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PubMed1997

Function of the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase in basal expression and integration host factor-mediated activation of the early promoter of bacteriophage Mu.

Integration host factor (IHF) can activate transcription from the early promoter (Pe) of bacteriophage Mu both directly and indirectly. Indirect activation occurs through alleviation of H-NS-mediated repression of the Pe promoter (P. Van Ulsen, M. Hillebrand, L. Zulianello, P. Van de Putte, and N. Goosen, Mol. Microbiol. 21:567-578, 1996). The direct activation involves the C-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase. We investigated which residues in the alphaCTD are important for IHF-mediated activation of the Pe promoter. Initial in vivo screening, using a set of substitution mutants derived from an alanine scan (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996; H. Tang, K. Severinov, A. Goldfarb, D. Fenyo, B. Chait, and R. H. Ebright, Genes Dev. 8:3058-3067, 1994), indicated that the residues, which are required for transcription activation by the UP element of the rrnB P1 promoter (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996), are also important for Pe expression in the presence of IHF. Two of the RNA polymerase mutants, alphaR265A and alphaG296A, that affected Pe expression most in vivo were subsequently tested in in vitro transcription experiments. Mutant RNA polymerase with alphaR265A showed no IHF-mediated activation and a severely reduced basal level of transcription from the Pe promoter. Mutant RNA polymerase with alphaG296A resulted in a slightly reduced transcription from the Pe promoter in the absence of IHF but could still be activated by IHF. These results indicate that interaction of the alphaCTD with DNA is involved not only in the IHF-mediated activation of Pe transcription but also in maintaining the basal level of transcription from this promoter. Mutational analysis of the upstream region of the Pe promoter identified a sequence, positioned from -39 to -51 with respect to the transcription start site, that is important for basal Pe expression, presumably through binding of the alphaCTD. The role of the alphaCTD in IHF-mediated stimulation of transcription from the Pe promoter is discussed.

Bacterial Proteins↗

PubMed1997

Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein nifA.

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupSp). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential -173 to -88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule.

Bacterial Proteins↗

PubMed1986

Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications.

The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.

Bacterial Proteins↗

PubMed1996

Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.

Attachment Sites, Microbiological↗

PubMed1993

Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides. Involvement of an integration host factor-binding sequence.

The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF. The binding of E. coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific. The recorded binding affinity was significantly lower than that of E. coli IHF to the lambda attP site. Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions. These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity. Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed. Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression.

Bacterial Proteins↗

PubMed1991

A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression.

A gene capable of encoding a protein sharing 45% identical amino acids with the alpha subunit of the integration host factor (IHF) of Escherichia coli was isolated from the photosynthetic bacterium Rhodobacter capsulatus strain B10 by complementation of a hydrogenase-deficient (Hup-) mutant, IR4. A DNA fragment of 274 base pairs containing an IHF binding consensus sequence, isolated from the promoter region of the hydrogenase structural genes (hupSL), was shown by gel retardation assays to bind the IHF protein from E. coli. The product of the R. capsulatus gene was shown to bind specifically to the 274-base-pair DNA fragment from the hupSL promoter. By analogy to the E. coli himA gene, which encodes the alpha subunit of IHF, the gene complementing the IR4 mutant was named himA of R. capsulatus. The wild-type himA gene, cloned in plasmid pBO2, was introduced into the IR4 strain and shown to restore, in trans, hydrogenase activity and autotrophic growth in the mutant. In IR4, a C----T transition mutation had replaced Arg-8 by Cys-8. Gel mobility shifts of the 274-base-pair DNA fragment, not observed with the himA gene product of IR4, were restored with extracts from IR4(pBO2) cells, containing the himA gene on the recombinant plasmid pBO2.

Amino Acid Sequence↗

PubMed2002

Direct atomic force microscopy visualization of integration host factor-induced DNA bending structure of the promoter regulatory region on the Pseudomonas TOL plasmid.

