Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.

Assessing Aedes aegypti candidate genes during viral infection and Wolbachia-mediated pathogen blocking.

One approach to control dengue virus transmission is the symbiont Wolbachia, which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti, Wolbachia's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha-mannosidase-2a (alpha-Mann-2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host-viral interactions; however, the function of alpha-mannosidases has not been described in mosquito-virus interactions. We examined alpha-Mann-2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha-Mann-2a in a tissue- and time-dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha-Mann-2a expression in Wolbachia-free mosquitoes, suggesting that alpha-Mann-2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha-Mann-2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia-infected mosquitoes. We also provide evidence that alpha-Mann-2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti.

Formation of light-harvesting complex II aggregates from LHCII-PSI-LHCI complexes in rice plants under high light.

During low light- (LL) induced state transitions in dark-adapted rice (Oryza sativa) leaves, light-harvesting complex (LHC) II become phosphorylated and associate with PSI complexes to form LHCII-PSI-LHCI supercomplexes. When the leaves are subsequently transferred to high light (HL) conditions, phosphorylated LHCII complexes are no longer phosphorylated. Under the HL-induced transition in LHC phosphorylation status, we observed a new green band in the stacking gel of native green-PAGE, which was determined to be LHCII aggregates by immunoblotting and 77K chlorophyll fluorescence analysis. Knockout mutants of protein phosphatase 1 (PPH1) which dephosphorylates LHCII failed to form these LHCII aggregates. In addition, the ability to develop non-photochemical quenching in the PPH1 mutant under HL was less than for wild-type plants. As determined by immunoblotting analysis, LHCII proteins present in LHCII-PSI-LHCI supercomplexes included the Lhcb1 and Lhcb2 proteins. In this study, we provide evidence suggesting that LHCII in the LHCII-PSI-LHCI supercomplexes are dephosphorylated and subsequently form aggregates to dissipate excess light energy under HL conditions. We propose that this LHCII aggregation, involving LHCII L-trimers, is a newly observed photoprotective light-quenching process operating in the early stage of acclimation to HL in rice plants.

Identification of the Optimal Light Harvesting Antenna Size for High-Light Stress Mitigation in Plants.

One of the major constraints limiting biomass production in autotrophs is the low thermodynamic efficiency of photosynthesis, ranging from 1 to 4%. Given the absorption spectrum of photosynthetic pigments and the spectral distribution of sunlight, photosynthetic efficiencies as high as 11% are possible. It is well-recognized that the greatest thermodynamic inefficiencies in photosynthesis are associated with light absorption and conversion of excited states into chemical energy. This is due to the fact that photosynthesis light saturates at one quarter full sunlight intensity in plants resulting in the dissipation of excess energy as heat, fluorescence and through the production of damaging reactive oxygen species. Recently, it has been demonstrated that it is possible to adjust the size of the light harvesting antenna over a broad range of optical cross sections through targeted reductions in chlorophyll b content, selectively resulting in reductions of the peripheral light harvesting antenna size, especially in the content of Lhcb3 and Lhcb6. We have examined the impact of alterations in light harvesting antenna size on the amplitude of photoprotective activity and the evolutionary fitness or seed production in Camelina grown at super-saturating and sub-saturating light intensities to gain an understanding of the driving forces that lead to the selection for light harvesting antenna sizes best fit for a range of light intensities. We demonstrate that plants having light harvesting antenna sizes engineered for the greatest photosynthetic efficiency also have the greatest capacity to mitigate high light stress through non-photochemical quenching and reduction of reactive oxygen associated damage. Under sub-saturating growth light intensities, we demonstrate that the optimal light harvesting antenna size for photosynthesis and seed production is larger than that for plants grown at super-saturating light intensities and is more similar to the antenna size of wild-type plants. These results suggest that the light harvesting antenna size of plants is designed to maximize fitness under low light conditions such as occurs in shaded environments and in light competition with other plants.

RNA Interference to Control Asian Corn Borer Using dsRNA from a Novel Glutathione-S-Transferase Gene of Ostrinia furnacalis (Lepidoptera: Crambidae).

