High-level Constitutive Expression Research Articles

Biochemical and Cellular Characterization of Helicobacter pylori RecA, a Protein with High-Level Constitutive Expression

Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonization. The biochemical characterization of the purified recombinase reveals activities similar to those of Escherichia coli RecA (EcRecA). We show that in H. pylori, HpRecA is present in about 80,000 copies per cell during exponential growth and decreases to about 50,000 copies in stationary phase. The amount of HpRecA remains unchanged after induction of DNA lesions, suggesting that HpRecA is always expressed at a high level in order to repair DNA damage or facilitate recombination. We performed HpRecA localization analysis by adding a Flag tag to the protein, revealing two different patterns of localization. During exponential growth, RecA-Flag presents a diffuse pattern, overlapping with the DAPI (4',6-diamidino-2-phenylindole) staining of DNA, whereas during stationary phase, the protein is present in more defined areas devoid of DAPI staining. These localizations are not affected by inactivation of competence or DNA recombination genes. Neither UV irradiation nor gamma irradiation modified HpRecA localization, suggesting the existence of a constitutive DNA damage adaptation system.

Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

BackgroundDesigning functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy.Methodology/Principal FindingsWe have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared.Conclusion and SignificanceWe propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells.

Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor-driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12-induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

Activation of NF-κB by TMPRSS2/ERG Fusion Isoforms through Toll-Like Receptor-4

The TMPRSS2/ERG (T/E) fusion gene is present and thought to be an oncogenic driver of approximately half of all prostate cancers. Fusion of the androgen-regulated TMPRSS2 promoter to the ERG oncogene results in constitutive high level expression of ERG which promotes prostate cancer invasion and proliferation. Here, we report the characterization of multiple alternatively spliced T/E fusion gene isoforms which have differential effects on invasion and proliferation. We found that T/E fusion gene isoforms differentially increase NF-κB-mediated transcription, which may explain in part the differences in biological activities of the T/E fusion isoforms. This increased activity is due to phosphorylation of NF-κB p65 on Ser536. Tissue microarray immunochemistry revealed that p65 phospho-Ser536 is present in the majority of prostate cancers where it is associated with ERG protein expression. The T/E fusion gene isoforms differentially increase expression of a number of NF-κB associated genes including PAR1, CCL2, FOS, TLR3, and TLR4 (Toll-like receptor). TLR4 activation is known to promote p65 Ser536 phosphorylation and knockdown of TLR4 with shRNA decreases Ser536 phosphorylation in T/E fusion gene expressing cells. TLR4 can be activated by proteins in the tumor microenvironment and lipopolysacharide from Gram (-) bacteria. Our findings suggest that bacterial infection of the prostate and/or endogenous microenvironment proteins may promote progression of high-grade prostatic intraepithelial neoplasia and/or prostate cancers that express the T/E fusion gene, where the NF-κB pathway might be targeted as a rational therapeutic approach.

Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels of constitutive expression of the NT/N gene. In this study, we demonstrated multiple cis-regulatory elements including a proximal region containing a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein (CREB)/ATF proteins (c-Jun, ATF-1, ATF-2, JunD, and CREB). Similar to the rat NT/N gene, this region is critical for constitutive hNT/N gene expression. Moreover, we identified a novel region that binds the orphan hormone receptor, NR2F2. We have demonstrated that the C terminus of NR2F2 strongly represses hNT/N transcription, whereas an N-terminal domain antagonizes this repressive effect. Regulation of NT/N expression by NR2F2 may have important consequences for lipid metabolism. We speculate that a complex interplay between the proximal CRE/AP-1-like motif and NR2F2 binding region exists to regulate hNT/N expression, which is critical for the high level of constitutive expression of NT/N in enteroendocrine cells. Finally, the BON cell line provides a unique model to characterize the factors regulating expression of the hNT/N gene and to better understand the mechanisms responsible for terminal differentiation of the N cell lineage in the gut.

DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

BackgroundWhen DNA double-strand breaks (DSB) are induced by ionizing radiation (IR) in cells, histone H2AX is quickly phosphorylated into γ-H2AX (p-S139) around the DSB site. The necessity of DNA-PKcs in regulating the phosphorylation of H2AX in response to DNA damage and cell cycle progression was investigated.ResultsThe level of γH2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy γ irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of γH2AX. An increased γH2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy γ rays, and this IR-increased γH2AX in ATM deficient cells was dramatically abolished by the PIKK inhibitor wortmannin and the DNA-PKcs specific inhibitor NU7026. A high level of constitutive expression of γH2AX was observed in another ATM deficient cell line ATS4. The alteration of γH2AX level associated with cell cycle progression was also observed. HeLa cells with siRNA-depressed DNA-PKcs (HeLa-H1) or normal level DNA-PKcs (HeLa-NC) were synchronized at the G1 phase with the thymidine double-blocking method. At ~5 h after the synchronized cells were released from the G1 block, the S phase cells were dominant (80%) for both HeLa-H1 and HeLa-NC cells. At 8 - 9 h after the synchronized cells released from the G1 block, the proportion of G2/M population reached 56 - 60% for HeLa-NC cells, which was higher than that for HeLa H1 cells (33 - 40%). Consistently, the proportion of S phase for HeLa-NC cells decreased to ~15%; while a higher level (26 - 33%) was still maintained for the DNA-PKcs depleted HeLa-H1 cells during this period. In HeLa-NC cells, the γH2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this γH2AX alteration associated with cell cycle progressing was remarkably suppressed in the DNA-PKcs depleted HeLa-H1 cells, while wortmannin and NU7026 could also suppress this cell cycle related phosphorylation of H2AX. Furthermore, inhibition of GSK3β activity with LiCl or specific siRNA could up-regulate the γH2AX level and prolong the time of increased γH2AX to 10 h or more after 4 Gy. GSK3β is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivation. Depression of DNA-PKcs in HeLa cells leads to a decreased phosphorylation of Akt on Ser473 and its target GSK3β on Ser9, which, in other words, results in an increased activation of GSK3β. In addition, inhibition of PDK (another up-stream regulator of Akt/GSK3β) by siRNA can also decrease the induction of γH2AX in response to both DNA damage and cell cycle progression.ConclusionDNA-PKcs plays a dominant role in regulating the phosphorylation of H2AX in response to both DNA damage and cell cycle progression. It can directly phosphorylate H2AX independent of ATM and indirectly modulate the phosphorylation level of γH2AX via the Akt/GSK3 β signal pathway.

Cardiac progenitor-stromal cell interaction: an important player in myocardial development

There is growing support to the notion that adult mammalian heart harbours a pool of resident cardiac stem cells (CSCs). This discovery has sparked new optimism in the field of cardiac regenerative medicine based on the idea that stimulating the myocardial endogenous repair may hold greater promises than reconstituting the post-infarcted heart by transplanted exogenous stem cells. However, before considering endogenous CSCs as a target of therapeutic options in the heart failure, we need to better understand the complex mechanisms controlling heart morphogenesis, paying attention to the cellular and molecular interactions that cardiomyocyte precursors establish with other cells of the stromal compartment. On these basis, in the present study we co-cultured mouse neonatal cardiomyocytes with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte development in vitro. We found that proliferation of cardiac precursors was dramatically enhanced by direct co-culture with MSC as compared with the single culture system, as judged by time lapse video-microscopy and cyclin A expression/nuclear translocation. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch1 receptor by its ligand Jagged 1 and that MSCs sustained this pathway through the constitutive expression of high levels of Jagged 1. This was supported by the observations that cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notchintracellular domain (Notch-ICD) as compared to those in single culture. In conclusion, these findings reveal a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation and development and suggest that stromal-cardiac muscle cell interactions, beside being crucial for correct myocardial morphogenesis, may represent an interesting target for innovative cell therapy strategies in cardiac regeneration.

CXCR2 Signals Act in Concert with CXCR4 to Regulate Neutrophil Release From the Bone Marrow.