Atomic force microscopy (AFM) was used to analyze DNA bending induced by integration host factor (IHF). The direct AFM visualization of IHF-DNA complexes on the OP1 promoter regulatory regions on the Pseudomonas TOL plasmid showed that there was no intrinsic DNA bend in the OP1 promoter region, but a sharp DNA bend was induced by binding of IHF to the region between the upstream regulatory sequence and the promoter sequence. The DNA bending angles were distributed with a mean bend angle of 123 degrees. The IHF-DNA complexes were shown to bend at the IHF binding site giving rise to an asymmetric structure. These results provide direct evidence that IHF is required functionally for activation of OP1 transcription and support the DNA-loop model that the sharp DNA bend induced by binding of IHF facilitates the contact between RNA polymerase bound by the promoter sequence and XylR protein attached to the upstream sequence in the OP1 promoter.

Bacterial Proteins↗

PubMed1997

The integration host factor-DNA complex upstream of the early promoter of bacteriophage Mu is functionally symmetric.

Inversion of the ihf site in the promoter region of the early promoter of bacteriophage Mu did not influence the integration host factor (IHF)-mediated functions. IHF bound to this inverted site could counteract H-NS-mediated repression, directly activate transcription, and support lytic growth of bacteriophage Mu. This implies that the IHF heterodimer and its asymmetrical binding site form a functionally symmetrical complex.

Bacterial Proteins↗

PubMed2000

Mobilization of chimeric oriT plasmids by F and R100-1: role of relaxosome formation in defining plasmid specificity.

Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

Bacterial Proteins↗

PubMed1991

Integration host factor-mediated expression of the ilvGMEDA operon of Escherichia coli.

The structural genes of the ilvGMEDA operon of Escherichia coli are preceded by two promoters, ilvPG1 and ilvPG2, and a leader-attenuator region. Alkylation protection and hydroxyl radical footprinting techniques have been used to demonstrate that integration host factor (IHF) interacts with the nucleotides in a consensus-like DNA sequence located immediately downstream of the RNA polymerase transcriptional pause site in the leader-attenuator region. In the presence of purified IHF protein, in vitro transcriptional pausing of RNA polymerase at the leader-attenuator pause site is increased 2-fold and, concomitantly, a 2-fold increase in transcriptional termination at the attenuator is observed. Strains containing chromosomal transcriptional fusions of various segments of the ilvGMEDA promoter-attenuator region to the galK gene were used to show that IHF also decreases the in vivo basal level of transcriptional readthrough at the attenuator 2-fold. The binding of IHF to another target site in the ilvPG1 promoter region represses transcription from this promoter and causes a 4-fold stimulation of transcription initiation from the downstream ilvPG2 promoter 4-fold. This IHF-mediated control of transcription initiation from the upstream promoter region is independent of the regulation of transcription termination effected by IHF interaction at the attenuator site. Thus, IHF is capable of regulating the expression of the ilvGMEDA operon in opposing manners; it can activate transcription initiation of this operon from the ilvPG2 promoter 4-fold and increase the termination of this transcription at the downstream attenuator 2-fold.

Amino Acids, Branched-Chain↗

PubMed1998

An AT-rich tract containing an integration host factor-binding domain and two UP-like elements enhances transcription from the pilEp1 promoter of Neisseria gonorrhoeae.

The pilE gene of Neisseria gonorrhoeae is transcribed from a sigma70 promoter (pilEp1) with an AT-rich tract extending 65 nucleotides upstream of the -35 box. Within this region is an integration host factor (IHF)-binding core consensus sequence. We have performed a detailed analysis to determine which upstream sequences are required for efficient transcription from pilEp1 in N. gonorrhoeae. Deletion of sequences upstream of the AT-rich tract had no effect on the level of transcription. However, the IHF-binding core consensus sequence and the AT-rich sequence further upstream were both required for enhanced levels of transcription from this promoter in both N. gonorrhoeae and an Escherichia coli strain producing IHF. In addition, an UP-like element positioned between the -35 box and the IHF-binding site was required for maximal transcription. The AT-rich region upstream of the IHF-binding core consensus sequence can also act as an UP-like element when appropriately repositioned upstream of the -35 box.

