cAMP Receptor Protein Complex Research Articles

Regulation of virulence-associated plasmid genes in enteroinvasive Escherichia coli.

The transposon TnphoA was used for construction of gene fusions and for studies of gene regulation in an enteroinvasive strain of Escherichia coli. Several plasmid-encoded virulence genes (e.g., the ipaB and virG operons) of such enteroinvasive strains are subject to coordinated thermoregulation involving both operon-specific (the VirB and VirF activators) and global regulators. The nucleoid-associated E. coli protein H-NS was shown to be a negative regulator as judged by studies using H-NS gene deletion mutations and by increasing the level of H-NS protein in the cells. An increased gene dosage of H-NS led to enhanced repression of the ipa and virG operons, particularly at low (30 degrees C) growth temperature. The cyclic AMP receptor protein complex, which is another global transcriptional regulator in E. coli, was not required for the regulation of ipa and virG expression. The virG operon was expressed in an activator-independent manner in cells lacking H-NS protein. We suggest that the role of the VirF activator is to counteract the silencing effect of H-NS.

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.

Control mechanism of the Escherichia coli K-12 cell cycle is triggered by the cyclic AMP-cyclic AMP receptor protein complex

The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains. One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter. In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed. The other strains were constructed as double mutants (NC6707 cya sulB [ftsZ(Ts)] and TR3318 crp sulB [ftsZ(Ts)]). In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318. These results indicate that lateral wall synthesis in the E. coli cell cycle is triggered by the cAMP-cAMP receptor protein complex.

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya+ crp+ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya+ crp+ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.

Regulation of expression of the ilvB operon in Salmonella typhimurium.

The ilvB gene of Salmonella typhimurium encodes the valine-sensitive form of acetohydroxy acid synthase, acetohydroxy acid synthase I, which catalyzes the first step in the parallel biosynthesis of isoleucine and valine. Although nearly all of the other genes involved in this pathway are clustered at minute 83, ilvB was found to lie at minute 80.5. Expression of ilvB was shown to be nearly completely repressed by the end products leucine and valine. Studies in which we used strains with mutations in cya (adenylate cyclase) and crp (cAMP receptor protein) demonstrated that synthesis of acetohydroxy acid synthase I is enhanced by the cAMP-cAMP receptor protein complex. Although no stimulation was achieved by growth on poor carbon sources, introduction of crp on a multicopy plasmid led to markedly increased expression. Strains of S. typhimurium lacking valine-resistant acetohydroxy acid synthase II (ilvG) are like Escherichia coli K-12 in that they are not able to grow in the presence of L-valine owing to a conditional isoleucine auxotrophy. The valine toxicity of these ilvG mutants of S. typhimurium was overcome by increasing the level of acetohydroxy acid synthase I. Enzyme activity could be elevated either by maximally derepressing expression with severe leucine limitation, by introduction of either ilvB or crp on a multicopy plasmid, or by the presence of the ilv-513 mutation. This mutation, which is closely linked to genes encoding the phosphoenol pyruvate:sugar phosphotransferase system (pts), causes highly elevated expression of ilvB that is refractory to repression by leucine and valine, as is the major ilv operon. The response of ilvB to the cAMP-cAMP receptor protein complex was not affected by this lesion. Data obtained by using this mutant led us to propose that the two modes of regulation act independently. We also present some evidence which suggests that ilvB expression may be affected by the phosphoenol pyruvate:sugar phosphotransferase system.

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy

The effect of specific DNA binding of the cAMP · cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP · cAMP receptor protein complex induces a kink of 30 to 45 ° in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.

On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions.

Using DNase footprinting and transcription assays in vitro we have probed the effect of the cAMP-cAMP receptor protein complex (cAMP-CRP) on the positioning of RNA polymerase and on the location of the transcription start point at the Escherichia coli gal and lac operon regulatory regions. In both cases, RNA polymerase can form two alternative complexes which promote transcription from two different start points, S1 and S2: pre-incubation of promoter DNA with cAMP-CRP results in a shift of the transcription start from S2 to S1 and in an increase in the rate of open complex formation. Moreover, the rate of formation of each heparin-resistant complex parallels the establishment of the corresponding footprint, showing that the stable binding corresponds to open complex formation. We show that, in the case of gal, RNA polymerase, which is bound so as to transcribe from S2, cannot be diverted to S1 by subsequent addition of cAMP-CRP. In contrast, in the case of lac, when cAMP-CRP is added after RNA polymerase, complexes which initiate transcription at S2 are rapidly converted to complexes which initiate at S1. Finally, we present data which suggest that protein-protein interactions are essential for CRP-induced activation at both the lac and gal promoters.

Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant

A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.

Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation

Using hydroxylamine mutagenesis in vitro, mutations were introduced into a short DNA fragment containing the two overlapping promoters of the Escherichia coli galactose operon and the start of the first gal gene, galE. The mutagenised fragment was inserted into a lac expression plasmid. In such a vector, lac expression is controlled by the gal promoter region. Amongst eighteen candidates in which expression was reduced due to mutations in the gal fragment, twelve contained promoter mutations and six carried mutations that reduce the initiation of galE translation. The candidates in which promoter activity was reduced contained mutations affecting the promoter PI, which is dependent on the cyclic AMP-receptor protein complex (cAMP-CRP) for activation. All carried mutations in the sequence 5'GTGA3' at the CRP binding site. One of the twelve also contained a second mutation affecting the second promoter, P2, which normally functions in the absence of cAMP-CRP. Amongst the six candidates affecting galE translation, two contained a mutation that changes the initiator codon from AUG to AUA and almost completely suppresses galE expression. The mutations in the other four candidates affect the ribosome binding sequence, 5'GGAG3'. However, multiple mutations that abolish this sequence do not totally suppress galE expression, showing that there must be another way to guide ribosomes to the correct initiation site.

Involvement of cyclic AMP and its receptor protein in filamentation of an Escherichia coli fic mutant.

Cyclic AMP (cAMP) inhibited septum formation in Escherichia coli PA3092 and induced cell filamentation at elevated temperatures. This phenomenon was first observed in E. coli PA3092 and is due to a temperature-sensitive mutation. We tentatively named this mutation fic (filamentation induced by cAMP). The fic gene was located near rpsL (formerly strA) on the E. coli K-12 map. the inhibitory effect of cAMP on cell division and filamentation in a fic mutant was not observed in a crp mutant. When cAMP was removed from the culture medium, filaments were divided into rods as the intracellular cAMP level decreased. These results suggest that the cAMP-cAMP receptor protein complex causes filamentation in the fic mutant, E. coli PA3092.

Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex.

The synthesis of the adenylate cyclase [ATP pyrophosphatelyase-(cyclizing), E.C. 4.6.1.1.] of Escherichia coli, appears to be regulated negatively by the cAMP receptor protein, CRP. This conclusion is based on a comparison of adenylate cyclase activities measured in vitro with the rates of cAMP synthesis by intact bacteria. The activity of adenylate cyclase, depending on conditions of growth, is also regulated by CRP; this effect, however, is indirect insofar as it is mediated by a protein or proteins under CRP control.

Catabolite repression in Escherichia coli mutants lacking cyclic AMP receptor protein.

Pleiotropic carbohydrate-positive pseudorevertants have been isolated from a specific class of rho-crp double mutants of Escherichia coli carrying both defective transcription termination protein, rho, and cyclic AMP receptor protein. The modulation of catabolite repression of beta-galactosidase, amylomaltase, and tryptophanase has been studied in the pseudorevertants. It has been found that these mutants exhibit catabolite repression. Because catabolite-sensitive operons can be expressed in the absence of functional cyclic AMP receptor protein, this would suggest on the one hand that the cyclic AMP-receptor protein complex is not the exclusive mediator of catabolite repression and on the other hand that rho might be involved in the regulation of catabolite-sensitive operons.

Control of minicell producing cell division by cAMP-receptor protein complex in Escherichia coli

It has been established that the strain CA8000 of Escherichia coli K12 produces minicells. This phenotype of CA8000 has been shown to be suppressed by additional mutations in cya or crp genes. Minicell production by cya+ crp+ min bacteria is probably a consequence of error, introduced by horizontal growth, in the selection of site on the envelope for initiation of hemispherical growth.