Abstract 235Truncation mutations of CXCR4 that cause increased receptor signaling are responsible for most cases of WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, which is characterized by the retention of mature neutrophils in the bone marrow despite peripheral neutropenia. This observation and others have established CXCR4 as a key regulator of neutrophil release from the bone marrow. However, it is unclear how modulation of neutrophil CXCR4 signaling is linked to their migration toward the vascular endothelium and subsequent entry into the circulation. Therefore, the question of whether neutrophil egress from the bone marrow is a passive, random process or actively directed and what (if any) signals regulate it remains unanswered. We recently analyzed a myelokathexis pedigree and discovered homozygous, loss-of-function mutations in CXCR2. Based on these observations, we developed a “tug-of-war” model in which opposing chemokine gradients, specifically release-inducing CXCR2 signals and retention-promoting CXCR4 signals, act antagonistically to regulate neutrophil release from the bone marrow. To test this model, we analyzed neutrophil trafficking in CXCR2−/− mice. These mice have a well-characterized defect in neutrophil emigration from the blood to sites of inflammation, leading to chronic subclinical infection and the systemic release of cytokines that stimulate granulopoiesis. To circumvent these potentially confounding effects, we generated mixed bone marrow chimeras reconstituted with a 1:1 ratio of wild-type and CXCR2−/− bone marrow. As expected, approximately 50% (46 ± 4%) of circulating B cells were derived from CXCR2−/− cells. In contrast, a significant decrease in the proportion of CXCR2−/− neutrophils in the blood was observed (23 ± 4%; P < 0.001). Consistent with a myelokathexis phenotype, there was a relative accumulation of mature CXCR2−/− neutrophils in the bone marrow (47 ± 9% of Gr-1hi SSChi cells in the bone marrow were derived from CXCR2−/− cells; P < 0.01). Neutrophil trafficking from the bone marrow was estimated by calculating the percentage of neutrophils in the blood out of the total amount in the blood and bone marrow (neutrophil distribution index or NDI). We estimated that 1.3 ± 0.2% of wild-type neutrophils were in the blood, but the percentage of CXCR2−/− neutrophils in the blood was reduced to 0.4 ± 0.1% (P < 0.01). These data provide genetic evidence that CXCR2 signals promote neutrophil release from the bone marrow in a cell-autonomous manner.To explore the epistatic relationship of CXCR2 and CXCR4 signals to neutrophil trafficking, the neutrophil response to AMD3100, a small molecule CXCR4 antagonist, was examined in the CXCR2−/− mixed chimeras. Consistent with previous reports, treatment with AMD3100 resulted in a 5.2 ± 0.7-fold increase in wild-type neutrophils in the blood one hour after administration. In contrast, AMD3100 induced release of CXCR2−/− neutrophils was impaired, with only a 2.8 ± 0.3-fold increase observed (P < 0.05). G-CSF treatment is thought to induce neutrophil release through disruption of CXCR4 signaling. Thus, we next characterized the neutrophil response to 5 days of G-CSF treatment. Wild-type neutrophils displayed a shift from the bone marrow to the blood, with an NDI of 5.0 ± 0.4%. The number of CXCR2−/−neutrophils in the blood increased after treatment, but the percentage in the blood (2.5 ± 0.7%) was less than wild-type (P < 0.05). These data show that maximal neutrophil release requires the coordinated regulation of CXCR2 and CXCR4 signals. Studies are underway to assess neutrophil trafficking of CXCR4−/− × CXCR2−/− neutrophils.The tug-of-war model of neutrophil trafficking in the bone marrow predicts that CXCR2 ligands will be highly expressed in bone marrow endothelial cells or other cells closely associated with the endothelium. To test this prediction, endothelial cells (CD45− Ter119− CD31+) were sorted from the bone marrow of wild-type mice at baseline or after 5 days of G-CSF treatment. RNA expression profiling showed constitutive high level expression of the CXCR2 ligands CXCL1 and CXCL2. Moreover, expression of CXCL2 was significantly induced after G-CSF treatment. Chemokine expression was confirmed by real time RT-PCR and ELISA. Taken together, our data suggest that CXCR2 signaling is a second chemokine pathway that, in coordination with CXCR4, controls neutrophil release from the bone marrow. Disclosures:No relevant conflicts of interest to declare.