Adenine↗

PubMed1984

Synergistic effect of himA and gyrB mutations: evidence that him functions control expression of ilv and xyl genes.

We have constructed Escherichia coli strains containing mutations at two different loci, both originally selected for failure to support lambda site-specific recombination: himA and gyrB-him(Ts). Although the gyrB-him(Ts) mutations by themselves reduce supercoiling at high temperature, the double mutants show a far greater effect on supercoiling. Our studies show that growth of phage lambda is severely inhibited and that maintenance of plasmid pBR322 is extremely unstable in the double mutants. Physiological studies also reveal that the double mutants are isoleucine auxotrophs at 42 degrees C. The fact that himA mutants are isoleucine auxotrophs at 42 degrees C in the presence of leucine suggests that a significant component of the isoleucine auxotrophy of the double mutants is a result of the himA mutation. The himA gene encodes the alpha subunit of a protein called the integration host factor. Since mutations in the hip or himD gene encoding beta, the other subunit of the integration host factor, also result in isoleucine auxotrophy in the presence of leucine, we suggest that the integration host factor regulates the synthesis of at least one of the enzymes in the ilv pathway, acetohydroxyacid synthase I, which is encoded by the ilvB gene. Studies of the utilization of various sugars as the sole carbon source suggest that the integration host factor controls expression of some gene(s) involved in the utilization of xylose.

Bacteriophage lambda↗

PubMed2003

Recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element.

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.

Base Sequence↗

PubMed1989

In vitro activity of the nitrogen fixation regulatory protein NIFA.

We have detected activity of the nitrogen fixation regulatory protein NIFA of Klebsiella pneumoniae in vitro. To do so we directed synthesis of NIFA in a coupled transcription-translation system and detected its ability to activate expression of a translational fusion between the nifH and lacZ genes. We infer that NIFA stimulates initiation of transcription by sigma 54 holoenzyme from the nifHDK promoter. The activity of NIFA was lost rapidly under both aerobic and anaerobic conditions at 30 degrees C and was lost somewhat less rapidly at 0 degrees C. Loss of activity was not accompanied by degradation of NIFA polypeptide. Loss of activity was approximately exponential and was not affected by NIFA concentration over a 5-fold range. Therefore, NIFA inactivation does not appear to be due to self-association. We found that the factor in crude extracts previously demonstrated to bind to the nifHDK promoter-regulatory region [Beynon, J., Cannon, M., Buchanan-Wollaston, V., and Cannon, F. (1983) Cell 34, 665-671] is the integration host factor, which is known to bend DNA. Since the binding site for integration host factor lies between the upstream binding site for NIFA and the nifHDK promoter, integration host factor may bend the DNA between these two sites to facilitate productive interactions between NIFA and sigma 54 holoenzyme.

Bacterial Proteins↗

PubMed1996

Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.

The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an E. coli sigma 70 promoter, with no similarities to binding sites for other sigma factors. Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site. In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter. The kpsF gene was 98.8% identical with the kpsF gene identified in the E. coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters. An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene. No essential role for KpsF in the expression of the K5 antigen could be established. The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C. Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression. Likewise, RfaH, which is known to regulate expression of E. coli group II capsules had no effect on the expression of region 1. Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.

Bacterial Capsules↗

PubMed1997

Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription.

