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L Søgaard-Andersen

Publications and source records attributed to L Søgaard-Andersen.

At least 19 recordsLinked to original sources
PubMed2001

C-signal: a cell surface-associated morphogen that induces and co-ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus.

In Myxococcus xanthus, morphogenesis of multicellular fruiting bodies and sporulation are co-ordinated temporally and spatially. csgA mutants fail to synthesize the cell surface-associated C-signal and are unable to aggregate and sporulate. We report that csgA encodes two proteins, a 25 kDa species corresponding to full-length CsgA protein and a 17 kDa species similar in size to C-factor protein, which has been shown previously to have C-signal activity. By systematically varying the accumulation of the csgA proteins, we show that overproduction of the csgA proteins results in premature aggregation and sporulation, uncoupling of the two events and the formation of small fruiting bodies, whereas reduced synthesis of the csgA proteins causes delayed aggregation, reduced sporulation and the formation of large fruiting bodies. These results show that C-signal induces aggregation as well as sporulation, and that an ordered increase in the level of C-signalling during development is essential for the spatial co-ordination of these events. The results support a quantitative model, in which aggregation and sporulation are induced at distinct threshold levels of C-signalling. In this model, the two events are temporally co-ordinated by the regulated increase in C-signalling levels during development. The contact-dependent C-signal transmission mechanism allows the spatial co-ordination of aggregation and sporulation by coupling cell position and signalling levels.

Bacterial Proteins↗
PubMed2000

Pattern formation: fruiting body morphogenesis in Myxococcus xanthus.

When Myxococcus xanthus cells are exposed to starvation, they respond with dramatic behavioral changes. The expansive swarming behavior stops and the cells begin to aggregate into multicellular fruiting bodies. The cell-surface-associated C-signal has been identified as the signal that induces aggregation. Recently, several of the components in the C-signal transduction pathway have been identified and behavioral analyses are beginning to reveal how the C-signal modulates cell behavior. Together, these findings provide a framework for understanding how a cell-surface-associated morphogen induces pattern formation.

Bacterial Proteins↗
PubMed1999

The cell surface-associated intercellular C-signal induces behavioral changes in individual Myxococcus xanthus cells during fruiting body morphogenesis.

Fruiting body formation in Myxococcus xanthus depends on ordered changes in cell movements from swarming to aggregation in response to starvation. We show that appropriately starved individual cells change behavior during fruiting body formation. Specifically, from the time of initiation of aggregation, individual wild-type cells began to move with increased gliding speeds, the duration of the mean gliding interval increased, and the stop frequency decreased whereas the duration of the mean stop interval and the reversal frequency remained unchanged. Mutants lacking the cell surface-associated, intercellular C-signal (csgA mutants) failed to aggregate. Likewise, appropriately starved individual csgA cells did not change their behavior during development. In the absence of other cell-cell interactions, the motility defect of individual csgA cells was corrected in a time- and concentration-dependent manner after C-signaling was reestablished by exogenous MalE-CsgA protein. The C-signal-induced stimulation of motility depended on the cytoplasmic Frz signal transduction system. We propose that C-signal instructs cells to move with high speed and low stop and reversal frequencies into aggregation centers during development.

Bacterial Proteins↗
PubMed1999

Dissection of a surface-exposed portion of the cAMP-CRP complex that mediates transcription activation and repression.

The Escherichia coli cAMP receptor protein (CRP) is essential for the activation and repression of transcription initiation at promoters in the CytR regulon. CRP performs these activities by making direct protein-protein interactions to the alpha-subunits of RNA polymerase and to the CytR regulator. Strikingly, it has been shown that amino acids of CRP that are critical for communication with the two partner proteins are located in close proximity on the surface of CRP. Here, we have dissected this surface in order to pinpoint the 'repression region' of CRP and to assess whether it overlaps with the characterized 'activating region'. Our results established that residues 12, 13, 17, 105, 108 and 110 are essential for the interaction with CytR and confirmed that 'activating region' 2 of CRP is made up of residues 19, 21 and 101. In the crystallographic structure of the CRP-DNA complex, the two sets of determinants are located immediately adjacent to each other forming a consecutive surface-exposed patch. The 'repression region' is chemically complementary to the characterized region on CytR that is essential for protein-protein communication to CRP. Moreover, the results provide insight into the mechanism by which CytR might prevent CRP-mediated transcription.

