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.

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.

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.

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.

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.

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.

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.

A mutation in the putative CRP binding site of the dctA promoter of Salmonella enterica serovar Typhimurium enables growth with low orotate concentrations.

Salmonella enterica and Escherichia coli use the inner membrane transporter DctA to import the pyrimidine biosynthetic pathway intermediate orotate from the environment. To study the regulation of dctA expression, we used an S. enterica serovar Typhimurium pyrimidine auxotroph to select a mutant that could grow in an otherwise nonpermissive culture medium containing glucose and a low concentration of orotate. Whole genome sequencing revealed a point mutation upstream of dctA in the putative cyclic AMP receptor protein (CRP) binding site. The C→T transition converted the least favourable base to the most favourable base for CRP-DNA affinity. A dctA::lux transcriptional fusion confirmed that the mutant dctA promoter gained responsiveness to CRP even in the presence of glucose. Moreover, dctA expression was higher in the mutant than the wild type in the presence of alternative carbon sources that activate CRP.

Plasticity of Promoter-Core Sequences Allows Bacteria to Compensate for the Loss of a Key Global Regulatory Gene.

Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.

Global Regulator of Rubber Degradation in Gordonia polyisoprenivorans VH2: Identification and Involvement in the Regulation Network.

A cAMP receptor protein (CRPVH2) was detected as a global regulator in Gordonia polyisoprenivorans VH2 and was proposed to participate in the network regulating poly(cis-1,4-isoprene) degradation as a novel key regulator. CRPVH2 shares a sequence identity of 79% with GlxR, a well-studied global regulator of Corynebacterium glutamicum Furthermore, CRPVH2 and GlxR have a common oligomerization state and similar binding motifs, and thus most likely have similar functions as global regulators. Size exclusion chromatography of purified CRPVH2 confirmed the existence as a homodimer with a native molecular weight of 44.1 kDa in the presence of cAMP. CRPVH2 bound to the TGTGAN6TCACT motif within the 131-bp intergenic region of divergently oriented lcp1VH2 and lcpRVH2, encoding a latex clearing protein and its putative repressor, respectively. DNase I footprinting assays revealed the exact operator size of CRPVH2 in the intergenic region (25 bp), which partly overlapped with the proposed promoters of lcpRVH2 and lcp1VH2 Our findings indicate that CRPVH2 represses the expression of lcpRVH2 while simultaneously directly or indirectly activating the expression of lcp1VH2 by binding the competing promoter regions. Furthermore, binding of CRPVH2 to upstream regions of additional putative enzymes of poly(cis-1,4-isoprene) degradation was verified in vitro. In silico analyses predicted 206 CRPVH2 binding sites comprising 244 genes associated with several functional categories, including carbon and peptide metabolism, stress response, etc. The gene expression regulation of several subordinated regulators substantiated the function of CRPVH2 as a global regulator. Moreover, we anticipate that the novel lcpR regulation mechanism by CRPs is widespread in other rubber-degrading actinomycetes.IMPORTANCE In order to develop efficient microbial recycling strategies for rubber waste materials, it is required that we understand the degradation pathway of the polymer and how it is regulated. However, only little is known about the transcriptional regulation of the rubber degradation pathway, which seems to be upregulated in the presence of the polymer. We identified a novel key regulator of rubber degradation (CRPVH2) that regulates several parts of the pathway in the potent rubber-degrader G. polyisoprenivorans VH2. Furthermore, we provide evidence for a widespread involvement of CRP regulators in the degradation of rubber in various other rubber-degrading actinomycetes. Thus, these novel insights into the regulation of rubber degradation are essential for developing efficient microbial degradation strategies for rubber waste materials by this group of actinomycetes.

Role of cAMP receptor protein in phenotype and stress tolerance in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is exposed to biotic and abiotic stresses. The survival of Salmonella in nature depends on the global regulators like cAMP receptor protein (CRP). The role of CRP in the phenotypic characteristics and stress tolerance was elucidated in S. Typhimurium using a crp gene null mutant (Δcrp). A 1.6-fold decrease in the cell size, a two-fold reduction in the colony size, and a 3.5-fold decrease in motility were observed in the Δcrp compared with the S. Typhimurium wild-type (WT). H2 S production on selective media was affected in the Δcrp. The utilisation of d-mannose, d-glycerol and d-mannitol was completely affected, whereas that of d-galactose and d-fructose was partially affected. The utilisation of d-arabinose was induced in the Δcrp. The growth rate of the Δcrp in Luria Bertani medium was unaffected. However, in the glucose-containing minimal medium, the growth rate of the Δcrp was reduced by 1.5-fold compared with the WT. The Δcrp was able to utilise ethanolamine as the sole carbon source similar to the WT. The Δcrp was more tolerant to heat and oxidative stress. Overexpression of heat and oxidative stress-related genes was observed in the Δcrp in the stationary phase. The Δcrp was less tolerant to radiation stress compared with the WT. The current findings decisively establish the CRP protein as a global regulator. The CRP affects multiple phenotypes, carbon metabolism and stress physiology of S. Typhimurium.

Tripartite Regulation of the glpFKD Operon Involved in Glycerol Catabolism by GylR, Crp, and SigF in Mycobacterium smegmatis.

The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism.IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.

Serotype-shifting gene rfbT is a direct transcriptional target of cAMP receptor protein (CRP) in V. cholerae O1.

