smaller amounts of the fusion protein. Since pAC-35cI-AsiA made insufficient levels of cI-AsiA to saturate the  operator present on the placO_R2-62 promoter construct, we also constructed a new test promoter bearing two relatively strong  operators in the upstream region (Fig. 6A; also see Materials and Methods). Figure 6B shows that the cI-AsiA chimera activated transcription from the modified test promoter in the presence of the -⁷⁰ chimera derived from ⁷⁰ of either E. coli or P. aeruginosa (similar findings with the -⁷⁰ chimera from E. coli have been obtained by S. Pande and D. Hinton [submitted for publication]). The data also show that substitution R596H in the E. coli  moiety or the corresponding R600H substitution in the P. aeruginosa  moiety had only a modest effect on the ability of the cI-AsiA chimera to activate transcription from placCOP-93+O_L2-62. Similarly, the R596A substitution in the E. coli  moiety did not reduce the stimulatory effect of the cI-AsiA chimera (data not shown). Since these substitutions do not appear to cause nonspecific defects in the abilities of the  moieties to interact with other proteins, we suggest that residue R596 of E. coli ⁷⁰ is at or near the contact surface for Rsd and the equivalent residue of P. aeruginosa ⁷⁰, R600, is at or near the contact surface for AlgQ (see Discussion). Furthermore, the finding that substitution R596A in E. coli ⁷⁰ results in a strong defect in the ability of  to interact with Rsd suggests that the arginine side chain may make an energetically significant contact with Rsd.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Transcriptional activation by cI-AsiA in the presence of the - chimeras. (A) Schematic diagram of test promoter placCOP-93+OL2-62 used to determine the effects of substitutions in the - chimeras on transcriptional activation by cI-AsiA. The test promoter placCOP-93+OL2-62 bears both a consensus  operator and OL2 centered 93 and 62 bp, respectively, upstream from the transcriptional start site of the lac core promoter. In reporter strain F'93+62 the placCOP-93+OL2-62 test promoter is located on an F' episome and drives expression of a linked lacZ gene. (B) Effect of cI-AsiA on transcription in vivo from placCOP-93+OL2-62 in the presence of the - chimeras. F'93+62 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for -galactosidase activity. cI refers to the fact that no cI was made by control plasmid pACcI.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Rsd and AlgQ can interact with region 4 of ⁷⁰ in vivo. We found that the E. coli Rsd protein and the P. aeruginosa AlgQ protein can interact in vivo with a C-terminal fragment of the ⁷⁰ subunit of E. coli and P. aeruginosa RNAP, respectively. This fragment of ⁷⁰ encompasses conserved region 4, which contains a DNA-binding domain that mediates recognition of the 35 element of ⁷⁰-dependent promoters (18). Furthermore, each protein can also interact with the corresponding ⁷⁰ fragment from the heterologous organism. We also identified a single amino acid substitution (R596H and the corresponding substitution R600H in E. coli and P. aeruginosa ⁷⁰, respectively) that inhibits the binding of both Rsd and AlgQ to either ⁷⁰ fragment. The interchangeability of the E. coli and P. aeruginosa ⁷⁰ fragments in our experiments suggests that regulators from P. aeruginosa or E. coli that interact with region 4 of ⁷⁰ might in general be expected to function in either organism. In fact, a previous in vitro study showed that the cI protein (which contacts region 4) can activate transcription from the  promoter P_RM by the P. aeruginosa RNAP (16).