Glutathione-S-transferases (GST) comprise a multifunctional protein superfamily, which plays important roles as detoxifiers and antioxidants in insects. The GST in Asian corn borer has not been previously characterized. In this study, we cloned, characterized, and expressed the complete GST genes from the midgut of Asian corn borer. Furthermore, we designed htL4440-OfGST vector to exploit this gene for RNA interference (RNAi) strategy to control this pest. A complete GST cDNA sequence in Asian corn borer was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends technology. The gene was 887bp in length and contained a 705bp open reading frame and 5' UTR and 3' UTR lengths of 89 and 93bp, respectively. The putative sequence encoded a putative 234 amino acid residue peptide and had a predicted molecular weight of ~26kDa. The GST protein of Asian corn borer is hydrophilic and may have a 30 amino acid signal peptide with a cleavage site between L30 and K31. A recombination vector pET28a-OfGST was constructed for purification and antibody preparation. Western blotting analysis showed that this protein reached the maximum expression level around 24 h in Asian corn borer larvae fed the plant toxin 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one. A second vector, htL4440-OfGST, was constructed to generate the dsRNA of the GST gene. A larval feeding bioassay showed that the expressed dsRNA significantly reduced the detoxification ability of Asian corn borer larvae and increased mortality rate up to 54%. Our data indicated that GST plays very important roles in detoxifying in Asian corn borer and can be used as an RNAi method to control this pest in the field.

Genome sequence and comparative analyses of atoxigenic Aspergillus flavus WRRL 1519.

Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus and a few other closely related species of Aspergillus. These highly toxigenic and carcinogenic mycotoxins contaminate global food and feed supplies, posing widespread health risks to humans and domestic animals. Field application of nonaflatoxigenic strains of A. flavus to compete against aflatoxigenic strains has emerged as one of the best management practices for reducing aflatoxins contamination, yielding successful commercial products for corn, cotton seed, and peanuts. In this study, we sequenced the genome and transcriptome of atoxigenic (does not produce aflatoxin or cyclopiazonic acid) A. flavus strain WRRL 1519 isolated from a tree nut orchard to define the genetic characteristics of the strain in relation to aflatoxigenic and other nonaflatoxigenic A. flavus strains. WRRL 1519 strain was similar to other strains in size (38.0 Mb), GC content (47.2%), number of predicted secondary metabolite gene clusters (46), and number of putative proteins (12 121). About 87.4% of the predicted proteome had high shared identity with protein sequences derived from other A. flavus genomes. However, the atoxigenic A. flavus strain WRRL 1519 had deletions, or low shared identity, for many genes in the clusters required for aflatoxins and cyclopiazonic acid (CPA) synthesis. Over half of the aflatoxin synthesis gene cluster was missing, and none of the components of the CPA gene cluster were identified with high sequence similarity. Importantly, the strain appeared to maintain functional sequences of several genes thought to be required for high infectivity. Since the ability to grow on target crop is an important attribute for a successful biocontrol agent, these results indicate that the nonaflatoxigenic A. flavus strain WRRL 1519 would be a good candidate as a biocontrol agent for reducing aflatoxin and CPA accumulation in high-value nut crops.

Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch.

Storage roots of cassava (Manihot esculenta Crantz), a major subsistence crop of sub-Saharan Africa, are calorie rich but deficient in essential micronutrients, including provitamin A ß-carotene. In this study, ß-carotene concentrations in cassava storage roots were enhanced by co-expression of transgenes for deoxy-d-xylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin-type 1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 µg/g DW, 15- to 20-fold increases relative to roots from nontransgenic plants. Approximately 85%-90% of these carotenoids accumulated as all-trans-ß-carotene, the most nutritionally efficacious carotenoid. ß-Carotene-accumulating storage roots displayed delayed onset of postharvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in ß-carotene-enhanced storage roots. Most significantly, an inverse correlation was observed between ß-carotene and dry matter content, with reductions of 50%-60% of dry matter content in the highest carotenoid-accumulating storage roots of different cultivars. Further analysis confirmed a concomitant reduction in starch content and increased levels of total fatty acids, triacylglycerols, soluble sugars and abscisic acid. Potato engineered to co-express DXS and crtB displayed a similar correlation between ß-carotene accumulation, reduced dry matter and starch content and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed a reduced expression of genes involved in starch biosynthesis including ADP-glucose pyrophosphorylase genes in transgenic, carotene-accumulating cassava roots relative to nontransgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development.

Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both ß-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, leaves had sufficient rhodanese activity to compete with cyanide assimilation into amino acids. Using transgenic low cyanogen plants, it was shown that reducing root cyanogen levels is associated with elevated root nitrate reductase activity, presumably to compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted in up to a 50% increase in root total amino acids and a 9% increase in root protein accumulation. However, plant growth and morphology was altered in plants overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth.

Possible role of interference, protein noise, and sink effects in nonphotochemical quenching in photosynthetic complexes.

