cAMP Receptor Research Articles

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.

The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP's recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

1,2-Dichloroethane Induces Reproductive Toxicity Mediated by the CREM/CREB Signaling Pathway in Male NIH Swiss Mice.

1,2-Dichloroethane (1,2-DCE) is a widely used chlorinated organic toxicant but little is known about the reproductive disorders induced by its excessive exposure. To reveal 1,2-DCE-induced male reproductive toxicity and to elucidate the underlying mechanisms, we exposed male National Institutes of Health Swiss mice to 1,2-DCE by inhalation at 0, 100, 350, and 700 mg/m3 for 6 h/day, for 1 and 4 weeks. Our findings showed a significant decrease in body weight with increased testis/body weight ratio, reduced sperm concentration and induced malformation of spermatozoa, and vacuolar degeneration of germ cells in the seminiferous tubules of testes in mice exposed to 1,2-DCE. Cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) and cAMP-response element modulator (CREM) were significantly inhibited by 1,2-DCE. This is consistent with the declines in the transducer of regulated CREB activity 1 and activator of CREM in testis, which results in the decrease in lactate dehydrogenase C and testis-specific kinase 1 in the testes. Moreover, the activation of p53 and Bax with the inhibition of Bcl-2 might be the reason for the upregulation of caspase-3 in the apoptosis, as detected by TdT-mediated dUTP nick-end labeling assay in the testes induced by 1,2-DCE. Finally, elevated testosterone levels were found along with increased levels of gonadotropin-releasing hormone, cAMP, luteinizing hormone (LH), and LH receptors in the testes. These findings suggest that 1,2-DCE inhibits CREM/CREB signaling cascade and subsequently induces apoptosis associated with p53 activation and mitochondrial dysfunction. This also results in induced malformation of spermatozoa, reduced sperm concentration, and pathological impairment of the testes.

CAMP receptor protein regulates mouse colonization, motility, fimbria-mediated adhesion, and stress tolerance in uropathogenic Proteus mirabilis

Cyclic AMP receptor protein (Crp) is a major transcriptional regulator in bacteria. This study demonstrated that Crp affects numerous virulence-related phenotypes, including colonization of mice, motility, fimbria-mediated adhesion, and glucose stress tolerance in uropathogenic Proteus mirabilis. Diabetic mice were more susceptible to kidney colonization by wild-type strain than nondiabetic mice, in which the crp mutant exhibited increased kidney colonization. Loss of crp or addition of 10% glucose increased the P. mirabilis adhesion to kidney cells. Direct negative regulation of pmpA (which encodes the major subunit of P-like fimbriae) expression by Crp was demonstrated using a reporter assay and DNase I footprinting. Moreover, the pmpA/crp double mutant exhibited reduced kidney adhesion comparable to that of the pmpA mutant, and mouse kidney colonization by the pmpA mutant was significantly attenuated. Hence, the upregulation of P-like fimbriae in the crp mutant substantially enhanced kidney colonization. Moreover, increased survival in macrophages, increased stress tolerance, RpoS upregulation, and flagellum deficiency leading to immune evasion may promote kidney colonization by the crp mutant. This is the first study to elucidate the role of Crp in the virulence of uropathogenic P. mirabilis, underlying mechanisms, and related therapeutic potential.

Characterization of a lactose-responsive promoter of ATP-binding cassette (ABC) transporter gene from Lactobacillus acidophilus 05-172.

A novel lactose-responsive promoter of the ATP-binding cassette (ABC) transporter gene Lba1680 of Lactobacillus acidophilus strain 05-172 isolated from a traditionally fermented dairy product koumiss was characterized. In L. acidophilus 05-172, expression of Lba1680 was induced by lactose, with lactose-induced transcription of Lba1680 being 6.1-fold higher than that induced by glucose. This is in contrast to L. acidophilus NCFM, a strain isolated from human feces, in which expression of Lba1680 and Lba1679 is induced by glucose. Both gene expression and enzyme activity assays in L. paracasei transformed with a vector containing the inducible Lba1680 promoter (PLba1680) of strain 05-172 and a heme-dependent catalase gene as reporter confirmed that PLba1680 is specifically induced by lactose. Its regulatory expression could not be repressed by glucose, and was independent of cAMP receptor protein. This lactose-responsive promoter might be used in the expression of functional genes in L. paracasei incorporated into a lactose-rich environment, such as dairy products.

The KP1_4563 gene is regulated by the cAMP receptor protein and controls type 3 fimbrial function in Klebsiella pneumoniae NTUH-K2044.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that can adhere to host cells or extracellular matrix via type 1 and type 3 fimbriae. KP1_4563 is a gene encoding a hypothetical protein in K. pneumoniae NTUH-K2044. KP1_4563 is located between the type 1 and type 3 fimbrial gene clusters and is likely associated with fimbrial function given its putative conserved domains of unknown function (DUF1471). Cyclic AMP receptor protein (CRP) regulates virulence-related gene expression and is a crucial transcriptional regulator in many bacteria. The predicted DNA recognition motif of CRP is present in the KP1_4563 promoter region. This study aimed to investigate the function of KP1_4563 in fimbriae and its transcriptional regulation mechanism by CRP. We generated Kp-Δ4563 mutant and complementation strains. We utilized phenotype and adhesion assays to evaluate the role of KP1_4563 in fimbriae. We conducted quantitative RT-PCR (qRT-PCR), LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays to study the transcriptional regulation of KP1_4563 gene by CRP. We found that KP1_4563 negatively regulates the function of type 3 fimbriae. Compared with NTUH-K2044, the absence of KP1_4563 enhanced the ability of Kp-Δ4563 to adhere to A549 cells. CRP negatively regulates KP1_4563 by directly binding to its promoter region. KP1_4563 plays an important role in type 3 fimbrial function. This novel insight will assist in the development of strategies for preventing K. pneumoniae infection.

Edwardsiella piscicida lacking the cyclic AMP receptor protein (Crp) is avirulent and immunogenic in fish

Edwardsiella piscicida is a Gram-negative pathogen that generally causes lethal septicemia in marine and freshwater fish. We generated a E. piscicida CK216 Δcrp mutant to investigate various biological roles related to this organism, including pathogenesis. Lack of Crp in CK216 was demonstrated by immunoblotting using a Crp-specific antibody. Compared to the parental strain, the mutant exhibited changes in three biochemical phenotypes, including ornithine decarboxylation, citrate utilization, and H2S production. Complementation of crp deletion in trans rescued the phenotype of the parental strain. This study proved that hemolytic activity in E. piscicida is controlled by Crp. In addition, significantly reduced motility of E. piscicida CK216 was observed, which resulted from a lack of flagella synthesis. To examine the virulence in fish, E. piscicida cells were injected into the goldfish (Carassius auratus) via intraperitoneal route. The LD50 of CK216 was 9.25 × 108 CFU, while that of the CK108 parental strain was 9.24 × 105 CFU, attenuated 1000 fold in goldfish. Fish immunized with CK216 elicited IgM responses. Moreover, 80% of goldfish immunized with 1 × 106 CFU survived after administration of a lethal dose (1 × 107 CFU) of virulent E. piscicida CK41, suggesting the potential for E. piscicida CK216 to serve as a live attenuated vaccine in aquaculture.

The Transcriptional Regulators of the CRP Family Regulate Different Essential Bacterial Functions and Can Be Inherited Vertically and Horizontally.

One of the best-studied transcriptional regulatory proteins in bacteria is the Escherichia coli catabolite repressor protein (CRP) that when complexed with 3′-5′-cyclic AMP (cAMP) changes its conformation and interacts with specific DNA-sequences. CRP DNA-binding can result in positive or negative regulation of gene expression depending on the position of its interaction with respect to RNA polymerase binding site. The aim of this work is to review the biological role and phylogenetic relations that some members of the CRP family of transcriptional regulators (also known as cAMP receptor protein family) have in different bacterial species. This work is not intended to give an exhaustive revision of bacterial CRP-orthologs, but to provide examples of the role that these proteins play in the expression of genes that are fundamental for the life style of some bacterial species. We highlight the conservation of their structural characteristics and of their binding to conserved-DNA sequences, in contrast to their very diverse repertoire of gene activation. CRP activates a wide variety of fundamental genes for the biological characteristic of each bacterial species, which in several instances form part of their core-genome (defined as the gene sequences present in all members of a bacterial species). We present evidence that support the fact that some of the transcriptional regulators that belong to the CRP family in different bacterial species, and some of the genes that are regulated by them, can be inherited by horizontal gene transfer. These data are discussed in the framework of bacterial evolution models.

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss).

Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic-3',5'-adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A.salmonicida virulent and non-virulent isolates. We found that A.salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild type. A.salmonicida ∆crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A.salmonicida ∆crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A.salmonicida wild type. We concluded that deletion of A.salmonicida crp influences sugar utilization, cell invasion and virulence. Deletion of crp in A.salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis.