Analysis of the interaction between Rsd and region 4 of ⁷⁰. Our bacterial two-hybrid results for Rsd and region 4 of ⁷⁰ are consistent with the biochemical data of Jishage and Ishihama (28). These authors found that Rsd could interact with ⁷⁰ in vitro but not with ³⁸ (or several other alternative  factors), and they showed that Rsd could interact with a tryptic fragment of ⁷⁰ composed of residues ~500 to 613. We found that Rsd can interact in vivo with an 86-amino-acid C-terminal fragment of ⁷⁰ that contains region 4 but cannot interact with the equivalent 88-amino-acid C-terminal fragment of ³⁸. Jishage et al. (30) have recently reported that alanine substitutions at two positions flanking residue 596 (L595 and L598) disrupt the association of Rsd with ⁷⁰ in vitro. However, in contrast with our results, they reported that substitution R596A in ⁷⁰ did not prevent association of Rsd with ⁷⁰ (in a glutathione S-transferase pulldown assay). To better compare the results of Jishage et al. with our results, we introduced substitutions L595A and L598A into the  moiety of the -⁷⁰ chimera and tested their effects on the interaction with Rsd in vivo. We found that both the L595A substitution and the L598A substitution strongly inhibited the interactions with Rsd and that their effects were more severe than that of the R596A substitution (data not shown). We suggest, therefore, that the in vivo assay may be more sensitive than the in vitro assay, allowing us to detect the less severe effect of the R596A substitution. Moreover, a structural model of region 4 of ⁷⁰ based on the crystal structure of the NarL protein (see below) suggests that the side chain of residue 595, at least, is likely to be partially buried. Substitutions at position 595 and possibly also at position 598 may affect the interaction with Rsd indirectly; consistent with this possibility, we found that substitutions L595A and L598A both completely eliminated the ability of cISa109 to activate transcription from our artificial test promoter in the presence of the -⁷⁰ chimera (data not shown).

Amino acid substitution R596H has been isolated previously. It was first identified based on its effect on expression of the arabinose operon (25, 53). Expression of the ara genes is positively controlled by two regulators, the global regulator cyclic AMP receptor protein (CRP) and the operon-specific regulator AraC (15). CRP is active only when it is complexed with the small molecule effector cyclic AMP, and consequently mutant strains that are not able to synthesize cyclic AMP (cya mutants) cannot induce expression of CRP-dependent operons, including the arabinose operon. A mutation in the ⁷⁰ (rpoD) gene specifying the R596H substitution was isolated as a suppressor that restored expression of the arabinose operon in a cya mutant background (25). It has been suggested that the R596H substitution enhances the ability of AraC to interact productively with RNAP (25, 37). This same amino acid substitution was subsequently isolated based on its ability to suppress the effect of a cI positive control mutation at the  promoter P_RM. Suppressor mutations in the rpoD gene were sought that would reverse the activation defect of a cI mutant bearing substitution D38N in its activating region, and a single mutation specifying the R596H change was obtained (35). Although the R596H substitution in ⁷⁰ reduces the ability of wild-type cI to activate transcription from P_RM, we have shown that this substitution actually enhances the ability of cISa109 (which bears a non-wild-type residue at position 38) to stimulate transcription from P_RM (unpublished data). Evidently, certain amino acid-amino acid combinations at position 38 of cI and position 596 of ⁷⁰ permit efficient activation, while others do not.

Taken together, the data obtained in previous studies and data obtained in this study suggest that R596 of ⁷⁰ is exposed on the surface of the polypeptide such that it can be contacted by interacting proteins. This suggestion is supported by the results of structural modeling. Region 4 of ⁷⁰ contains a putative helix-turn-helix motif, and the structure of this DNA-binding domain has been modeled based on the three-dimensional crystal structures of two related helix-turn-helix proteins, the E. coli NarL protein and the bacteriophage 434 Cro protein (4, 37). In both structural models, residue 596 is solvent exposed and accessible when the protein domain is bound to DNA.

AlgQ and regulation of gene expression in P. aeruginosa. Our findings with AlgQ may be relevant to understanding the mechanism by which it regulates gene expression in P. aeruginosa. The finding that AlgQ, like Rsd, can interact with a C-terminal fragment of the ⁷⁰ subunit of RNAP provides strong support for the proposal that it is a functional homolog of Rsd. This conclusion is reinforced by our finding that the interactions of Rsd and AlgQ with region 4 of ⁷⁰ are both weakened by the same substitution in ⁷⁰.

The algQ (algR2) gene was originally identified as a regulatory gene implicated in activation of the algD promoter in experimentally derived mucoid strains of P. aeruginosa (8, 32). In particular, a putative algQ mutation was found to eliminate transcription from the algD promoter in a laboratory strain of P. aeruginosa (8). Furthermore, overexpression of algQ was shown to reverse the nonmucoid phenotype of spontaneous Alg mutants derived from mucoid cystic fibrosis isolates of P. aeruginosa (32). Interestingly, expression of algQ was also shown to mediate strong activation of the algD promoter in E. coli cells grown under high-osmolarity conditions (32). Activation of the algD promoter has been shown to occur by at least two different mechanisms involving one of two alternative sigma factors, ^E and ⁵⁴ (3, 9, 11, 21, 38, 44, 49, 59). In particular, mutations that activate ^E (encoded by the algU gene) lead to increased expression of algD (reviewed in reference 9), but algD expression can also be activated by a ⁵⁴-dependent pathway (3). Our results support the idea that the effect of AlgQ on algD expression is likely to be indirect since there is no evidence that the ⁷⁰ form of RNAP can recognize the algD promoter. Possibly AlgQ functions as an anti- factor, increasing the amount of RNAP core that is available to bind the relevant alternative  factor (either ^E or ⁵⁴), thereby increasing the occupancy of the algD promoter. It is interesting that algD gene expression is negatively controlled by an anti- factor (the product of the mucA gene), which is specific for ^E (44, 48, 58). Thus, most mucoid cystic fibrosis isolates of P. aeruginosa have been found to bear mutations in the mucA gene, which result in constitutive alginate production (2).

Our finding that AlgQ can bind to ⁷⁰ from E. coli as well as to ⁷⁰ from P. aeruginosa could be relevant to its ability to activate the algD promoter in E. coli (32). Nevertheless, it is possible that the role of AlgQ in activation of the algD promoter is unrelated to its ability to bind to ⁷⁰ in either P. aeruginosa or E. coli. Although AlgQ was originally reported to have a kinase activity (45), the subsequent finding that it regulates production of a kinase (nucleoside diphosphate kinase) which has a similar molecular weight suggests that AlgQ is not itself a kinase (47).

The regulatory effects of AlgQ are not limited to alginate production. For example, AlgQ regulates production of a variety of secretable virulence factors, up-regulating a neuraminidase and a siderophore and down-regulating extracellular proteases and a rhamnolipid biosurfactant (6, 46, 54). AlgQ also regulates production of Ndk (see above) and succinyl coenzyme A synthetase, an enzyme of the tricarboxylic acid cycle that forms a complex with Ndk in P. aeruginosa (33, 46, 47). Finally, characterization of an algQ null mutant revealed a dramatic loss of viability in the stationary phase of growth, as well as reductions in the intracellular concentrations of GTP, ppGpp, and inorganic polyphosphate (34). Although the molecular basis for these regulatory effects has not been defined, the pleiotropic nature of AlgQ-dependent phenotypes and our results support the suggestion that AlgQ is a global regulator of transcription in P. aeruginosa. In addition to its similarity to Rsd, AlgQ exhibits 58% identity with PfrA, a positive regulator of siderophore biosynthetic genes in Pseudomonas putida (54). Interestingly, both PfrA and a putative member of the extracytoplasmic function family of alternative  factors (PfrI) are required for transcriptional activation of siderophore biosynthetic genes under iron limitation conditions in P. putida (54, 55).

Two-hybrid assay for the interaction of transcriptional regulators with region 4 of ⁷⁰. The two-hybrid system that we used to study the interactions of Rsd and AlgQ with region 4 of ⁷⁰ should facilitate studies of other regulators that interact with this region of ⁷⁰ from E. coli, P. aeruginosa, or other bacteria. Use of the -⁷⁰ chimeras, in particular, could facilitate genetic analysis of these interactions by providing a convenient vehicle for mutagenesis of region 4 of ⁷⁰. Whereas isolation and analysis of rpoD mutations are complicated by the fact that ⁷⁰ is an essential protein that exerts global effects on cellular transcription, our - chimeras exert their effects at specifically designed test promoters. Moreover, mutant chimeras can be assayed to determine their abilities to interact with a number of different regulators so that the specificities of their effects can be assessed.

	ACKNOWLEDGMENTS

We thank Arne Rietsch for providing the P. aeruginosa PAO1 chromosomal DNA and Debbie Hinton for the gift of plasmid pEG-AsiA as a source of the asiA gene. We also thank Debbie Hinton for communicating results prior to publication and Cliff Boucher for helpful discussions.

This work was supported by National Institutes of Health grant GM44025, by an established investigatorship from the American Heart Association (to A.H.), and by a Charles A. King Trust postdoctoral fellowship (to S.L.D.).

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 432-1986. Fax: (617) 738-7664. E-mail: ahochschild{at}hms.harvard.edu.

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Journal of Bacteriology, November 2001, p. 6413-6421, Vol. 183, No. 21
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.21.6413-6421.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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