Ogawa and Inaba are two main serotypes of O1 V. cholerae and alternate among cholera epidemics. The rfbT gene encodes a methyltransferase and is required for Ogawa serotype. The Inaba serotype is the consequence of genetic alterations in rfbT gene which results in loss-of-function enzyme product. However, the expression and regulation of rfbT has not been understood yet. Here we demonstrated that a global regulator, cAMP receptor protein (CRP), positively regulates rfbT transcription through a non-canonical CRP binding site (CBS) in its promoter region. This finding is supported by the analyses of rfbT mRNA abundance, rfbT-lacZ fusions and electrophoretic mobility shift assay (EMSA). The analyses of rfbT mRNA level in wild type (WT), Δcrp, and lower or higher level of cAMP backgrounds revealed that CRP is required for rfbT expression in response to intracellular cAMP level. Subsequent rfbT-lacZ fusions and EMSA collectively displayed that cAMP-CRP complex transcriptionally activates rfbT through directly binding to CBS in rfbT promoter region. Consistently, serological microagglutination test showed that crp deletion resulted in at least 4-fold decrease in titer of Ogawa serum compared to its WT. These results expanded our knowledge of understanding the genetic determinants and probable regulatory mechanism of V. cholerae O1 serotype shift between Ogawa and Inaba.

Sycrp2 Is Essential for Twitching Motility in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

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.

Escherichia coli OxyS RNA triggers cephalothin resistance by modulating the expression of CRP-associated genes.

Antibiotics have been one of the most successful forms of therapy in medicine. However, the efficiency of antibiotics is compromised by the emergence of antibiotic-resistant pathogens. To reduce antibiotic resistance, complete understanding of bacterial tactics to defend themselves against antibiotics is necessary. Small-noncoding RNAs (sRNAs) modulate gene expression by base-pairing with multiple target mRNAs. Cellular levels of Hfq-dependent sRNAs influence antibiotic resistance by modulating expression of specific target genes; therefore, such sRNAs could be a good tool to identify target mRNAs that modulate antibiotic susceptibility and may themselves be used as druggable molecules. Here, we report the identification of genes and pathways associated with OxyS RNA-mediated cephalothin resistance using phenotypic and expression analyses of OxyS-regulated genes identified by RNA-seq, literature mining, or predictions. From our studies we found that the differential expression of 27 OxyS-regulated genes was involved in cephalothin susceptibility. Among them, 17 gene knockouts showed resistance to the drug and nine from them is associated with cAMP receptor protein (CRP), a transcriptional dual regulator in E. coli. Moreover, levels of OxyS and OxyS-modulated genes (cycA and cysH) were also altered in multidrug-resistant (MDR) E. coli strains. Together, our data suggest that OxyS extensively modulates gene expression in multiple pathways to develop cephalothin resistance. In addition, OxyS and its regulated target genes, either individually or in combination, could be used as molecular markers and targets for the identification and eradication of cephalothin-resistant strains.

Engineering E. coli for simultaneous glucose-xylose utilization during methyl ketone production.

BACKGROUND: We previously developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. RESULTS: In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ketone-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. CONCLUSIONS: This work demonstrated a strategy for engineering simultaneous utilization of C6 and C5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.

CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1.

Cyclic 3',5'-adenosine monophosphate (cAMP) phosphodiesterase (CPD) is an enzyme that catalyzes the hydrolysis of cAMP, a signaling molecule affecting diverse cellular and metabolic processes in bacteria. Some CPDs are also known to function in cAMP-independent manners, while their physiological roles remain largely unknown. Here, we investigated physiological roles of CPD in Shewanella oneidensis MR-1, a model environmental bacterium, and report that CPD is involved in amino-acid metabolism. We found that a CPD-deficient mutant of MR-1 (ΔcpdA) showed decreased expression of genes for the synthesis of methionine, S-adenosylmethionine, and histidine and required these three compounds to grow in minimal media. Interestingly, deletion of adenylate cyclases in ΔcpdA did not restore the ability to grow in minimal media, indicating that the amino acid requirements were not due to the accumulation of cAMP. These results suggest that CPD is involved in the regulation of amino acid metabolism in MR-1 in a cAMP-independent manner.

Large-Scale Functional Analysis of CRP-Mediated Feed-Forward Loops.

Feed-forward loops (FFLs) represent an important and basic network motif to understand specific biological functions. Cyclic-AMP (cAMP) receptor protein (CRP), a transcription factor (TF), mediates catabolite repression and regulates more than 400 genes in response to changes in intracellular concentrations of cAMP in Escherichia coli. CRP participates in some FFLs, such as araBAD and araFGH operons and adapts to fluctuating environmental nutrients, thereby enhancing the survivability of E. coli. Although computational simulations have been conducted to explore the potential functionality of FFLs, a comprehensive study on the functions of all structural types on the basis of in vivo data is lacking. Moreover, the regulatory role of CRP-mediated FFLs (CRP-FFLs) remains obscure. We identified 393 CRP-FFLs in E. coli using EcoCyc and RegulonDB. Dose⁻response genomic microarray of E. coli revealed dynamic gene expression of each target gene of CRP-FFLs in response to a range of cAMP dosages. All eight types of FFLs were present in CRP regulon with various expression patterns of each CRP-FFL, which were further divided into five functional groups. The microarray and reported regulatory relationships identified 202 CRP-FFLs that were directly regulated by CRP in these eight types of FFLs. Interestingly, 34% (147/432) of genes were directly regulated by CRP and CRP-regulated TFs, which indicates that these CRP-regulated genes were also regulated by other CRP-regulated TFs responding to environmental signals through CRP-FFLs. Furthermore, we applied gene ontology annotation to reveal the biological functions of CRP-FFLs.

Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.