Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

INTRODUCTION: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. RESULTS: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. CONCLUSION: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant.

As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.

Mutations upstream from sdaC and malT in Escherichia coli uncover a complex interplay between the cAMP receptor protein and different sigma factors.

In Escherichia coli, one of the best understood microorganisms, much can still be learned about the basic interactions between transcription factors and promoters. When a cAMP-deficient cya mutant is supplied with maltose as the main carbon source, mutations develop upstream from the two genes malT and sdaC. Here, we explore the regulation of the two promoters, using fluorescence-based genetic reporters in combination with both spontaneously evolved and systematically engineered cis-acting mutations. We show that in the cya mutant, regulation of malT and sdaC evolves toward cAMP-independence and increased expression in the stationary phase. Furthermore, we show that the location of the cAMP receptor protein (Crp) binding site upstream of malT is important for alternative sigma factor usage. This provides new insights into the architecture of bacterial promoters and the global interplay between Crp and sigma factors in different growth phases.IMPORTANCEThis work provides new general insights into (1) the architecture of bacterial promoters, (2) the importance of the location of Class I Crp-dependent promoters, and (3) the global interplay between Crp and sigma factors in different growth phases.

The interplay between metabolic stochasticity and cAMP-CRP regulation in single E. coli cells.

The inherent stochasticity of metabolism raises a critical question for understanding homeostasis: are cellular processes regulated in response to internal fluctuations? Here, we show that, in E. coli cells under constant external conditions, catabolic enzyme expression continuously responds to metabolic fluctuations. The underlying regulatory feedback is enabled by the cyclic AMP (cAMP) and cAMP receptor protein (CRP) system, which controls catabolic enzyme expression based on metabolite concentrations. Using single-cell microscopy, genetic constructs in which this feedback is disabled, and mathematical modeling, we show how fluctuations circulate through the metabolic and genetic network at sub-cell-cycle timescales. Modeling identifies four noise propagation modes, including one specific to CRP regulation. Together, these modes correctly predict noise circulation at perturbed cAMP levels. The cAMP-CRP system may thus have evolved to control internal metabolic fluctuations in addition to external growth conditions. We conjecture that second messengers may more broadly function to achieve cellular homeostasis.

A simple mechanism for integration of quorum sensing and cAMP signalling in Vibrio cholerae.

Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived 'autoinducer' signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In Vibrio cholerae, quorum-sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in V. cholerae. Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other Vibrio species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows V. cholerae to regulate subsets of genes during the transition between aquatic environments and the human host.

Designing glucose utilization "highway" for recombinant biosynthesis.

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from "glucose repression" are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Unleashing the Influence of cAMP Receptor Protein: The Master Switch of Bacteriocin Export in Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a Gram-negative phytopathogenic bacterium that produces carocin, a low-molecular-weight bacteriocin that can kill related strains in response to factors in the environment such as UV exposure or nutritional deficiency. The function of the catabolite activator protein (CAP), also known as the cyclic AMP receptor protein (CRP), as a regulator of carocin synthesis was examined. The crp gene was knocked out as part of the investigation, and the outcomes were assessed both in vivo and in vitro. Analysis of the DNA sequence upstream of the translation initiation site of carocin S3 revealed two putative binding sites for CRP that were confirmed using a biotinylated probe pull-down experiment. This study revealed that the deletion of crp inhibited genes involved in extracellular bacteriocin export via the flagellar type III secretion system and impacted the production of many low-molecular-weight bacteriocins. The biotinylated probe pull-down test demonstrated that when UV induction was missing, CRP preferentially attached to one of the two CAP sites while binding to both when UV induction was present. In conclusion, our research aimed to simulate the signal transduction system that controls the expression of the carocin gene in response to UV induction.

Quantitative model for genome-wide cyclic AMP receptor protein binding site identification and characteristic analysis.