Pseudomonas aeruginosa strains infecting patients with cystic fibrosis (CF) acquire a mucoid phenotype due to overproduction of alginate. The key enzyme in alginate synthesis is AlgD, whose promoter is transcriptionally active in mucoid strains and under the control of several trans-acting factors, including the integration host factor (IHF). The algD promoter (palgD) contains two IHF-binding sites (ihf1 and ihf2). Study of IHF binding to ihf2 of palgD, by electrophoretic mobility-shift assays, led to the discovery of a protein of 36 kDa (p36) able to bind downstream from ihf2, to the 3' region of palgD. The gene encoding p36 was isolated from the mucoid strain CHA of P. aeruginosa and sequenced. It can encode a 324-amino-acid protein, which shares a high degree of sequence identity (63%) with CysB from Escherichia coli and from Salmonella typhimurium, a transcriptional factor of the LysR superfamily. Furthermore, both p36 and S. typhimurium CysB bind the same site of palgD; p36 was therefore termed CysB and its structural gene was called cysB. Next to cysB, on the opposite DNA strand, cysH was capable of encoding a protein sharing 26% identity with CysH (PAPS reductase) of E. coli and an even greater identity (54%) with the nucleotide-deduced protein from Arabidopsis. A CysB-deficient mutant of CHA, constructed by insertional inactivation of cysB, was a cysteine auxotroph and was unable to form a specific complex with palgD in vitro. Activity of palgD in the cysB mutant, in CHA and in the non-mucoid strain PAO was assessed by the use of a transcriptional algD-xylE fusion. Cells of PAO and of the cysB mutant grown in minimal media in the presence of 0.3 M NaCl exhibited a palgD activity, which was 10% or less that of the mucoid strain CHA. Thus, P. aeruginosa CysB can act as an activator of algD expression.

Bacterial Proteins↗

PubMed1994

Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12.

The promoter-operator region of the aroL gene of Escherichia coli K-12 contains three TYR R boxes and one TrpR binding site. Mutational analysis showed that TYR R boxes 1 and 3 are essential for TyrR-mediated regulation of aroL expression, while a fully functional TYR R box 2 does not appear to be essential for regulation. Regulation mediated by the TrpR protein required the TYR R boxes and TrpR site to be functional and was observed in vivo only with a tyrR+ strain. Under conditions favoring the formation of TyrR hexamers, DNase I protection experiments revealed the presence of phased hypersensitive sites, indicative of DNA backbone strain. This suggests that TyrR-mediated repression involves DNA looping. Purified TrpR protein protected the putative TrpR binding site in the presence of tryptophan, and this protection was slightly enhanced in the presence of TyrR protein. This result along with the in vivo findings implies that TyrR and TrpR are able to interact in some way. Inserting 4 bp between TYR R box 1 and the TrpR binding site results in increased tyrosine repression and the abolition of the tryptophan effect. Identification of a potential integration host factor binding site and repression studies of a himA mutant support the notion that integration host factor binding normally exerts a negative effect on tyrosine-mediated repression.

Bacterial Proteins↗

PubMed1989

Nucleotide sequence and expression of the gene for the site-specific integration protein from bacteriophage HP1 of Haemophilus influenzae.

The nucleotide sequence of the leftmost 2,363 base pairs of the HP1 genome, which includes the attachment site (attP) and the integration region, was determined. This sequence contained an open reading frame encoding a 337-residue polypeptide, which is a member of the integrase family of site-specific recombination proteins as judged by sequence comparison. The open reading frame was located immediately adjacent to the att site and was oriented so that initiation of translation would begin distal to the att site and end in its immediate vicinity. Expression of this DNA segment in Escherichia coli provided extracts which promoted site-specific recombination between plasmids containing cloned HP1 attP and Haemophilus influenzae attB sites. This recombination was directional, since no reaction was observed between plasmids containing attR and attL sites. The reaction was stimulated by the accessory protein integration host factor of E. coli. Evidence was also obtained that the integration host factor influenced the levels of HP1 integrase expression. The deduced amino acid sequence of HP1 integrase has remarkable similarity to that deduced for the integrase of coliphage 186.

Amino Acid Sequence↗