Alanine↗
PubMed1998

The FruA signal transduction protein provides a checkpoint for the temporal co-ordination of intercellular signals in Myxococcus xanthus development.

During fruiting body morphogenesis in Myxococcus xanthus, the intercellular C-signal induces aggregation, sporulation and developmental gene expression. To understand how a single signal system may induce temporally separated processes, we have focused on the class II gene, which codes for an essential component in the C-signal transduction pathway. We report that class II is identical to fruA and codes for a DNA binding response regulator. Transcription of fruA is developmentally regulated and depends on the early acting intercellular A- and E-signals. However, fruA transcription is independent of C-signal. Rather, genetic evidence suggests that C-signal controls FruA activity post-translationally. Genetic evidence strongly indicates that FruA is activated by phosphorylation. We propose that C-signalling results in the phosphorylation of FruA, thus activating FruA to interact with downstream targets. In the motility branch of the C-signalling pathway, FruA interacts with the Frz motility system; in the sporulation branch, we show that FruA is required for transcription of the sporulation locus devRS. On the basis of the two levels of control of FruA activity, we propose that FruA serves as a control point for the temporal co-ordination of intercellular signals during M. xanthus development.

Amino Acid Sequence↗
PubMed1997

Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression.

In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required. The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.

Base Sequence↗
PubMed1996

CytR/cAMP-CRP nucleoprotein formation in E. coli: the CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP.

The CytR repressor protein relies on protein-protein interactions to the cAMP-CRP complex to bind its operators with sufficiently high affinity to repress transcription. Here, the quaternary structure of CytR and the mechanism underlying the cooperative binding of CytR and cAMP-CRP have been analyzed. Using a modified Ferguson analysis in which protein-DNA complexes are separated in a non-denaturing gel system, we show that CytR binds its operators as a dimer alone as well as in a ternary complex with cAMP-CRP. Analyses of DNA binding of CytR at low protein concentrations indicate that CytR is a dimer in solution at physiological concentrations. Moreover, the CytR inducer cytidine was found not to have any effect on the oligomerization of free CytR or DNA bound CytR. Thus, these data rule out the possibility that the cooperative DNA binding of CytR and cAMP-CRP involves induced dimerization of CytR, and they suggest that cytidine interrupts the cooperative binding of CytR and cAMP-CRP solely by perturbing the protein-protein interactions between the two proteins.

Bacterial Proteins↗
PubMed1996

C factor, a cell-surface-associated intercellular signaling protein, stimulates the cytoplasmic Frz signal transduction system in Myxococcus xanthus.

C factor, an intercellular signaling protein, is required for aggregation and sporulation of the social bacterium, Myxococcus xanthus. We report that C factor, which normally is associated with the cell surface, provides input to the Frz signal transduction cascade. Elements of this cascade have sequence homology to bacterial chemotaxis systems and are known to control the frequency of gliding reversal. Exposure of developing cells of a C-factor-less mutant (csgA) to purified C factor increases the ratio of methylated to nonmethylated FrzCD protein, the Frz homolog of the methyl-accepting chemotaxis proteins. Methylation depends on the cognate methyltransferase FrzF, and its extent increases with the concentration of C factor. C-factor-induced methylation also depends on the product of a gene, called class II, which is necessary in vivo for all known responses to C factor. A model for aggregation is proposed in which C factor stimulates the Frz cascade and thereby decreases cell reversals in a way that preferentially leads cells into an aggregate.

Bacterial Proteins↗
PubMed1996

Intercellular C-signaling in Myxococcus xanthus involves a branched signal transduction pathway.

C-factor, the product of the csgA gene, is a cell-surface associated short-range intercellular signaling protein in Myxococcus xanthus. C-factor is required for at least four responses during starvation-induced fruiting body morphogenesis: rippling, aggregation, sporulation, and full expression of the csgA gene, all of which fail in a csgA mutant. To analyze the C-factor signaling pathway, eight Tn5 lac insertion mutants that began but failed to complete fruiting body aggregation were characterized. Seven of the insertions identified genes whose products function in the csgA signaling pathway. The seven mutants were differentially deficient in the C-factor responses, and could be divided into two classes on the basis of those differences. On one hand, the four mutants in class I were deficient in rippling and aggregation, but sporulated and produced C-factor at wild-type levels. The Tn5 lac insertions in the class I mutants mapped to the frz locus, which encodes a signal transduction system that controls the frequency of single cell reversals. On the other hand, mutants carrying any of the three closely linked class II Tn5 lac insertions had deficiencies in all four C-factor responses. Because the sporulation defect in the class 11 mutants is cell autonomous, the data suggest that the primary defect in these mutants is an inability to respond to the C-factor signal. All the data can be explained by a model in which the first part of the C-factor signaling pathway is common to all four C-factor-dependent responses. The genes identified by the class 11 insertions would function in the common part. Downstream of class II, the pathway branches. One branch includes the frz genes and leads to aggregation and rippling; the second branch leads to sporulation and controls the level of csgA gene expression. This model was confirmed in epistasis tests with characterized frz mutations, a csgA null mutation, and a class II mutation.

Bacterial Proteins↗
PubMed1996

A flexible partnership: the CytR anti-activator and the cAMP-CRP activator protein, comrades in transcription control.

A vital point in gene regulation is control at the level of transcription initiation. Recent research has established that this regulation can involve sophisticated networks of interacting proteins that modulate the activity of the transcription machinery by DNA looping, direct protein-protein interactions or changing DNA topology in the promoter region. This Micro-Review focuses on our investigations of a relatively simple prokaryotic gene regulatory system, the Escherichia coli CytR regulon, which exhibits a number of these features. This work has opened the door to the molecular understanding of how a prokaryotic repressor can be correctly positioned at specific DNA sequences with the help of a global activator, and how the repressor subsequently inhibits factor-dependent transcription initiation.

Bacterial Proteins↗
PubMed1994

CRP induces the repositioning of MalT at the Escherichia coli malKp promoter primarily through DNA bending.

Activation of transcription initiation at the Escherichia coli malKp promoter requires the repositioning of MalT, the primary activator, from a set of non-productive sites to a set of productive sites, which is staggered by 3 bp. Occupation of the latter relies on the formation of a higher order structure involving distal MalT sites and the binding of CRP (cAMP receptor protein) to three sites located in the intervening region. We show here that one can successfully replace all of the CRP sites by the binding site of another DNA-bending protein, integration host factor, or by a sequence-directed bend without altering the process of malKp activation. This observation indicates that CRP action at malKp does not involve critical interactions with MalT and that CRP promotes MalT repositioning primarily through DNA bending. This structural role of CRP differs markedly from its role in the activation of the lac promoter.

Bacterial Proteins↗
PubMed1994

The gene encoding the periplasmic cyclophilin homologue, PPIase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP-CRP complex and negatively regulated by the CytR repressor.

The rot gene in Escherichia coli encodes PPIase A, a periplasmic peptidyl-prolyl cis-trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA-binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP-CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP-CRP to two sites separated by 53 bp. Moreover, one of the cAMP-CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP-CRP/DNA nucleoprotein complex consisting of the two cAMP-CRP molecules and CytR bound in between. The two regulators bind co-operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP-CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo- and deoxyribonucleoside catabolism or transport.

Amino Acid Isomerases↗
PubMed1993

Protein-protein interactions in gene regulation: the cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor.

Maximal repression by the CytR protein depends on the formation of nucleoprotein complexes in which CytR interacts with DNA and with cAMP-cAMP receptor protein (CRP). Here we demonstrate that CytR regulates transcription from deoP2 promoters in which the entire CytR recognition sequence has been eliminated. Furthermore, CytR proteins deleted for the DNA-binding domain repress deoP2 in vivo and interact with deoP2 in vitro in a strictly cAMP-CRP-dependent fashion. These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CytR-DNA interactions may primarily serve to stabilize the nucleo-protein complex. This type of specificity mechanism may represent a general concept in the recruitment of DNA-binding proteins in combinatorial regulatory systems.

Amino Acid Sequence↗
PubMed1993

Identification of the nucleotide sequence recognized by the cAMP-CRP dependent CytR repressor protein in the deoP2 promoter in E. coli.

In E. coli repression of transcription initiation by the CytR protein relies on CytR-DNA interactions as well as on interactions between CytR and the cAMP-CRP activator complex. To identify the nucleotide sequence recognized by CytR, mutants of the deoP2 promoter with a reduced regulatory response to CytR have been isolated. Five single bp mutation derivatives of deoP2 with a 2-5-fold decrease in CytR regulation have been characterized. In vitro, the only effect of the mutations was a decrease in the binding affinity of CytR, and a clear correlation was observed between the reduction in CytR regulation in vivo and the reduction in CytR binding in vitro. The mutations all reside in a sequence element that contains an imperfect direct as well as an imperfect inverted repeat. As the active form of CytR, most likely, is an oligomer with two-fold rotational symmetry, CytR probably interacts with the inverted repeat. Degenerate versions of the inverted repeat are present in all CytR binding sites characterized so far, however, the distance between the half-sites varies.

Bacterial Proteins↗
PubMed1992

cAMP-CRP activator complex and the CytR repressor protein bind co-operatively to the cytRP promoter in Escherichia coli and CytR antagonizes the cAMP-CRP-induced DNA bend.

Initiation of transcription from the cytRP promoter in Escherichia coli is activated by the cAMP-CRP complex and negatively regulated by the CytR repressor protein. By combining gel retardation and footprinting assays, we show that cAMP-CRP binds to a single site centered at position -64 and induces a considerable bend in the DNA. CytR binds to a region immediately downstream from, and partially overlapping, the CRP site, and induces a modest bend into the DNA. In combination, cAMP-CRP and CytR bind co-operatively to cytRP forming a nucleoprotein complex in which the proteins directly interact with each other and bind to the same face of the DNA helix. CytR binding concomitantly antagonizes the cAMP-CRP-induced bend. This study indicates that the minimal DNA region required to obtain CytR regulation consists of a single binding site for each of cAMP-CRP and CytR. The case described here, in which a protein-induced DNA bend is modulated by a second protein, may illustrate a mechanism that applies to other regulatory systems.

Bacterial Proteins↗
PubMed1992

The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions -41, -91 and -93, respectively. In the absence of CytR, cAMP-CRP interacts at one set of sites (CRP-1 and CRP-2) and both of these binding sites are required for full promoter activation. In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP-CRP binds to CRP-1 and CRP-3 and CytR occupies the sequence between these sites. Thus, association of the two regulators involves a repositioning of the cAMP-CRP complex. Moreover, mutant cdd promoters in which CRP-2 and CRP-3 have been deleted are partially regulated by CytR, and cAMP-CRP and CytR still bind cooperatively to these promoters. These findings provide clues to an understanding of how cAMP-CRP and CytR interact at a structurally diverse set of promoters.

Bacterial Proteins↗
PubMed1991

Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter.

Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode two binding sites for the cAMP.CRP complex. Biochemical and genetic studies have shown that CytR relies on interactions with the cAMP.CRP complex in order to bind promoter DNA and repress transcription. Here we have purified CytR to near homogeneity and addressed the question of how it interacts with the deoP2 promoter. Gel retardation and DNase I footprinting analyses show that CytR is a true sequence-specific DNA-binding protein that binds to the sequence between the two CRP sites in deoP2 with a relatively low affinity. In the presence of the cAMP.CRP complex the two protein species bind cooperatively to deoP2, forming a complex in which CytR occupies the sequence between the two DNA bound cAMP.CRP complexes. Furthermore, the inducer (cytidine) does not affect independent DNA binding of CytR, rather the CytR/cAMP.CRP cooperativity is perturbed. These results indicate that CytR binding to deoP2 relies on both repressor-DNA interactions and protein-protein interactions to cAMP.CRP. This combinatorial repression mechanism, in which an activator functions as an adaptor for a repressor that is not capable of blocking transcription on its own, is unprecedented in prokaryotes; it is, however, reminiscent of repression mechanisms found in eukaryotes.

Bacterial Proteins↗