Transcriptional effects of CRP* expression in Escherichia coli

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native <it>crp </it>gene with a catabolite repression mutant (referred to as <it>crp</it>*) enables co-utilization of glucose and other sugars in <it>E. coli</it>. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of <it>E. coli </it>W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.</p> <p>Results</p> <p>The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.</p> <p>We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP⁺ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.</p> <p>Conclusion</p> <p>While the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of <it>crp* </it>strains that extend beyond relief of catabolite repression.</p

Single-cell characterization of autotransporter mediated Escherichia coli surface display of disulfide-bond containing proteins

Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal ?-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal ?-barrel and the threaded ?-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering

Functional enrichment by direct plasmid recovery after Fluorescence Activated Cell Sorting

Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) typically involves culturing the pooled clones after each sort. In these experiments, if cell viability is compromised by the sort conditions and/or expression of the target protein(s), rescue PCR provides an alternative to culturing but requires re-cloning and can introduce amplification bias. We haveoptimized a simple protocol using commercially available reagents to directly recover plasmid DNA from sorted cells for subsequenttransformation. We tested our protocol with 2 different screening systems in which 60% of the sorted cell population was recovered

The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2′3’ - cyclic Guanosine Monophosphate–Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate–Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with &lt;20 EU/mL (&lt;0.3 EU/μg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes

Novel functions and regulation of cryptic cellobiose operons in Escherichia coli

Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (beta-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolismopen1

Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197-203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197-203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197-203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production

Measurement of the Forward-Backward Asymmetry in the B -> K(*) mu+ mu- Decay and First Observation of the Bs -> phi mu+ mu- Decay

We reconstruct the rare decays $B^+ \to K^+\mu^+\mu^-$, $B^0 \to K^{*}(892)^0\mu^+\mu^-$, and $B^0_s \to \phi(1020)\mu^+\mu^-$ in a data sample corresponding to $4.4 {\rm fb^{-1}}$ collected in $p\bar{p}$ collisions at $\sqrt{s}=1.96 {\rm TeV}$ by the CDF II detector at the Fermilab Tevatron Collider. Using $121 \pm 16$ $B^+ \to K^+\mu^+\mu^-$ and $101 \pm 12$ $B^0 \to K^{*0}\mu^+\mu^-$ decays we report the branching ratios. In addition, we report the measurement of the differential branching ratio and the muon forward-backward asymmetry in the $B^+$ and $B^0$ decay modes, and the $K^{*0}$ longitudinal polarization in the $B^0$ decay mode with respect to the squared dimuon mass. These are consistent with the theoretical prediction from the standard model, and most recent determinations from other experiments and of comparable accuracy. We also report the first observation of the $B^0_s \to \phi\mu^+\mu^- decay and measure its branching ratio${\mathcal{B}}(B^0_s \to \phi\mu^+\mu^-) = [1.44 \pm 0.33 \pm 0.46] \times 10^{-6}$using$27 \pm 6$signal events. This is currently the most rare$B^0_s$ decay observed.Comment: 7 pages, 2 figures, 3 tables. Submitted to Phys. Rev. Let

Measurements of the properties of Lambda_c(2595), Lambda_c(2625), Sigma_c(2455), and Sigma_c(2520) baryons

We report measurements of the resonance properties of Lambda_c(2595)+ and Lambda_c(2625)+ baryons in their decays to Lambda_c+ pi+ pi- as well as Sigma_c(2455)++,0 and Sigma_c(2520)++,0 baryons in their decays to Lambda_c+ pi+/- final states. These measurements are performed using data corresponding to 5.2/fb of integrated luminosity from ppbar collisions at sqrt(s) = 1.96 TeV, collected with the CDF II detector at the Fermilab Tevatron. Exploiting the largest available charmed baryon sample, we measure masses and decay widths with uncertainties comparable to the world averages for Sigma_c states, and significantly smaller uncertainties than the world averages for excited Lambda_c+ states.Comment: added one reference and one table, changed order of figures, 17 pages, 15 figure

Search for a New Heavy Gauge Boson Wprime with Electron + missing ET Event Signature in ppbar collisions at sqrt(s)=1.96 TeV

We present a search for a new heavy charged vector boson $W^\prime$ decaying to an electron-neutrino pair in $p\bar{p}$ collisions at a center-of-mass energy of 1.96\unit{TeV}. The data were collected with the CDF II detector and correspond to an integrated luminosity of 5.3\unit{fb}^{-1}. No significant excess above the standard model expectation is observed and we set upper limits on $\sigma\cdot{\cal B}(W^\prime\to e\nu)$. Assuming standard model couplings to fermions and the neutrino from the $W^\prime$ boson decay to be light, we exclude a $W^\prime$ boson with mass less than 1.12\unit{TeV/}c^2 at the 95\unit{%} confidence level.Comment: 7 pages, 2 figures Submitted to PR