We analyze theoretically a simple and consistent quantum mechanical model that reveals the possible role of quantum interference, protein noise, and sink effects in the nonphotochemical quenching (NPQ) in light-harvesting complexes (LHCs). The model consists of a network of five interconnected sites (excitonic states of light-sensitive molecules) responsible for the NPQ mechanism. The model also includes the "damaging" and the dissipative channels. The damaging channel is responsible for production of singlet oxygen and other destructive outcomes. In our model, both damaging and "dissipative" charge transfer channels are described by discrete electron energy levels attached to their sinks, that mimic the continuum part of electron energy spectrum. All five excitonic sites interact with the protein environment that is modeled using a stochastic process. Our approach allowed us to derive the exact and closed system of linear ordinary differential equations for the reduced density matrix and its first momentums. These equations are solved numerically including for strong interactions between the light-sensitive molecules and protein environment. As an example, we apply our model to demonstrate possible contributions of quantum interference, protein noise, and sink effects in the NPQ mechanism in the CP29 minor LHC. The numerical simulations show that using proper combination of quantum interference effects, properties of noise, and sinks, one can significantly suppress the damaging channel. Our findings demonstrate the possible role of interference, protein noise, and sink effects for modeling, engineering, and optimizing the performance of the NPQ processes in both natural and artificial light-harvesting complexes.

Comparative energetics and kinetics of autotrophic lipid and starch metabolism in chlorophytic microalgae: implications for biomass and biofuel production.

Due to the growing need to provide alternatives to fossil fuels as efficiently, economically, and sustainably as possible there has been growing interest in improved biofuel production systems. Biofuels produced from microalgae are a particularly attractive option since microalgae have production potentials that exceed the best terrestrial crops by 2 to 10-fold. In addition, autotrophically grown microalgae can capture CO2 from point sources reducing direct atmospheric greenhouse gas emissions. The enhanced biomass production potential of algae is attributed in part to the fact that every cell is photosynthetic. Regardless, overall biological energy capture, conversion, and storage in microalgae are inefficient with less than 8% conversion of solar into chemical energy achieved. In this review, we examine the thermodynamic and kinetic constraints associated with the autotrophic conversion of inorganic carbon into storage carbohydrate and oil, the dominant energy storage products in Chlorophytic microalgae. We discuss how thermodynamic restrictions including the loss of fixed carbon during acetyl CoA synthesis reduce the efficiency of carbon accumulation in lipids. In addition, kinetic limitations, such as the coupling of proton to electron transfer during plastoquinone reduction and oxidation and the slow rates of CO2 fixation by Rubisco reduce photosynthetic efficiency. In some cases, these kinetic limitations have been overcome by massive increases in the numbers of effective catalytic sites, e.g. the high Rubisco levels (mM) in chloroplasts. But in other cases, including the slow rate of plastoquinol oxidation, there has been no compensatory increase in the abundance of catalytically limiting protein complexes. Significantly, we show that the energetic requirements for producing oil and starch relative to the recoverable energy stored in these molecules are very similar on a per carbon basis. Presently, the overall rates of starch and lipid synthesis in microalgae are very poorly characterized. Increased understanding of the kinetic constraints of lipid and starch synthesis, accumulation and turnover would facilitate the design of improved biomass production systems.

Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1.

We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants.

Retention during processing and bioaccessibility of ß-carotene in high ß-carotene transgenic cassava root.

Cassava is a root crop that serves as a primary caloric source for many African communities despite its low content of ß-carotene (ßC). Carotenoid content of roots from wild type (WT) and three transgenic lines with high ßC were compared after cooking and preparation of nonfermented and fermented flours according to traditional African methods. The various methods of processing all decreased ßC content per gram dry weight regardless of genotype. The greatest loss of ßC occurred during preparation of gari (dry fermentation followed by roasting) from WT and transgenic lines. The quantities of ßC in cooked transgenic cassava root that partitioned into mixed micelles during in vitro digestion and transported into Caco-2 cells were significantly greater than those for identically processed WT root. These results suggest that transgenic high ßC cassava will provide individuals with greater quantities of bioaccessible ßC.

Iron and protein biofortification of cassava: lessons learned.

Over two hundred and fifty million Africans rely on the starchy root crop cassava (Manihot esculenta) as their primary source of calories. Cassava roots, however, have the lowest protein:energy ratio of all the world's major staple crops. Furthermore, a typical cassava-based diet provides less than 10-20% of the required amounts of iron, zinc, vitamin A and vitamin E. The BioCassava Plus program employed modern biotechnologies to improve the health of Africans through development and delivery of novel cassava germplasm with increased nutrient levels. Here we describe the development of molecular strategies and their outcomes to meet minimum daily allowances for protein and iron in cassava based diets. We demonstrate that cyanogens play a central role in cassava nitrogen metabolism and that strategies employed to increase root protein levels result in reduced cyanogen levels in roots. We also demonstrate that enhancing root iron uptake has an impact on the expression of genes that regulate iron homeostasis in multiple tissues. These observations demonstrate the complex metabolic interactions involved in enhancing targeted nutrient levels in plants and identify potential new strategies for further enhancing nutrient levels in cassava.

Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement.

Comparing photosynthetic and photovoltaic efficiencies is not a simple issue. Although both processes harvest the energy in sunlight, they operate in distinctly different ways and produce different types of products: biomass or chemical fuels in the case of natural photosynthesis and nonstored electrical current in the case of photovoltaics. In order to find common ground for evaluating energy-conversion efficiency, we compare natural photosynthesis with present technologies for photovoltaic-driven electrolysis of water to produce hydrogen. Photovoltaic-driven electrolysis is the more efficient process when measured on an annual basis, yet short-term yields for photosynthetic conversion under optimal conditions come within a factor of 2 or 3 of the photovoltaic benchmark. We consider opportunities in which the frontiers of synthetic biology might be used to enhance natural photosynthesis for improved solar energy conversion efficiency.

Removal of mercury from sediment by ultrasound combined with biomass (transgenic Chlamydomonas reinhardtii).

As one of the most pervasive environmental problems, Hg pollution in sediment is particularly difficult to remediate because it cannot be decomposed. The application of ultrasound combined with biomass (transgenic Chlamydomonas reinhardtii (C. reinhardtii), a green alga) for the removal of Hg from model and contaminated sediments (Al(2)O(3), α-HgS, and PACS-2 marine sediment) was investigated in this study. Ultrasound was found to enhance Hg release from Al(2)O(3), α-HgS, and PACS-2 marine sediment into the aqueous phase compared to mechanical shaking. A transgenic C. reinhardtii (2AMT-2) expressing a plasmamembrane-anchored metallothionein polymer effectively recovered Hg(II) released into the aqueous phase by sonication over a broad pH range from 2.0 to 9.0. The results showed that this combined technique of ultrasound and alga biomass (2AMT-2) engineered for enhanced metal recovery was effective to remove Hg from solids and sediments, especially from Al(2)O(3) and α-HgS with no natural organic matter. The results of this study are discussed with respect to the development of in situ remediation techniques for Hg-contaminated sediments.

The Iron Assimilatory Protein, FEA1, from Chlamydomonas reinhardtii Facilitates Iron-Specific Metal Uptake in Yeast and Plants.

We demonstrate that the unique green algal iron assimilatory protein, FEA1, is able to complement the Arabidopsis iron-transporter mutant, irt1, as well as enhance iron accumulation in FEA1 expressing wild-type plants. Expression of the FEA1 protein reduced iron-deficient growth phenotypes when plants were grown under iron limiting conditions and enhanced iron accumulation up to fivefold relative to wild-type plants when grown in iron sufficient media. Using yeast iron-uptake mutants, we demonstrate that the FEA1 protein specifically facilitates the uptake of the ferrous form of iron. Significantly, the FEA1 protein does not increase sensitivity to toxic concentrations of competing, non-ferrous metals nor facilitate their (cadmium) accumulation. These results indicate that the FEA1 protein is iron specific consistent with the observation the FEA1 protein is overexpressed in cadmium stressed algae presumably to facilitate iron uptake. We propose that the FEA1 iron assimilatory protein has ideal characteristics for the iron biofortification of crops and/or for facilitated iron uptake in plants when they are grown in low iron, high pH soils, or soils that may be contaminated with heavy metals.

N-ACYL HOMOSERINE LACTONe LACTONASE, AiiA, INACTIVATION OF QUORUM-SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE(1).

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N-acyl homoserine lactone (AHL) bacterial quorum-sensing (QS) signals and alter QS-regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal-mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR-stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal-mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL-producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.

A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi.

Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-L-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~38kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~28kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two fluorophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN(-) (defective in AI-1), luxS(-) (defective in BAI-2), and luxN(-)/luxS(-) (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS(-) mutant (MM30) and luxN(-)/luxS(-) double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating extracellular protease production in V. harveyi.