Integration Host Factor Binding Research Articles

Integration host factor and cyclic AMP receptor protein are required for TyrR-mediated activation of tpl in Citrobacter freundii.

The tpl gene of Citrobacter freundii encodes an enzyme that catalyzes the conversion of L-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates -272.5, -158.5, and -49.5, respectively, were identified in the upstream region of the tpl promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of tpl required integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate -85 and that there are CRP binding sites centered at coordinates -220 and -250. Mutational alteration of the IHF binding site reduced the efficiency of the tpl promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into tpl promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased when tpl DNA was present. The participation of both IHF and CRP in the activation of the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of the tpl promoter is presented.

Maximal transcriptional activation by the IHF protein of Escherichia coli depends on optimal DNA bending by the activator.

Transcriptional activation in prokaryotes can be mediated by at least two different mechanisms: direct contacts between the activator and RNA polymerase or modulation of the overall geometry of DNA. In the latter case, an activator protein that bends DNA favours contacts between the DNA upstream of the activator binding site and the back of RNA polymerase. The architectural protein integration host factor (IHF) of Escherichia coli bends DNA and activates transcription at several promoters. We have isolated mutants of IHF that maximize transcriptional activation by adjusting the bending angle of the DNA. The amino acid residues of IHF that adjust the bending angle are close to the DNA and probably make electrostatic interactions with the DNA. We show that transcriptional activation is maintained when the IHF binding site is moved further upstream or when its orientation is inverted, and we conclude from these data that direct interactions between IHF and RNA polymerase do not participate in activation. IHF acts merely by bending DNA; weaker bending leading to stronger activation. We propose that wild-type IHF induces too strong a DNA bend (180 degrees) for optimal interactions between DNA upstream of the IHF binding site and the back of RNA polymerase.

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site.

Analysis of the puc operon promoter from Rhodobacter capsulatus.

Expression of the Rhodobacter capsulatus puc operon, which codes for structural polypeptides of the light-harvesting-II peripheral antenna complex, is highly regulated in response to alterations in oxygen tension and light intensity. To obtain an understanding of the puc promoter region we report the high-resolution 5' mapping of the puc mRNA transcriptional start site and DNA sequence analysis of the puc upstream regulatory sequence (pucURS). A sigma70-type promoter sequence was identified (pucP1) which has a high degree of sequence similarity with carotenoid and bacteriochlorophyll biosynthesis promoters. Inspection of the DNA sequence also indicated the presence of two CrtJ and four integration host factor (IHF) binding sites. Transcriptional fusions of the pucURS fused to lacZ also confirmed that puc promoter activity is regulated by the transcriptional regulators IHF, CrtJ, and RegA. Gel retardation analysis using cell extracts indicates that mutations in IHF and RegA disrupt protein binding to DNA fragments containing the pucURS.

Activation of Gene Expression by a Novel DNA Structural Transmission Mechanism That Requires Supercoiling-induced DNA Duplex Destabilization in an Upstream Activating Sequence

We have previously demonstrated that integration host factor (IHF)-mediated activation of transcription from the ilvPG promoter of Escherichia coli requires a supercoiled DNA template and occurs in the absence of specific interactions between IHF and RNA polymerase. In this report, we describe a novel, supercoiling-dependent, DNA structural transmission mechanism for this activation. We provide theoretical evidence for a supercoiling-induced DNA duplex destabilized (SIDD) structure in the A + T-rich, ilvPG regulatory region between base pair positions +1 and -160. We show that the region of this SIDD sequence immediately upstream of an IHF binding site centered at base pair position -92 is, in fact, destabilized by superhelical stress and that this duplex destabilization is inhibited by IHF binding. Thus, in the presence of IHF, the negative superhelical twist normally absorbed by this DNA structure in the promoter distal half of the SIDD sequence is transferred to the downstream portion of the SIDD sequence containing the ilvPG promoter site. This IHF-mediated translocation of superhelical energy facilitates duplex destabilization in the -10 region of the downstream ilvPG promoter and activates transcription by increasing the rate of open complex formation.

Activation and repression of transcription at the double tandem divergent promoters for the xylR and xylS genes of the TOL plasmid of Pseudomonas putida.

The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a sigma54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma54 modulates expression from the downstream Ps2 sigma70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the sigma54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing sigma54 at the upstream promoter may modulate expression from the Ps2 promoter.

GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition.

As part of our analysis of the role of a uniquely clustered set of dam methylation sites (the motif GATC) within the origin of DNA replication in Escherichia coli, we have studied the effect of GATCs in various methylation states on the intrinsic curvature of DNA. We have designed a set of DNA linkers and used commercially available linkers containing GATC motifs. The linkers were ligated and the electrophoretic mobility of the resulting multimers in different states of methylation was tested relative to reference fragments. We report that properly phased GATCs in certain sequence environments modulate DNA curvature and that these effects may be enhanced by N6-adenine methylation of the GATCs. These structural alterations may in turn affect DNA-protein interactions, especially those involving proteins that rely on both primary sequence and structure for recognition. We present an example, where introduction of a GATC within an integration host factor (IHF) binding site, which does not alter the consensus sequence, reduces the binding affinity of the protein for the modified site.

Integration host factor affects the oxygen-regulated expression of photosynthesis genes in Rhodobacter capsulatus.

The puf and puc operons of Rhodobacter capsulatus, which encode pigment binding proteins, are regulated by oxygen tension. Strain IR4 which carries a point mutation in the himA gene for integration host factor (IHF) shows a delayed increase in the amount of photosynthetic complexes after reduction of oxygen tension when compared to the isogenic wild-type strain. This delay in the formation of photosynthetic complexes correlates with a delay in increasing the level of transcription of the puf and puc operons, whereas the transcription of the bchD genes is not affected by IHF. Several IHF binding sites can be identified upstream of the puf and puc transcriptional starts by in vitro footprint analysis. Our results suggest that IHF facilitates the binding of other trans-acting factors, e.g. RegA, which in turn stimulate transcription of the puf and puc genes under low oxygen tension.

Involvement of the RpoN protein in the transcription of the oprE gene in Pseudomonas aeruginosa

OprE is a channel-forming outer membrane protein of Pseudomonas aeruginosa, the expression of which is induced under anaerobic conditions. We constructed various mutants and observed the effects on oprE expression. Deficiency in RpoN, an alternative sigma factor for RNA polymerase, abolished oprE expression under aerobic conditions, but did not affect the expression under anaerobic conditions. One mutation on the putative RpoN recognition site also caused reduction of oprE expression. The region 500 nucleotides upstream of the mRNA start site was required for optimal oprE transcription, which contains an AT-rich region including a putative integration host factor binding site. These results indicate that OprE production is directly or indirectly controlled by RpoN but also require some other regulatory proteins bound to the upstream region.

Involvement of the RpoN protein in the transcription of the oprE gene in Pseudomonas aeruginosa.

OprE is a channel-forming outer membrane protein of Pseudomonas aeruginosa, the expression of which is induced under anaerobic conditions. We constructed various mutants and observed the effects on oprE expression. Deficiency in RpoN, an alternative sigma factor for RNA polymerase, abolished oprE expression under aerobic conditions, but did not affect the expression under anaerobic conditions. One mutation on the putative RpoN recognition site also caused reduction of oprE expression. The region 500 nucleotides upstream of the mRNA start site was required for optimal oprE transcription, which contains an AT-rich region including a putative integration host factor binding site. These results indicate that OprE production is directly or indirectly controlled by RpoN but also require some other regulatory proteins bound to the upstream region.

Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE.

Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate. These changes are mediated by the transcription factors Fnr and NarL respectively. Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon. To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA-lacZ operon fusions were constructed and analysed. The pattern of dmsA-lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE. In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E. coli ModE operator sites. Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation. In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site. We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity. Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA-lacZ expression in vivo. This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.

A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365.

We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

Integration Host Factor and Cyclic AMP Receptor Protein Are Required for TyrR-Mediated Activation of tpl inCitrobacter freundii

The tpl gene of Citrobacter freundiiencodes an enzyme that catalyzes the conversion ofl-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates −272.5, −158.5, and −49.5, respectively, were identified in the upstream region of the tpl promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of tplrequired integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate −85 and that there are CRP binding sites centered at coordinates −220 and −250. Mutational alteration of the IHF binding site reduced the efficiency of the tpl promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into tpl promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased whentpl DNA was present. The participation of both IHF and CRP in the activation of the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of thetpl promoter is presented.

In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site.

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis.

Fis is a small DNA-binding and -bending protein in Escherichia coli that is involved in several different biological processes, including stimulation of specialized DNA recombination events and regulation of gene expression. fis protein and mRNA levels rapidly increase during early logarithmic growth phase in response to a nutritional upshift but become virtually undetectable during late logarithmic and stationary phases. We present evidence that the growth phase-dependent fis expression pattern is not determined by changes in mRNA stability, arguing in favor of regulation at the level of transcription. DNA deletion analysis of the fis promoter (fis P) region indicated that DNA sequences from -166 to -81, -36 to -26, and +107 to +366 relative to the transcription start site are required for maximum expression. A DNA sequence resembling the integration host factor (IHF) binding site centered approximately at -114 showed DNase I cleavage protection by IHF. In ihf cells, maximum cellular levels of fis mRNA were decreased more than 3-fold and transcription from fis P on a plasmid was decreased about 3.8-fold compared to those in cells expressing wild-type IHF. In addition, a mutation in the ihf binding site resulted in a 76 and 61% reduction in transcription from fis P on a plasmid in the presence or absence of Fis, respectively. Insertions of 5 or 10 bp between this ihf site and fis P suggest that IHF functions in a position-dependent manner. We conclude that IHF plays a role in stimulating transcription from fis P by interacting with a site centered approximately at -114 relative to the start of transcription. We also showed that although the fis P region contains six Fis binding sites, Fis site II (centered at -42) played a predominant role in autoregulation, Fis sites I and III (centered at +26 and -83, respectively) seemingly played smaller roles, and no role in negative autoregulation could be attributed to Fis sites IV, V, and VI (located upstream of site III). The fis P region from -36 to +7, which is not directly regulated by either IHF or Fis, retained the characteristic fis regulation pattern in response to a nutritional upshift.