CRP Regulates D-Lactate Oxidation in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a heterotrophic facultative anaerobe that respires using various organic and inorganic compounds. This organism has served as a model to study bacterial metabolic and regulatory systems that facilitate their survival in redox-stratified environments. The expression of many anaerobic respiratory genes in MR-1, including those for the reduction of fumarate, dimethyl sulfoxide, and metal oxides, is regulated by cyclic AMP receptor protein (CRP). However, relatively little is known about how this organism regulates the expression of catabolic enzymes catalyzing the oxidation of organic compounds, including lactate. Here, we investigated transcriptional mechanisms for the lldP (SO_1522) and dld (SO_1521) genes, which encode putative lactate permease and D-lactate dehydrogenase, respectively, and demonstrate that CRP regulates their expression in MR-1. We found that a crp-deletion mutant of MR-1 (Δcrp) showed impaired growth on D-lactate. Complementary expression of dld in Δcrp restored the ability to grow on D-lactate, indicating that the deficient growth of Δcrp on D-lactate is attributable to decreased expression of dld. In vivo transcription and in vitro electrophoretic mobility shift assays reveal that CRP positively regulates the expression of the lldP and dld genes by directly binding to an upstream region of lldP. Taken together, these results indicate that CRP is a global transcriptional regulator that coordinately regulates the expression of catabolic and respiratory pathways in MR-1, including D-lactate dehydrogenase and anaerobic terminal reductases.

Identification of bacterial guanylate cyclases.

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRPG, constructed here. While wild-type CRP is activated exclusively by cAMP,_CRPG can be activated by either cAMP or cGMP. Using CRP- and CRPG-dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties.

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals.

C-terminal dimerization of apo-cyclic AMP receptor protein validated in solution.

Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo-CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP.

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

Involvement of cAMP receptor protein in biofilm formation, fimbria production, capsular polysaccharide biosynthesis and lethality in mouse of Klebsiella pneumoniae serotype K1 causing pyogenic liver abscess.

The global regulator cAMP receptor protein (CRP) has been shown to be required for the full virulence and/or for the expression of virulence determinants in a wide set of bacterial pathogens. In this work, the crp mutant as well as the complemented mutant was constructed from a wild-type Klebsiella pneumoniae capsular serotype K1 strain causing the primary pyogenic liver abscess. The phenotypes of wild-type strain, crp mutant and complemented mutant were characterized systematically. It was disclosed that K. pneumoniae CRP was required for the in vitro growth, fimbria production, biofilm formation and lethality in mouse, but it inhibited the capsular polysaccharide biosynthesis. These indicated the important roles of CRP in regulating the expression of virulence and biofilm genes in K. pneumoniae.

Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium.

Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.

Disrupting the Allosteric Interaction between the Plasmodium falciparum cAMP-dependent Kinase and Its Regulatory Subunit

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth.

Spot 42 sRNA regulates arabinose-inducible araBAD promoter activity by repressing synthesis of the high-affinity low-capacity arabinose transporter.

The bacterial arabinose inducible system is widely used for titratable control of gene expression. We demonstrate here that a post-transcriptional mechanism mediated by the Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system, and has implications for improving its usage for tunable gene expression.

A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis

Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.

Protein-induced DNA linking number change by sequence-specific DNA binding proteins and its biological effects.

Sequence-specific DNA-binding proteins play essential roles in many fundamental biological events such as DNA replication, recombination, and transcription. One common feature of sequence-specific DNA-binding proteins is to introduce structural changes to their DNA recognition sites including DNA-bending and DNA linking number change (ΔLk). In this article, I review recent progress in studying protein-induced ΔLk by several sequence-specific DNA-binding proteins, such as E. coli cAMP receptor protein (CRP) and lactose repressor (LacI). It was demonstrated recently that protein-induced ΔLk is an intrinsic property for sequence-specific DNA-binding proteins and does not correlate to protein-induced other structural changes, such as DNA bending. For instance, although CRP bends its DNA recognition site by 90°, it was not able to introduce a ΔLk to it. However, LacI was able to simultaneously bend and introduce a ΔLk to its DNA binding sites. Intriguingly, LacI also constrained superhelicity within LacI-lac O1 complexes if (-) supercoiled DNA templates were provided. I also discuss how protein-induced ΔLk help sequence-specific DNA-binding proteins regulate their biological functions. For example, it was shown recently that LacI utilizes the constrained superhelicity (ΔLk) in LacI-lac O1 complexes and serves as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop. These constrained (-) supercoils enhance LacI's binding affinity and therefore the repression of the lac promoter. Other biological functions include how DNA replication initiators λ O and DnaA use the induced ΔLk to open/melt bacterial DNA replication origins.