Cyclic AMP receptor proteins (CRPs) are important transcription regulators in many species. The prediction of CRP-binding sites was mainly based on position-weighted matrixes (PWMs). Traditional prediction methods only considered known binding motifs, and their ability to discover inflexible binding patterns was limited. Thus, a novel CRP-binding site prediction model called CRPBSFinder was developed in this research, which combined the hidden Markov model, knowledge-based PWMs and structure-based binding affinity matrixes. We trained this model using validated CRP-binding data from Escherichia coli and evaluated it with computational and experimental methods. The result shows that the model not only can provide higher prediction performance than a classic method but also quantitatively indicates the binding affinity of transcription factor binding sites by prediction scores. The prediction result included not only the most knowns regulated genes but also 1089 novel CRP-regulated genes. The major regulatory roles of CRPs were divided into four classes: carbohydrate metabolism, organic acid metabolism, nitrogen compound metabolism and cellular transport. Several novel functions were also discovered, including heterocycle metabolic and response to stimulus. Based on the functional similarity of homologous CRPs, we applied the model to 35 other species. The prediction tool and the prediction results are online and are available at: https://awi.cuhk.edu.cn/∼CRPBSFinder.

Structural Basis of Dual Specificity of Sinorhizobium meliloti Clr, a cAMP and cGMP Receptor Protein.

In bacteria, the most prevalent receptor proteins of 3',5'-cyclic AMP (cAMP) and 3',5'-cyclic GMP (cGMP) are found among transcription factors of the Crp-Fnr superfamily. The prototypic Escherichia coli catabolite activator protein (CAP) represents the main Crp cluster of this superfamily and is known to bind cAMP and cGMP but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by Sinorhizobium meliloti Clr, mapping to cluster G of Crp-like proteins. We present crystal structures of Clr-cAMP and Clr-cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr-cNMP-CBS-DNA complexes (where cNMP is cyclic nucleotide monophosphate) to almost identical active conformations, unlike the situation known for the E. coli CAP-cNMP complex. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in the presence of CBS core motif DNA (equilibrium dissociation constant for cNMP (KDcNMP], ~7 to 11 µM). However, different affinities were determined in the absence of this DNA (KDcGMP, ~24 µM; KDcAMP, ~6 µM). Sequencing of Clr-coimmunoprecipitated DNA as well as electrophoretic mobility shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases that are consistent with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr-cNMP-CBS-DNA crystal structures. IMPORTANCE Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'-GMP (cGMP) are both long known as important nucleotide secondary messengers in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main Crp cluster, binds both cyclic mononucleotides, but only the CAP-cAMP complex promotes transcription activation. In contrast, Crp cluster G proteins studied so far are activated by cGMP or by both cAMP and cGMP. Here, we report a structural analysis of the cAMP- and cGMP-activatable cluster G member Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

The cAMP receptor protein (CRP) enhances the competitive nature of Salmonella Typhimurium.

The cAMP receptor protein (CRP) is a global regulatory protein. We evaluated the role of CRP in starvation physiology in Salmonella Typhimurium. The Δcrp mutant survived 10 days of starvation. However, in a co-culture with the wild type in nutrient-rich medium, Δcrp died within 48 h. Similar co-culture results were observed with Escherichia coli and Staphylococcus aureus. Our study showed that the Δcrp mutant was not killed by toxins and the Type IV secretion system of the WT. The possibility of viable but non-culturable cells (VBNC) was also ruled out. However, when the overall metabolism of the co-culture was slowed down (anaerobic condition, inhibition by antibiotics and low temperature) that improved the survival of Δcrp in co-culture. But one more significant observation was that the Δcrp mutant survived in nutrient-free co-culture conditions. These two observations suggest that CRP protein is essential for efficient nutrient assimilation in a competitive environment. The cells without CRP protein are unable to evaluate the energy balance within the cell, and the cell spends energy to absorb nutrients. But the wild type cell absorbs nutrients at a faster rate than Δcrp mutant. This leads to a situation wherein the Δcrp is spending energy to absorb the nutrients but is unable to compete with the wild type. This futile metabolism leads to death. Hence, this study shows that CRP is a metabolism modulator in a complex nutrient environment. This study also highlights the need for innovative growth conditions to understand the unique function of a gene.

