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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 sigma 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 sigma 70 subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of sigma 70 that harbors conserved region 4. This region of sigma  contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma 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-sigma factor Rsd and the sigma 70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of sigma 70 and also that amino acid substitution R596H, within region 4 of sigma 70, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between sigma  and two other regulators shown previously to contact region 4 of sigma 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 sigma 70 subunit of RNAP from this organism. We found that amino acid substitution R600H in sigma 70 from P. aeruginosa, corresponding to the R596H substitution in E. coli sigma 70, specifically weakens the interaction between AlgQ and sigma 70. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of sigma 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 sigma  factors, and sigma 70 is the principal sigma  factor (27). The presence of different types of sigma  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 sigma  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-sigma factors, which are regulatory proteins that bind sigma  factors and often prevent their association with the RNAP core enzyme (19, 26). Anti-sigma factors ultimately inhibit transcription from the class of promoters recognized by the sigma  factors that they sequester.

The sigma 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 sigma 70 often contact a region of sigma  that contains a putative helix-turn-helix DNA-binding motif responsible for contacting the -35 elements of sigma 70-dependent promoters (18, 23, 37). This DNA-binding region of sigma  is conserved in members of the sigma 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 sigma 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 sigma 70 (but not with several alternative sigma  factors) and was shown to be capable of inhibiting sigma 70-dependent transcription from certain promoters in vitro (28). The binding site for Rsd on sigma 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 sigma 70-dependent transcription that occurs as cells enter the stationary phase of growth, Jishage and Ishihama suggested that Rsd might be an anti-sigma factor (28). Subsequent work has shown that consistent with this idea, Rsd may facilitate the replacement of sigma 70 by the stationary-phase-specific sigma  factor sigma 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-sigma 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 sigma  factors; transcription initiates from two superimposed promoters, one of which is recognized by RNAP containing sigma E (AlgU/AlgT) and the other of which is recognized by RNAP containing sigma 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 sigma 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 sigma 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 sigma  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 placOR2-62 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 OR2-55/Cons -35 driving expression of a linked lacZ reporter gene and has also been described previously (13).

Plasmids pAClambda cI, pACDelta cI, pBRalpha , pBRalpha -sigma 38, pBRalpha -sigma 70, and pBRalpha -sigma 70 (R596H) have all been described previously (13, 14). Plasmid pAClambda cI-Rsd encodes lambda cI (residues 1 to 236) fused to Rsd (residues 1 to 158) via three alanine residues. pAClambda cI-Rsd was made by cloning the appropriate NotI-BamHI-digested PCR product into NotI-BstYI-digested pAClambda cI32 (24); expression of the cI-rsd fusion gene was therefore under the control of the lacUV5 promoter. Plasmid pAClambda cI-AlgQ encodes lambda 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 pAClambda cI-Rsd was made. Expression of the cI-algQ fusion gene on pAClambda cI-AlgQ is also under the control of the lacUV5 promoter. Plasmid pAClambda cI-AsiA encodes lambda 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 pAClambda cI-Rsd was made. Plasmid pACDelta -35lambda cI-AsiA contains the cI-asiA fusion gene from plasmid pAClambda cI-AsiA under the control of a lacUV5 promoter variant in which the -35 element of the promoter has been deleted. Plasmid pACDelta -35lambda cI-AsiA, therefore, expresses less of the lambda cI-AsiA fusion protein than plasmid pAClambda cI-AsiA expresses under identical conditions. pACDelta -35lambda cI-AsiA was made by cloning the appropriate HindIII-BstYI fragment from pAClambda cI-AsiA into plasmid pA3B2 (57) cut with both HindIII and BstYI. Plasmid pBRalpha -sigma 70PA encodes residues 1 to 248 of the alpha  subunit of E. coli RNAP fused to residues 532 to 617 of the sigma 70 subunit of P. aeruginosa RNAP. The hybrid alpha -sigma 70PA gene was made by performing PCR and was cloned into HindIII-BamHI-digested pBRalpha . Expression of the chimeric gene on pBRalpha -sigma 70PA is, therefore, under the control of tandem lpp and lacUV5 promoters. pBRalpha -sigma 70PA (R600H) is a derivative of pBRalpha -sigma 70PA in which the R600H substitution in the sigma  moiety of the chimera was introduced by PCR.