Recombinant dengue virus-like particles from Pichia pastoris: efficient production and immunological properties

The envelope glycoprotein (E) of flavivirus is the major structural protein on the surface of the mature virions. The complexes of premembrane (prM) and E play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. In the present study, the cDNA encoding prM and E proteins of dengue virus type 2 (DENV-2) was subcloned into the pGAPZalphaA vector and further integrated into the genome of Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The high-level constitutive expression of recombinant E antigen was achieved in P. pastoris. Both the cell lysate and the culture supernatant, examined by electron microscopy, were found to contain DENV-2 virus-like particles (VLPs) with diameters of about 30 nm. After immunization of BALB/c mice, the VLPs exhibited similar efficacies as inactivated virus in terms of antibody induction and neutralization titer. These results suggest that recombinant DENV VLPs can be efficiently produced in the GAP promoter-based P. pastoris expression system. This system may be useful for the development of effective and economic dengue subunit vaccine.

A role for Bach1 and HO-2 in suppression of basal and UVA-induced HO-1 expression in human keratinocytes

Ultraviolet A (UVA) radiation is an oxidizing agent that strongly induces the heme oxygenase 1 (HO-1) gene and expression of the protein in cultured human skin fibroblasts but weakly induces it in skin keratinocytes. Lower basal levels of HO-1 and much higher basal levels of HO-2 protein are observed in keratinocytes compared with fibroblasts. Using both overexpression and knockdown approaches, we demonstrate that HO-2 modulates basal and UVA-induced HO-1 protein levels, whereas HO-1 levels do not affect HO-2 levels in skin fibroblasts and keratinocytes. Silencing of Bach1 strongly increases HO-1 levels in transformed HaCaT keratinocytes and these HO-1 levels are not further increased by either UVA irradiation or silencing of HO-2. This is consistent with the conclusion that high constitutive levels of HO-2 expression in keratinocytes are responsible for the resistance of these cells to HO-1 induction by UVA radiation and that Bach1 plays a predominant role in influencing the lack of HO-1 expression in keratinocytes. Bach1 inhibition leading to HO-1 induction reduced UVA-irradiation-induced damage as monitored both by the extent of LDH release and by nuclear condensation, so that Bach1 inhibition seems to protect against UVA-irradiation-induced damage in keratinocytes.

Increased expression of Mg 2+ transport proteins enhances the survival of Salmonella enterica at high temperature

Mg(2+) homeostasis is important for Salmonella pathogenesis. In Salmonella enterica, the transcription of the mgtA gene, which encodes a Mg(2+) transporter, is regulated by a Mg(2+)-sensing riboswitch [Cromie MJ, Shi Y, Latifi T, Groisman EA (2006) Cell 125:71-84]. In a genetic analysis of the determinants of thermotolerance in S. enterica serovar Typhimurium, we isolated the chr-1 mutation that increased the resistance of exponential phase cells to killing by high temperature. This mutation is a single base change in the mgtA riboswitch that causes high-level constitutive expression of mgtA. We showed that another mgtA riboswitch mutation, DeltaUTR(re-100), which had been constructed by Cromie et al., also confers similar increased thermotolerance. Surprisingly, the chr-1 mutation is located at a position that would not be predicted to be important for the regulatory function of the riboswitch. We obtained physiological evidence suggesting that the chr-1 mutation increases the cytosolic free Mg(2+) concentration. High-level expression of the heterologous MgtE Mg(2+) transport protein of Bacillus subtilis also enhanced the thermotolerance of S. enterica. We hypothesize that increased Mg(2+) accumulation might enhance thermotolerance by protecting the integrity of proteins or membranes, by mitigating oxidative damage or acting as an inducer of thermoprotective functions.

Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc

Activation of E2F transcription factors at the G1-to-S phase boundary, with the resultant expression of genes needed for DNA synthesis and S-phase, is due to phosphorylation of the retinoblastoma-related (RBR) protein by cyclin D-dependent kinase (CYCD-CDK), particularly CYCD3-CDKA. Arabidopsis has three canonical E2F genes, of which E2Fa and E2Fb are proposed to encode transcriptional activators and E2Fc a repressor. Previous studies have identified genes regulated in response to high-level constitutive expression of E2Fa and of CYCD3;1, but such plants display significant phenotypic abnormalities. We have sought to identify targets that show responses to lower level induced changes in abundance of these cell cycle regulators. Expression of E2Fa, E2Fc or CYCD3;1 was induced using dexamethasone and the effects analysed using microarrays in a time course allowing short and longer term effects to be observed. Overlap between CYCD3;1 and E2Fa modulated genes substantiates their action in a common pathway with a key role in controlling the G1/S transition, with additional targets for CYCD3;1 in chromatin modification and for E2Fa in cell wall biogenesis and development. E2Fc induction led primarily to gene downregulation, but did not antagonise E2Fa action and hence E2Fc appears to function outside the CYCD3-RBR pathway, does not have a direct effect on cell cycle genes, and promoter analysis suggests a distinct binding site preference.

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.

Translational Control of the Antibiotic Inducibility of the PA5471 Gene Required for mexXY Multidrug Efflux Gene Expression in Pseudomonas aeruginosa

The PA5471 gene required for induction of the MexXY multidrug efflux system in response to ribosome-targeting antimicrobials was itself shown to be inducible by ribosome-targeting antimicrobials (Y. Morita, M. L. Sobel, and K. Poole, J. Bacteriol. 188:1847-1855, 2006). Using a lacZ transcriptional reporter, drug inducibility of PA5471 was shown to require the entirety of the 367-bp PA5472-PA5471 intergenic region. A constitutive promoter activity was, however, localized to the first 75 bp of this region, within which a single PA5471 transcription initiation site was mapped. That 3' sequences of the intergenic region blocked PA5471 expression and made it antibiotic dependent was suggestive of an attenuation mechanism of control. A 13-amino-acid leader peptide (LP)-encoding open reading frame preceded by a Shine-Dalgarno sequence was identified ca. 250 bp upstream of the PA5471 coding sequence, and its expression and translation were confirmed using a lacZ translational reporter. Alteration of the initiation codon (M1T) or introduction of translational stop signals at codons 3 (Q3Am) and 8 (C8Op) of this LP sequence (PA5471.1) yielded high-level constitutive expression of PA5471, suggesting that interference with LP translation was linked to PA5471 gene expression. Consistent with this, a Q3K mutation in the LP sequence maintained the drug inducibility of PA5471 expression. Introduction of the LP Q3Am mutation into the chromosome of Pseudomonas aeruginosa yielded stronger expression of PA5471 than did antibiotic (chloramphenicol) exposure of wild-type P. aeruginosa, in agreement with lacZ transcriptional fusion data. Still, the Q3Am mutation yielded modest expression of mexXY, less than that seen for antibiotic-treated wild-type P. aeruginosa. These data suggest that PA5471 is not sufficient for MexXY recruitment in response to antibiotic exposure and that additional antibiotic-dependent effects are needed.

Discovery of Anas platyrhynchos avian β-defensin 2 (Apl_AvBD2) with antibacterial and chemotactic functions

The cationic, cysteine-rich peptides called β-defensins play a major role in the innate immune response. Here, we describe the identification and characterization of the duck β-defensin-2 homologue, Anas platyrhynchos avian β-defensin 2 (Apl_AvBD2). The 195 base pair open reading frame (ORF) of Apl_AvBD2 has 83% identity with Gga_AvBD2 (chicken) and 85% identity with Mga_AvBD2 (turkey) at nucleotide level. The gene corresponding to the coding region is comprised of three exons and two introns in both Apl_AvBD2 and Gga_AvBD2. The predicted secondary structure of Apl_AvBD2 has the classical “β-defensin core motif” formed by the β-sheet rich structure. Apart from mild expression in tissues like kidney, lung, brain, bursa of Fabricious and ovary, Apl_AvBD2 mRNA show a very high level constitutive expression in bone marrow and spleen, indicating that it is a myeloid defensin. Purified recombinant Apl_AvBD2 demonstrated in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria, with a minimum bactericidal concentration (MBC) of 3.7μM against Micrococcus luteus NCIM 2871 and Escherichia coli NCIM 2685, and of 2.2μM against Reimerella anatipestifer. The immunomodulatory potential of Apl_AvBD2 was shown by chemotaxis of DT-40 chicken B-lymphocytes. The widespread tissue distribution and the potent bactericidal and chemotactic activity make Apl_AvBD2 an important molecule in the innate immune response in ducks. It may play a vital role in the immune response of these birds against bacterial and viral pathogens.

Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice

To identify minimal effective promoters for driving abiotic stress-inducible transgene expression in rice, we selected promoter elements of three stress-responsive genes, viz. rab16A coding for dehydrin, OsABA2 coding for zeaxanthin epoxidase, and a gene coding for a hypothetical protein (HP1) based on the presence of ABA-, salt- and drought-responsive cis-acting elements. These were translationally fused to the gusA reporter gene and introduced into rice to study their effect on heterologous gene expression. The OsABA2 promoter was found to be the most effective and desirable promoter among the three in terms of driving a low constitutive transgene expression under normal conditions and high induction in response to ABA, salt and drought stress, the highest being a 12-fold induction in response to ABA. The rab16A and HP1 promoters resulted in high levels of constitutive expression. While induction of GUS activity was generally two- to threefold for all the treatments in roots for both the promoters, induction in leaves was generally insignificant, the exceptions being rab16A in response to continuous salt stress and HP1 in response to water deficit. It was also observed that the three promoters, in general, resulted in lower constitutive expression, but higher induction in roots as compared to leaves.

Protein Kinase D Is an Essential Regulator of C. elegans Innate Immunity

Protein kinase D (PKD) mediates signal transduction downstream from phospholipase C and diacylglycerol (DAG). PKDs are activated by hormones and stress in cell lines, but little is known about PKD functions and regulation in vivo. Here, we show that DKF-2, a C. elegans PKD, regulates innate immunity. Animals lacking DKF-2 were hypersensitive to killing by bacteria that are pathogens of C. elegans and humans. DKF-2 induced 85 mRNAs, which encode antimicrobial peptides and proteins that sustain intestinal epithelium. Induction of immune effector mRNAs by DKF-2 proceeded via PMK-1 (p38 Map-kinase)-dependent and -independent pathways. TPA-1, a PKCdelta homolog, regulated activation and functions of DKF-2 in vivo. Therefore, DKF-2 provides a molecular link that couples DAG signaling to regulation of immunity. This intersection between DAG-TPA-1-DKF-2 and PMK-1 pathways enables integrated immune responses to multiple stimuli. Thus, a PKD mobilizes activation of host immune defenses against pathogens by previously unappreciated signaling pathways and mechanisms.

Genetic characterization of oligopeptide uptake systems inSinorhizobium meliloti

The genetic characterization of three ABC transport systems involved in oligopeptide uptake by Sinorhizobium meliloti is reported. Oligopeptide permease (Opp) encoded by the pSymB oppABCD operon, is required for uptake of tetrapeptides and certain tripeptides like 3Ala and bialaphos. The chromosomally encoded dipeptide permease (Dpp1), also able to import the toxic tripeptide bialaphos, is required for utilization of dipeptides and tripeptides like 3Gly and GlyGlyAla, with minor importance for utilization of 3Ala and tetrapeptides. The ttp (tri and tetrapeptide uptake) operon, encodes a third ABC system (Ttp) unable of transporting bialaphos and with minor role in the utilization of tetrapeptides and tripeptides like 3-Ala. Despite the overlapping substrate specificities of these ABC transporters, the corresponding gene operons displayed distinct expression profiles: dpp1 showed high constitutive expression levels under all conditions tested, in contrast to the low expression levels of ttp, whereas opp was maximally expressed upon entry into stationary phase. Nevertheless, complex interactions among the three systems at the transcriptional level were observed: opp was negatively autoregulated via OppA and positively regulated via DppA1, whereas dpp1 seems negatively autoregulated via DppA1. The expression of both opp and dppl was reduced in Ttp mutants. The ABC transport systems characterized in this work are not essential for the establishment of nitrogen-fixing symbiosis with alfalfa.

A soybean (Glycine max) polyubiquitin promoter gives strong constitutive expression in transgenic soybean

The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.

An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli

The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli.