Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.Classic experiments in microbial bioenergetics used light-driven reactions from halobacterial bacteriorhodopsin or the photosynthetic reaction center to provide a temporary driving force for understanding transport and chemiosmotic coupling (6, 7, 19, 35). However, light-driven reactions have not been used in metabolic engineering to alter microbial physiology and production of chemicals. The recent discovery of proteorhodopsin (PR) in ocean microorganisms and the ease with which this membrane protein can be functionally expressed by recombinant bacteria have made possible many engineering strategies previously not available (1, 16). In this paper, we describe progress toward the goal of integrating light-driven reactions with biocatalysis.In contrast to the situation for established industrial microorganisms, such as Escherichia coli, our current understanding of less-studied algal and phototrophic bacteria may limit metabolic engineering strategies which require genetic manipulation. Metabolic engineering strategies using photosynthetic bacteria have focused largely on methods to increase hydrogen production, and improvements rely mainly on engineering of nitrogenase and hydrogenase to produce H₂. Algae appear to be suited to large-scale cultivation for lipid production, but so far little has been done to engineer these organisms (36). In principle, platform microbial hosts capable of producing a diverse range of products could be boosted by addition of light-driven processes from phototrophic metabolism.To demonstrate the feasibility of transferring a light-driven process into a nonphotosynthetic bacterium, we chose to study proteorhodopsin (PR) first because it is one of the simplest mechanisms for harnessing the energy from light. The proteorhodopsins are a group of transmembrane proteins that use the light-induced isomerization of retinal, the oxidative cleavage product of the carotenoid β-carotene, either to initiate signaling pathways or to catalyze the transfer of ions across cell membranes (8). PR was discovered by metagenomic analysis of marine samples (1) and is related to the well-studied bacteriorhodopsin of archaea (33) and rhodopsin (34), a eukaryotic light-sensing protein. The membrane potential generated by light-driven proton pumping by PR has been confirmed to drive ATP synthesis in a heterologous system (25). However, bacteria expressing heterologous PR were shown not to benefit from this pumping activity, as no significant increases in growth rates were observed (9). This led to the suggestion that PR may benefit the organism only under starvation conditions. In agreement with this hypothesis, Gomez-Consarnau et al. (10) have reported that the light-dependent growth rates of a marine flavobacterium that has a native PR are increased only when the organism is cultured under energy-limited conditions.Studies of both native and recombinant systems in which rhodopsins are expressed have generated light-dependent membrane potentials. In membrane vesicles isolated from a native host, the light-dependent membrane potential generated by bacteriorhodopsin provides the driving force for ATP synthesis (35) and uptake of leucine and glutamate (20, 22). More recently, studies of recombinant systems have coupled the membrane potential to other transport processes. In one example, the membrane potential-dependent export of specific toxic molecules increased when E. coli cells expressing both an archaeal rhodopsin and a specific efflux pump were exposed to light (17). In another experiment, starved E. coli cells expressing PR increased the swimming motion of their flagella when they were illuminated (44). Based upon measurements of flagellar motion as a function of light intensity and azide concentration, the proton motive force generated by PR was estimated to be −0.2 V, a value similar to the value for aerobic respiration in E. coli (42).As a nonphotosynthetic host for recombinant PR expression, we chose the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1, which is genetically tractable for engineering and is able to use a variety of terminal electron acceptors, including insoluble metal oxides (11, 30). Key to the ability of this bacterium to reduce metal oxides is a multicomponent extracellular respiratory pathway that transports electrons from menaquinol to cytochromes in the outer membrane. This pathway is composed of a cytoplasmic membrane tetraheme protein (CymA), a periplasmic decaheme protein (MtrA), an integral outer membrane protein (MtrB), and a decaheme lipoprotein (MtrC) that is associated with MtrB (14, 37, 40). The ability of S. oneidensis to reduce extracellular metal oxides has made it possible to harvest electrons from this organism by coupling it to an electrode which serves as the electron acceptor (21). The electron flow to the outer surface allows respiration rates to be measured directly by electrochemistry.In the current work, we introduced PR into an electricity-generating bacterium, S. oneidensis strain MR-1, and demonstrated that there was integration of a light-driven process into the metabolism of a previously nonphotosynthetic organism that resulted in a useful output. We show here that PR allows cells to survive for extended periods in stationary phase and that the presence of light results in an increase in electricity generation. A possible physiological model to explain these effects is discussed.     