The lac promoter derivative placCOP-93+OL2-62 was made by the PCR and contains two lambda  operators; a near-consensus lambda  operator (TACCACCGGCGGTGATA) and OL2 (CAACACCGCCAGAGATA) are centered 93 and 62 bp, respectively, upstream of the transcriptional start site of the lac core promoter. Plasmid pFW11-COP-93+OL2-62 was constructed by cloning an EcoRI-HindIII-cut PCR product containing placCOP-93+OL2-62 into pFW11 (56) cut with EcoRI and HindIII. Plasmid pFW11-COP-93+OL2-62 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-beta -D-thiogalactoside (IPTG) at the concentration indicated. Cells were permeabilized with sodium dodecyl sulfate-CHCl3 and assayed for beta -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

lambda cI-Rsd activates transcription from a test promoter in the presence of a chimeric alpha -subunit harboring region 4 of E. coli sigma 70. We sought to detect an interaction between Rsd and region 4 of sigma 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 sigma 70 involved the use of two chimeric proteins, one comprising Rsd fused to the repressor of bacteriophage lambda  (lambda cI) and the other comprising a modified form of the alpha -subunit of RNAP in which the C-terminal domain (CTD) of alpha  has been replaced by a C-terminal fragment of sigma 70. We reasoned that RNAP containing the resulting alpha -sigma 70 chimera would display a target for Rsd that could be contacted by a DNA-bound lambda cI-Rsd dimer (Fig. 1A). Having fused the entire Rsd protein (residues 1 to 158) to the C terminus of lambda cI, we placed the gene encoding this chimeric protein on a plasmid vector downstream of the IPTG-inducible lacUV5 promoter, thus creating plasmid pAClambda cI-Rsd. We used plasmid pBRalpha -sigma 70 as a source of the alpha -sigma 70 chimera. This plasmid encodes a chimera in which residues 528 to 613 of E. coli sigma 70 are fused to residues 1 to 248 of the alpha -subunit of RNAP (13). We introduced plasmids pAClambda cI-Rsd and pBRalpha -sigma 70 into E. coli KS1 (14), which harbors on its chromosome the lac promoter derivative placOR2-62 (bearing a single lambda  operator centered 62 bp upstream of the transcriptional start site) linked to a lacZ reporter gene. We then tested the ability of the lambda cI-Rsd chimera to activate transcription from the placOR2-62 test promoter in the presence of the alpha -sigma 70 chimera. Figure 1B shows that lambda cI-Rsd activated transcription from the test promoter up to ~24-fold in cells containing the alpha -sigma 70 chimera compared to control cells containing only wild-type alpha . An additional control revealed that lambda cI (lacking the fused Rsd moiety) did not activate transcription from the test promoter in the presence of the alpha -sigma 70 chimera (Fig. 1B). We also found that lambda cI-Rsd did not activate transcription from the test promoter in the presence of an alpha -sigma 38 chimera (comprising residues 1 to 248 of alpha  fused to residues 243 to 330 of sigma 38) encoded by plasmid pBRalpha -sigma 38 (13; data not shown).


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  		FIG. 1.   Transcriptional activation by lambda cI-Rsd in the presence of the alpha -sigma 70 chimera. (A) Replacement of the RNAP alpha -CTD by a C-terminal fragment of E. coli sigma 70 (residues 528 to 613) permits interaction with the Rsd moiety of a lambda cI-Rsd chimera bound to DNA. The diagram depicts the test promoter placOR2-62, which bears the lambda  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 alpha  is designated alpha NTD. (B) Effect of lambda cI-Rsd on transcription in vivo from placOR2-62 in the presence of the alpha -sigma 70 or alpha -sigma 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 beta -galactosidase activity.