Impact of the cAMP-cAMP Receptor Protein Regulatory Complex on Lipopolysaccharide Modifications and Polymyxin B Resistance in Escherichia coli.

Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, cyaA, fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ptsI) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. IMPORTANCE Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance.

Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase.

cAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor.

The active and inactive structures of the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcription factor, are compared to generate a paradigm in the cAMP-induced activation of CRP. The resulting paradigm is shown to be consistent with numerous biochemical studies of CRP and CRP*, a group of CRP mutants displaying cAMP-free activity. The cAMP affinity of CRP is dictated by two factors: (i) the effectiveness of the cAMP pocket and (ii) the protein equilibrium of apo-CRP. How these two factors interplay in determining the cAMP affinity and cAMP specificity of CRP and CRP* mutants are discussed. Both the current understanding and knowledge gaps of CRP-DNA interactions are also described. This review ends with a list of several important CRP issues that need to be addressed in the future.

A Class IV Adenylate Cyclase, CyaB, Is Required for Capsule Polysaccharide Production and Biofilm Formation in Vibrio parahaemolyticus.

Cyclic AMP (cAMP) receptor protein (CRP), encoded by crp, is a global regulator that is activated by cAMP, a second messenger synthesized by a class I adenylate cyclase (AC-I) encoded by cyaA in Escherichia coli. cAMP-CRP is required for growth on nonpreferred carbon sources and is a global regulator. We constructed in-frame nonpolar deletions of the crp and cyaA homologs in Vibrio parahaemolyticus and found that the Δcrp mutant did not grow in minimal media supplemented with nonpreferred carbon sources, but the ΔcyaA mutant grew similarly to the wild type. Bioinformatics analysis of the V. parahaemolyticus genome identified a 181-amino-acid protein annotated as a class IV adenylate cyclase (AC-IV) named CyaB, a member of the CYTH protein superfamily. AC-IV phylogeny showed that CyaB was present in Gammaproteobacteria and Alphaproteobacteria as well as Planctomycetes and Archaea. Only the bacterial CyaB proteins contained an N-terminal motif, HFxxxxExExK, indicative of adenylyl cyclase activity. Both V. parahaemolyticus cyaA and cyaB genes functionally complemented an E. coli ΔcyaA mutant. The Δcrp and ΔcyaB ΔcyaA mutants showed defects in growth on nonpreferred carbon sources and in swimming and swarming motility, indicating that cAMP-CRP is an activator. The ΔcyaA and ΔcyaB single mutants had no defects in these phenotypes, indicating that AC-IV complements AC-I. Capsule polysaccharide and biofilm production assays showed significant defects in the Δcrp, ΔcyaBΔcyaA, and ΔcyaB mutants, whereas the ΔcyaA strain behaved similarly to the wild type. This is consistent with a role of cAMP-CRP as an activator of these phenotypes and establishes a cellular role for AC-IV in capsule and biofilm formation, which to date has been unestablished. IMPORTANCE Here, we characterized the roles of CRP and CyaA in V. parahaemolyticus, showing that cAMP-CRP is an activator of metabolism, motility, capsule production, and biofilm formation. These results are in contrast to cAMP-CRP in V. cholerae, which represses capsule and biofilm formation. Previously, only an AC-I CyaA had been identified in Vibrio species. Our data showed that an AC-IV CyaB homolog is present in V. parahaemolyticus and is required for optimal growth. The data demonstrated that CyaB is essential for capsule production and biofilm formation, uncovering a physiological role of AC-IV in bacteria. The data showed that the cyaB gene was widespread among Vibrionaceae species and several other Gammaproteobacteria, but in general, its phylogenetic distribution was limited. Our phylogenetic analysis also demonstrated that in some species the cyaB gene was acquired by horizontal gene transfer.