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca²⁺/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.     

Dynamics of plasmodesmal connectivity in successive interfaces of the cambial zone

Frequency, density and branching of plasmodesmata were counted in successive tangential and transverse walls in the cambial zone of tomato stems in order to examine development of the plasmodesmal network in a chronological order. Coincident with progress of cell development, plasmodesmal connectivity increased, both at the xylem- and phloem-side. In transverse walls, the number of secondary plasmodesmata enhanced considerably. The same held for tangential walls, with a superimposed plasmodesmal doubling during the first phase of phloem development. This plasmodesmal doubling was interpreted to result from the deposition of wall material between branched plasmodesmal strands. Structural plasmodesmal development was correlated with production of hydroxyl radicals which control local cell wall alterations. Successive phases of plasmodesmal deployment and modification were distinguished which may coincide with differential functional capacities as documented by intracellular injection of fluorochromes. Diffusion-driven symplasmic transport appeared to be transiently interrupted during cell maturation.     

Validation of QTLs for Orobanche crenata resistance in faba bean (Vicia faba L.) across environments and generations

Broomrape (Orobanche crenata Forsk.) is a major root–parasite of faba bean (Vicia faba L.), that seriously limits crop cultivation in the whole Mediterranean area. This parasitic weed is difficult to control, difficult to evaluate and the resistance identified so far is of polygenic nature. This study was conducted to identify genetic regions associated with broomrape resistance in recombinant inbred lines (RILs) and to validate their previous location in the original F₂ population derived from the cross between lines Vf6 and Vf136. A progeny consisting of 165 F₆ RILs was evaluated in three environments across two locations in 2003 and 2004. Two hundred seventy seven molecular markers were assigned to 21 linkage groups (9 of them assigned to specific chromosomes) that covered 2,856.7 cM of the V. faba genome. The composite interval mapping on the F₆ map detected more quantitative trait loci (QTL) than in the F₂ analysis. In this sense, four QTLs controlling O. crenata resistance (Oc2–Oc5) were identified in the RI segregant population in three different environments. Only Oc1, previously reported in the F₂ population, was not significant in the advanced lines. Oc2 and Oc3 were found to be associated with O. crenata resistance in at least two of the three environments, while the remaining two, Oc4 and Oc5, were only detected in Córdoba-04 and Mengíbar-04 and seemed to be environment dependent.     

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia

Background

Although some epidemiologic studies found inverse associations between alcohol drinking and Parkinson's disease (PD), the majority of studies found no such significant associations. Additionally, there is only limited research into the possible interactions of alcohol intake with aldehyde dehydrogenase (ALDH) 2 activity with respect to PD risk. We examined the relationship between alcohol intake and PD among Japanese subjects using data from a case-control study.

Methods

From 214 cases within 6 years of PD onset and 327 controls without neurodegenerative disease, we collected information on "peak", as opposed to average, alcohol drinking frequency and peak drinking amounts during a subject's lifetime. Alcohol flushing status was evaluated via questions, as a means of detecting inactive ALHD2. The multivariate model included adjustments for sex, age, region of residence, smoking, years of education, body mass index, alcohol flushing status, presence of selected medication histories, and several dietary factors.

Results

Alcohol intake during peak drinking periods, regardless of frequency or amount, was not associated with PD. However, when we assessed daily ethanol intake separately for each type of alcohol, only Japanese sake (rice wine) was significantly associated with PD (adjusted odds ratio of ≥66.0 g ethanol per day: 3.39, 95% confidence interval: 1.10-11.0, P for trend = 0.001). There was no significant interaction of alcohol intake with flushing status in relation to PD risk.