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

Substitution R596H in the sigma  moiety of the alpha -sigma 70 chimera does not compromise the ability of a superactivating variant of lambda cI to stimulate transcription from an appropriate test promoter. To determine whether the R596H substitution affects the interaction of sigma 70 with Rsd specifically, we tested its effect on the ability of another protein to interact with the sigma  moiety of the chimera. We showed recently that the lambda cI protein (a transcriptional activator as well as a repressor) can interact specifically with the sigma  moiety of the alpha -sigma 70 chimera and stabilize its binding to a promoter -35 element (13). The in vivo assay which we designed to detect the interaction between lambda cI and region 4 of sigma 70 is shown in Fig. 2A. In this experimental setup a DNA-bound lambda cI dimer activates transcription from test promoter placOR2-55/Cons-35 by stabilizing the binding of the sigma  moiety of the alpha -sigma 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 lambda cI and the tethered sigma  moiety but also on the protein-DNA interaction between the tethered sigma  moiety and the ectopic -35 element (13).


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  		FIG. 2.   Transcriptional activation by lambda cI superactivator in the presence of the alpha -sigma 70 chimera. (A) lambda cISa109 stabilizes the binding of region 4 of sigma 70 to an ectopic -35 element. The diagram depicts the test promoter placOR2-55/Cons-35, which bears an ectopic -35 element and the lambda  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 sigma  moiety of the alpha -sigma 70 chimera on the ability of lambda 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 beta -galactosidase activity.

We tested whether the R596H substitution in the alpha -sigma 70 chimera has an effect on the ability of a superactiviating variant of lambda cI to activate transcription from test promoter placOR2-55/Cons-35 (Fig. 2A). Reporter strain SF1 carries promoter placOR2-55/Cons-35 fused to the lacZ gene in single copy on an F' episome (13). We assayed the ability of lambda cI superactivator 109 (lambda cISa109) (5, 13) to activate transcription from this reporter in the presence of the alpha -sigma 70 chimera with or without the R596H substitution in the sigma  moiety. lambda cISa109 activated transcription a maximum of ~5.5-fold from the test promoter in the presence of the alpha -sigma 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 lambda cI to activate transcription from PRM [35], we have observed that this substitution does not inhibit the ability of lambda cISa109 to activate transcription from PRM [see below].) We concluded that substitution R596H in the sigma  moiety of the alpha -sigma 70 chimera does not result in a general defect in the ability of the sigma  moiety to interact with other proteins.

We also tested the effect of the R596A substitution in the sigma  moiety of the alpha -sigma 70 chimera on the ability of lambda cISa109 to activate transcription from the same test promoter. Unlike the R596H substitution, the R596A substitution significantly reduced the magnitude of the activation by lambda 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).

lambda cI-AlgQ activates transcription from a test promoter in the presence of a chimeric alpha -subunit harboring region 4 of P. aeruginosa sigma 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 sigma 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 alpha -sigma 70 chimera that contained region 4 of sigma 70 from P. aeruginosa. We fused the entire AlgQ protein (residues 1 to 160) to the C terminus of lambda cI. We placed the gene encoding this chimeric protein on a plasmid vector downstream of the IPTG-inducible lacUV5 promoter, creating plasmid pAClambda cI-AlgQ. We then fused residues 532 to 617 of P. aeruginosa sigma 70 (equivalent to residues 528 to 613 of E. coli sigma 70) to residues 1 to 248 of alpha . The resulting chimera was called alpha -sigma 70PA. The hybrid alpha -sigma 70PA gene encoding this chimera was placed on a plasmid vector downstream of tandemly arranged lpp and lacUV5 promoters, creating plasmid pBRalpha -sigma 70PA. We introduced plasmids pAClambda cI-AlgQ and pBRalpha -sigma 70PA into E. coli KS1. We then tested the ability of the lambda cI-AlgQ chimera to activate transcription from placOR2-62 in the presence of the alpha -sigma 70PA chimera (Fig. 3A). Figure 3B shows that lambda cI-AlgQ activated transcription from the test promoter a maximum of ~17-fold in cells containing the alpha -sigma 70PA chimera compared to control cells containing only wild-type alpha . An additional control revealed that lambda cI without the fused AlgQ moiety did not activate transcription from the test promoter in the presence of the alpha -sigma 70PA chimera (Fig. 3B).


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  		FIG. 3.   Transcriptional activation by lambda cI-AlgQ in the presence of the alpha -sigma 70PA chimera. (A) Replacement of the RNAP alpha -CTD by a C-terminal fragment of P. aeruginosa sigma 70 (residues 532 to 617) permits interaction with the AlgQ moiety of a lambda cI-AlgQ chimera bound to DNA. The diagram depicts the test promoter placOR2-62, which bears the lambda  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 lambda cI-AlgQ on transcription in vivo from placOR2-62 in the presence of the alpha -sigma 70PA or alpha -sigma 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 beta -galactosidase activity.

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

Substitution R600H in the sigma  moiety of the alpha -sigma 70PA chimera does not compromise the ability of a superactivating variant of lambda 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 sigma 70PA and AlgQ, we first tested whether lambda cISa109 could interact with the sigma  moiety of the alpha -sigma 70PA chimera. We found that lambda cISa109 stimulated transcription from test promoter placOR2-55/Cons-35 up to ~3.5-fold specifically in the presence of the alpha -sigma 70PA chimera (Fig. 4). Furthermore, introduction of the R600H substitution into the sigma  moiety of the alpha -sigma 70PA chimera did not abrogate the stimulatory effect of lambda cISa109 (instead it resulted in a modest increase in the observed activation), suggesting that the effect of the R600H substitution is specific for the lambda cI-AlgQ chimera (Fig. 4B).


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  		FIG. 4.   Transcriptional activation by lambda cI superactivator in the presence of the alpha -sigma 70PA chimera. (A) lambda cISa109 stabilizes the binding of region 4 of sigma 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 lambda  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 sigma  moiety of the alpha -sigma 70PA chimera on the ability of lambda 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 beta -galactosidase activity.

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


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  		FIG. 5.   Rsd can interact with region 4 of sigma 70 from P. aeruginosa, and AlgQ can interact with region 4 of sigma 70 from E. coli. (A) Effect of lambda cI-Rsd on transcription in vivo from placOR2-62 in the presence of the alpha -sigma 70PA, alpha -sigma 70PA(R600H), or alpha -sigma 70 chimera. KS1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for beta -galactosidase activity. (B) Effect of lambda cI-AlgQ on transcription in vivo from placOR2-62 in the presence of the alpha -sigma 70, alpha -sigma 70(R596H), or alpha -sigma 70PA chimera. KS1 cells harboring compatible plasmids expressing the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for beta -galactosidase activity.

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

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

We therefore sought to detect the interaction between AsiA and region 4 of sigma 70 by fusing AsiA to lambda cI and measuring the ability of the resulting lambda cI-AsiA chimera to activate transcription from placOR2-62 in the presence of either the alpha -sigma 70 chimera or the alpha -sigma 70PA chimera. We fused the entire AsiA protein (residues 1 to 90) to the C terminus of lambda cI. We placed the gene encoding this chimeric protein on a plasmid vector downstream of the IPTG-inducible lacUV5 promoter, creating plasmid pAClambda cI-AsiA. Initial experiments demonstrated that the lambda cI-AsiA chimera was extremely toxic to cells at the levels provided by the expression vector pAClambda cI-AsiA (data not shown). For subsequent experiments we constructed a different expression vector, pACDelta -35lambda cI-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 pACDelta -35lambda cI-AsiA made insufficient levels of lambda cI-AsiA to saturate the lambda  operator present on the placOR2-62 promoter construct, we also constructed a new test promoter bearing two relatively strong lambda  operators in the upstream region (Fig. 6A; also see Materials and Methods). Figure 6B shows that the lambda cI-AsiA chimera activated transcription from the modified test promoter in the presence of the alpha -sigma 70 chimera derived from sigma 70 of either E. coli or P. aeruginosa (similar findings with the alpha -sigma 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 sigma  moiety or the corresponding R600H substitution in the P. aeruginosa sigma  moiety had only a modest effect on the ability of the lambda cI-AsiA chimera to activate transcription from placCOP-93+OL2-62. Similarly, the R596A substitution in the E. coli sigma  moiety did not reduce the stimulatory effect of the lambda cI-AsiA chimera (data not shown). Since these substitutions do not appear to cause nonspecific defects in the abilities of the sigma  moieties to interact with other proteins, we suggest that residue R596 of E. coli sigma 70 is at or near the contact surface for Rsd and the equivalent residue of P. aeruginosa sigma 70, R600, is at or near the contact surface for AlgQ (see Discussion). Furthermore, the finding that substitution R596A in E. coli sigma 70 results in a strong defect in the ability of sigma  to interact with Rsd suggests that the arginine side chain may make an energetically significant contact with Rsd.