Acetylation of Cyclic AMP Receptor Protein by Acetyl Phosphate Modulates Mycobacterial Virulence.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen is partly attributed to its ability to sense and respond to dynamic host microenvironments. The cyclic AMP (cAMP) receptor protein (CRP) is closely related to the pathogenicity of Mtb and plays an important role in this process. However, the molecular mechanisms guiding the autoregulation and downstream target genes of CRP while Mtb responds to its environment are not fully understood. Here, it is demonstrated that the acetylation of conserved lysine 193 (K193) within the C-terminal DNA-binding domain of CRP reduces its DNA-binding ability and inhibits transcriptional activity. The reversible acetylation status of CRP K193 was shown to significantly affect mycobacterial growth phenotype, alter the stress response, and regulate the expression of biologically relevant genes using a CRP K193 site-specific mutation. Notably, the acetylation level of K193 decreases under CRP-activating conditions, including the presence of cAMP, low pH, high temperature, and oxidative stress, suggesting that microenvironmental signals can directly regulate CRP K193 acetylation. Both cell- and murine-based infection assays confirmed that CRP K193 is critical to the regulation of Mtb virulence. Furthermore, the acetylation of CRP K193 was shown to be dependent on the intracellular metabolic intermediate acetyl phosphate (AcP), and deacetylation was mediated by NAD+-dependent deacetylases. These findings indicate that AcP-mediated acetylation of CRP K193 decreases CRP activity and negatively regulates the pathogenicity of Mtb. We believe that the underlying mechanisms of cross talk between transcription, posttranslational modifications, and metabolites are a common regulatory mechanism for pathogenic bacteria. IMPORTANCE Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, and the ability of Mtb to survive harsh host conditions has been the subject of intensive research. As a result, we explored the molecular mechanisms guiding downstream target genes of CRP when Mtb responds to its environment. Our study makes a contribution to the literature because we describe the role of acetylated K193 in regulating its binding affinity to target DNA and influencing the virulence of mycobacteria. We discovered that mycobacteria can regulate their pathogenicity through the reversible acetylation of CRP K193 and that this reversible acetylation is mediated by AcP and a NAD+-dependent deacetylase. The regulation of CRPMtb by posttranslational modifications, at the transcriptional level, and by metabolic intermediates contribute to a better understanding of its role in the survival and pathogenicity of mycobacteria.

Nitration of cAMP-Response Element Binding Protein Participates in Myocardial Infarction-Induced Myocardial Fibrosis via Accelerating Transcription of Col1a2 and Cxcl12.

Aims: Myocardial fibrosis after myocardial infarction (MI) leads to heart failure. Nitration of protein can alter its function. cAMP-response element binding protein (CREB) is a key transcription factor involved in fibrosis. However, little is known about the role of nitrated CREB in MI-induced myocardial fibrosis. Meanwhile, downstream genes of transcription factor CREB in myocardial fibrosis have not been identified. This study aims to verify the hypothesis that nitrated CREB promotes MI-induced myocardial fibrosis via regulating the transcription of Col1a2 and Cxcl12. Results: Our study showed that (1) the level of nitrative stress was elevated and nitrated CREB was higher in the myocardium after MI. Tyr182, 307, and 336 were the nitration sites of CREB; (2) with the administration of peroxynitrite (ONOO-) scavengers, CREB phosphorylation, nuclear translocation, and binding activity to TORC2 (transducers of regulated CREB-2) were attenuated; (3) the expressions of extracellular matrix (ECM) proteins were upregulated and downregulated in accordance with the expression alteration of CREB both in vitro and in vivo; (4) CREB accelerated transcription of Col1a2 and Cxcl12 after MI directly. With the administration of ONOO- scavengers, ECM protein expressions were attenuated; meanwhile, the messenger RNA (mRNA) levels of Col1a2 and Cxcl12 were alleviated as well. Innovation and Conclusion: Nitration of transcription factor CREB participates in MI-induced myocardial fibrosis through enhancing its phosphorylation, nuclear translocation, and binding activity to TORCs, among which CREB transcripts Col1a2 and Cxcl12 directly. These data indicated that nitrated CREB might be a potential therapeutic target against MI-induced myocardial fibrosis. Antioxid. Redox Signal. 38, 709-730.