Conclusions

We did not find significant associations between alcohol intake and PD, except for the daily amount of Japanese sake. Effect modifications by alcohol flushing status were not observed.     

Short term hypothyroidism affects ovarian function in the cycling rat

Background  

Rats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.     

Cocoa flavonoids up-regulate antioxidant enzyme activity via the ERK1/2 pathway to protect against oxidative stress-induced apoptosis in HepG2 cells

Oxidative stress is widely recognized as an important mediator of apoptosis in liver cells and plays a pivotal role in the pathogenesis of several diseases. Cocoa flavonoids have shown a powerful antioxidant activity providing protection against oxidation and helping prevent oxidative stress-related diseases. However, the molecular mechanisms responsible for this protection are not fully understood. Thus, in this study we investigated the protective effect of a cocoa polyphenolic extract (CPE) against tert-butyl hydroperoxide (t-BOOH)-induced apoptosis and the molecular mechanisms involved in this process. Incubation of HepG2 cells with t-BOOH induced apoptosis as evidenced by caspase-3 activation. This effect was accompanied by increased reactive oxygen species formation and by transient activation of the extracellular regulated kinases (ERKs) as well as sustained activation of the c-Jun N-terminal kinases (JNKs). On the contrary, pretreatment of HepG2 cells with CPE prevented apoptosis through the reduction of reactive oxygen species generation and the modulation of the apoptotic pathways activated by t-BOOH. CPE treatment also activated survival signaling proteins, such as protein kinase B (AKT) and ERKs, and increased the activities of two antioxidant enzymes, glutathione peroxidase (GPx) and glutathione reductase (GR). ERK's implication on GPx and GR induction and the protective effect of CPE against t-BOOH-induced oxidative stress and apoptosis were confirmed through experiments with selective inhibitors. These findings suggest that CPE is an effective inductor of GPx and GR activities via ERK activation and that this up-regulation seems to be required to attenuate t-BOOH-induced injury.     

Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms

Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24 degrees C and 14 degrees C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14 degrees C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14 degrees C was more favorable for the survival of this pathogen than 24 degrees C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.     

Spatial distribution and species composition of prosthecate bacteria in Lake Baikal

From the water column of Lake Baikal, several strains of prosthecate bacteria belonging to the genera Caulobacter and Brevundimonas were isolated. In this article, the methods applied for their isolation and cell number determination are described; the occurrence frequency and spatial distribution of these microorganisms in the lake are demonstrated. Characterization of the species composition of cultivable and uncultivable prosthecate bacteria was carried out using the methods of traditional and molecular microbiology, respectively. A comparative phylogenetic analysis of the DNA sequences of uncultivable bacteria, which showed homology to the members of the alpha subclass of proteobacteria, was carried out. It was demonstrated that the lake water column is inhabited by uncultivable alpha-proteobacteria of uncertain phylogenetic affinity, in addition to representatives of the species Caulobacter vibrioides and C. leidyi, which were detected by traditional microbiological methods.     

Generation,annotation, and analysis of ESTs from four different <Emphasis Type="Italic">Trichoderma</Emphasis> strains grown under conditions related to biocontrol

The functional genomics project “TrichoEST” was developed focused on different taxonomic groups of Trichoderma with biocontrol potential. Four cDNA libraries were constructed, using similar growth conditions, from four different Trichoderma strains: Trichoderma longibrachiatum T52, Trichoderma asperellum T53, Trichoderma virens T59, and Trichoderma sp. T78. In this study, we present the analysis of the 8,160 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1,000–1,300 unique sequences were identified in each strain. First, we queried our collection of ESTs against the NCBI nonredundant database using the BLASTX algorithm. Moreover, using the Gene Ontology hierarchy, we performed the annotation of 40.9% of the unique sequences. Later, based on the EST abundance, we examined the highly expressed genes in the four strains. A hydrophobin was found as the gene expressed at the highest level in two of the strains, but we also found that other unique sequences similar to the HEX1, QID3, and NMT1 proteins were highly represented in at least two of the Trichoderma strains. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.