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  		FIG. 6.   Transcriptional activation by lambda cI-AsiA in the presence of the alpha -sigma chimeras. (A) Schematic diagram of test promoter placCOP-93+OL2-62 used to determine the effects of substitutions in the alpha -sigma chimeras on transcriptional activation by lambda cI-AsiA. The test promoter placCOP-93+OL2-62 bears both a consensus lambda  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 lambda cI-AsiA on transcription in vivo from placCOP-93+OL2-62 in the presence of the alpha -sigma 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 beta -galactosidase activity. Delta cI refers to the fact that no lambda cI was made by control plasmid pACDelta cI.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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

Analysis of the interaction between Rsd and region 4 of sigma 70. Our bacterial two-hybrid results for Rsd and region 4 of sigma 70 are consistent with the biochemical data of Jishage and Ishihama (28). These authors found that Rsd could interact with sigma 70 in vitro but not with sigma 38 (or several other alternative sigma  factors), and they showed that Rsd could interact with a tryptic fragment of sigma 70 composed of residues ~500 to 613. We found that Rsd can interact in vivo with an 86-amino-acid C-terminal fragment of sigma 70 that contains region 4 but cannot interact with the equivalent 88-amino-acid C-terminal fragment of sigma 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 sigma 70 in vitro. However, in contrast with our results, they reported that substitution R596A in sigma 70 did not prevent association of Rsd with sigma 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 sigma  moiety of the alpha -sigma 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 sigma 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 lambda cISa109 to activate transcription from our artificial test promoter in the presence of the alpha -sigma 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 sigma 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 lambda cI positive control mutation at the lambda  promoter PRM. Suppressor mutations in the rpoD gene were sought that would reverse the activation defect of a lambda 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 sigma 70 reduces the ability of wild-type lambda cI to activate transcription from PRM, we have shown that this substitution actually enhances the ability of lambda cISa109 (which bears a non-wild-type residue at position 38) to stimulate transcription from PRM (unpublished data). Evidently, certain amino acid-amino acid combinations at position 38 of lambda cI and position 596 of sigma 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 sigma 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 sigma 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 sigma 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 sigma 70 are both weakened by the same substitution in sigma 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, sigma E and sigma 54 (3, 9, 11, 21, 38, 44, 49, 59). In particular, mutations that activate sigma 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 sigma 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 sigma 70 form of RNAP can recognize the algD promoter. Possibly AlgQ functions as an anti-sigma factor, increasing the amount of RNAP core that is available to bind the relevant alternative sigma  factor (either sigma E or sigma 54), thereby increasing the occupancy of the algD promoter. It is interesting that algD gene expression is negatively controlled by an anti-sigma factor (the product of the mucA gene), which is specific for sigma 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 sigma 70 from E. coli as well as to sigma 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 sigma 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 sigma  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 sigma 70. The two-hybrid system that we used to study the interactions of Rsd and AlgQ with region 4 of sigma 70 should facilitate studies of other regulators that interact with this region of sigma 70 from E. coli, P. aeruginosa, or other bacteria. Use of the alpha -sigma 70 chimeras, in particular, could facilitate genetic analysis of these interactions by providing a convenient vehicle for mutagenesis of region 4 of sigma 70. Whereas isolation and analysis of rpoD mutations are complicated by the fact that sigma 70 is an essential protein that exerts global effects on cellular transcription, our alpha -sigma 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.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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