Sequestration of a dual function DNA-binding protein by Vibrio cholerae CRP.

Although the mechanism by which the cyclic AMP receptor protein (CRP) regulates global gene transcription has been intensively studied for decades, new discoveries remain to be made. Here, we report that, during rapid growth, CRP associates with both the well-conserved, dual-function DNA-binding protein peptidase A (PepA) and the cell membrane. These interactions are not present under nutrient-limited growth conditions, due to post-translational modification of three lysines on a single face of CRP. Although coincident DNA binding is rare, dissociation from CRP results in increased PepA occupancy at many chromosomal binding sites and differential regulation of hundreds of genes, including several encoding cyclic dinucleotide phosphodiesterases. We show that PepA represses biofilm formation and activates motility/chemotaxis. We propose a model in which membrane-bound CRP interferes with PepA DNA binding. Under nutrient limitation, PepA is released. Together, CRP and free PepA activate a transcriptional response that impels the bacterium to seek a more hospitable environment. This work uncovers a function for CRP in the sequestration of a regulatory protein. More broadly, it describes a paradigm of bacterial transcriptome modulation through metabolically regulated association of transcription factors with the cell membrane.

Negative regulation of the acsA1 gene encoding the major acetyl-CoA synthetase by cAMP receptor protein in Mycobacterium smegmatis.

Acetyl-CoA synthetase (ACS) is the enzyme that irreversibly catalyzes the synthesis of acetyl-CoA from acetate, CoA-SH, and ATP via acetyl-AMP as an intermediate. In this study, we demonstrated that AcsA1 (MSMEG_6179) is the predominantly expressed ACS among four ACSs (MSMEG_6179, MSMEG_0718, MSMEG_3986, and MSMEG_5650) found in Mycobacterium smegmatis and that a deletion mutation of acsA1 in M. smegmatis led to its compromised growth on acetate as the sole carbon source. Expression of acsA1 was demonstrated to be induced during growth on acetate as the sole carbon source. The acsA1 gene was shown to be negatively regulated by Crp1 (MSMEG_6189) that is the major cAMP receptor protein (CRP) in M. smegmatis. Using DNase I footprinting analysis and site-directed mutagenesis, a CRP-binding site (GGTGA-N6-TCACA) was identified in the upstream regulatory region of acsA1, which is important for repression of acsA1 expression. We also demonstrated that inhibition of the respiratory electron transport chain by inactivation of the major terminal oxidase, aa3 cytochrome c oxidase, led to a decrease in acsA1 expression probably through the activation of CRP. In conclusion, AcsA1 is the major ACS in M. smegmatis and its gene is under the negative regulation of Crp1, which contributes to some extent to the induction of acsA1 expression under acetate conditions. The growth of M. smegmatis is severely impaired on acetate as the sole carbon source under respiration-inhibitory conditions.

RedB, a Member of the CRP/FNR Family, Functions as a Transcriptional Redox Brake.