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli.

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication (oriC) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC. GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis-acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.

Kinetic characterization of the strand separation ("helicase") activity of the DNA packaging enzyme from bacteriophage lambda.

Bacteriophage lambda is assembled from preformed viral capsids (proheads), tails, and genomes that are excised from a concatemeric DNA precursor. The enzyme responsible for insertion of the genome into the precapsid is known as terminase. This enzyme possesses site-specific endonuclease, ATPase, and DNA strand separation ("helicase") catalytic activities, which work in concert to excise and package a single viral genome during phage assembly. We have previously characterized the endonuclease [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065] and ATPase [Tomka, M. A., & Catalano, C. E. (1993) Biochemistry 32, 11992-11997] catalytic activities of lambda terminase and present here similar studies on the strand separation activity of the enzyme. Strand separation requires terminase, divalent metal, and adenosine nucleotides with a hydrolyzable beta,gamma-phosphate bond. Two apparent binding sites for ATP-mediated strand separation were identified, one of which appears to be distinct from the high- and low-affinity sites previously observed for ATP hydrolysis [Hwang, Y., Catalano, C. E., & Feiss, M. (1995) Biochemistry 35, 2796-2803]. Salt stimulates the reaction at low concentrations but is strongly inhibitory at elevated concentrations, presumably due to impaired DNA binding. The above results are identical with either a complex DNA mixture (a nicked, annealed DNA duplex in the presence of excess nonspecific DNA) or a purified DNA substrate; however, a kinetic analysis of the reaction revealed that the observed rate was approximately 5-fold greater with the purified DNA substrate. Moreover, while Escherichia coli integration host factor (IHF) stimulates terminase-mediated strand separation with both substrates, the observed stimulation is more pronounced with the complex DNA mixture (10-fold rate increase) than the purified DNA substrate (5-fold rate increase). Our data are consistent with a model where IHF binding to the terminase assembly site forms a binary protein.DNA complex readily distinguishable from bulk DNA. The implications of these results to the process of DNA packaging in bacteriophage lambda are discussed.

PspF and IHF bind co-operatively in the psp promoter-regulatory region of Escherichia coli.

PspF bound to the psp enhancer activates E sigma54 holoenzyme-dependent transcription of the Escherichia coli phage-shock protein (psp) operon and autogenously represses its own sigma70-dependent transcription, thereby keeping its concentration at a low level. It has been demonstrated previously that integration host factor (IHF) bound to a DNA site located between the psp core promoter and the PspF binding sites stimulates psp expression. We show here that wild-type IHF strongly retards DNA containing the psp promoter region. In vitro, PspF binding to the psp enhancer facilitates IHF binding, while IHF binding to the pspF-pspA-E promoter-regulatory region increases the efficacy of PspF binding to the upstream activating sequences (UASs). This is the first demonstration of co-operative binding of an activator and IHF in a sigma54-dependent system. In the absence of IHF, in vivo autoregulation of pspF transcription is lifted and, consequently, PspF production is increased, indicating that IHF enhances PspF binding to the psp enhancer in vivo.

Identification of a maintenance system on rolling circle replicating plasmid pVT736-1. dgalli@iusd.iupui.edu.

Distribution of plasmid molecules to the two daughter cells at cell division is of major importance for their stable inheritance. Several mechanisms that control equipartitioning of low-copy-number plasmids have been described in molecular terms. However, no homologous or analogous systems have been identified for intermediate or high-copy-number plasmids, including rolling circle replicating (RCR) plasmids. It has been suggested that distribution of such plasmids at cell division relies solely on random segregation. Plasmid pVT736-1 is a 2 kb RCR plasmid that was isolated from the Gram-negative capnophilic coccobacillus Actinobacillus actinomycetemcomitans. The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stability. An operon that consists of two genes (orf3 and orf2) is followed by a putative cis-acting site that contains an integration host factor (IHF) binding site, flanked by several repeats. Mutations in orf2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or analogues of the pVT736-1 stabilization system have been detected on numerous plasmid and bacterial genomes.

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.