Phylogenetic and sequence similarity network analyses of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family of transcription factors indicate the presence of numerous subgroups, many of which have not been analyzed. Five homologs of the CRP/FNR family are present in the Rhodobacter capsulatus genome. One is a member of a broadly disseminated, previously uncharacterized CRP/FNR family subgroup encoded by the gene rcc01561. In this study, we utilize mutational disruption, transcriptome sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the role of RCC01561 in regulating R. capsulatus physiology. This analysis shows that a mutant strain disrupted for rcc01561 exhibits altered expression of 451 genes anaerobically. A detailed analysis of the affected loci shows that RCC01561 represses photosynthesis and favors catabolism over anabolism and the use of the Entner-Doudoroff shunt and glycolysis over that of the tricarboxylic acid (TCA) cycle to limit NADH and ATP formation. This newly characterized CRP/FNR family member with a predominant role in reducing the production of reducing potential and ATP is given the nomenclature RedB as it functions as an energy and redox brake. Beyond limiting energy production, RedB also represses the expression of numerous genes involved in protein synthesis, including those involved in translation initiation, tRNA synthesis and charging, and amino acid biosynthesis. IMPORTANCE CRP and FNR are well-characterized members of the CRP/FNR family of regulatory proteins that function to maximize cellular energy production. In this study, we identify several new subgroups of the CRP/FNR family, many of which have not yet been characterized. Using Rhodobacter capsulatus as a model, we have mutationally disrupted the gene rcc01561, which codes for a transcription factor that is a member of a unique subgroup of the CRP/FNR family. Transcriptomic analysis shows that the disruption of rcc01561 leads to the altered expression of 451 genes anaerobically. Analysis of these regulated genes indicates that RCC01561 has a novel role in limiting cellular energy production. To our knowledge, this is first example of a member of the CRP/FNR family that functions as a brake on cellular energy production.

Redox Brake Regulator RedB and FnrL Function as Yin-Yang Regulators of Anaerobic-Aerobic Metabolism in Rhodobacter capsulatus.

We recently described a new member of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family called RedB, an acronym for redox brake, that functions to limit the production of ATP and NADH. This study shows that the RedB regulon significantly overlaps the FnrL regulon, with 199 genes being either directly or indirectly regulated by both of these global regulatory proteins. Among these 199 coregulated genes, 192 are divergently regulated, indicating that RedB functions as an antagonist of FnrL. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicates that RedB and Fnr directly coregulate only 4 out of 199 genes. The primary mechanism for the divergent regulation of target genes thus involves indirect regulation by both RedB and FnrL (156 cases). Additional regulation involves direct binding by RedB and indirect regulation by FnrL (36 cases) or direct binding by FnrL and indirect regulation by RedB (3 cases). Analysis of physiological pathways under direct and indirect control by these global regulators demonstrates that RedB functions primarily to limit energy production, while FnrL functions to enhance energy production. This regulation includes glycolysis, gluconeogenesis, photosynthesis, hydrogen oxidation, electron transport, carbon fixation, lipid biosynthesis, and protein synthesis. Finally, we show that 75% of genomes from diverse species that code for RedB proteins also harbor genes coding for FNR homologs. This cooccurrence indicates that RedB likely has an important role in buffering FNR-mediated energy production in a broad range of species. IMPORTANCE The CRP/FNR family of regulatory proteins constitutes a large collection of related transcription factors, several of which globally regulate cellular energy production. A well-characterized example is FNR (called FnrL in Rhodobacter capsulatus), which is responsible for regulating the expression of numerous genes that promote maximal energy production and growth under anaerobic conditions. In a companion article (N. Ke, J. E. Kumka, M. Fang, B. Weaver, et al., Microbiol Spectr 10:e02353-22, 2022, https://doi.org/10.1128/Spectrum02353-22), we identified a new subgroup of the CRP/FNR family and demonstrated that a member of this new subgroup, called RedB, has a role in limiting cellular energy production. In this study, we show that numerous genes encompassing the RedB regulon significantly overlap genes that are members of the FnrL regulon. Furthermore, 97% of the genes that are members of both the RedB and FnrL regulons are divergently regulated by these two transcription factors. RedB thus functions as a buffer limiting the amount of energy production that is promoted by FnrL.