Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

Increasing the efficiency of the conversion of light energy to products by photosynthesis represents a grand challenge in biotechnology. Photosynthesis is limited by the carbon-fixing enzyme Rubisco resulting in much of the absorbed energy being wasted as heat or fluorescence or lost as excess reductant via alternative electron dissipation pathways. To harness this wasted reductant, we engineered the model cyanobacterium Synechococcus PCC 7002 to express the mammalian cytochrome P450 CYP1A1 to serve as an artificial electron sink for excess electrons derived from light-catalyzed water-splitting. This improved photosynthetic efficiency by increasing the maximum rate of photosynthetic electron flow by 31.3%. A simple fluorescent assay for CYP1A1 activity demonstrated that the P450 was functional in the absence of its native reductase, that activity was light-dependent and scaled with irradiance. We show for the first time in live cells that photosynthetic reductant can be redirected to power a heterologous cytochrome P450. Furthermore, Synechococcus PCC 7002 expressing CYP1A1 degraded the herbicide atrazine, which is a widespread environmental pollutant

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes.

This is the final version of the article. Available from Rockefeller University Press via the DOI in this record.Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3- and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein.This work was supported by Wellcome Trust (097835/Z/11/Z) and the Biotechnology and Biological Sciences Research Council (BB/J009903/1)

Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture

Epidemiology analysis of bovine acute mastitis

從83年7月至85年6月自台灣中,南部地區奶牛場,收集罹患急性乳房炎 牛隻之乳樣進行細菌分離並分析發生情形(臨床症狀,乳房之肉眼病變,乳 汁之肉眼變化等),結果:由收集108 頭發病乳牛採到 117 個分房乳樣,分 離出 110 株細菌( 9 個分房未分離出細菌,2 個分房分離出2 種細 菌),110 株病原菌的分佈上以Coliform bacteria 53 株(48.2%)和 Staphlococci 27 株 (24.5%) 佔絕大多數,其次為 Streptococci 9 株 (8.2%),Bacillus spp 6株(5.5%),Actinomyces pyogenes 4株(3.6%), Pseudomona aeruginosa3 株(2.7%)其他病原菌 8 株(7.3). 在53 株 Coliform bacteria(包括E.coli 37 株, Klebsiella spp15 株及 Enterobacter spp 1 株)取22種抗生素進行抗生素感受性試驗,結果抗藥 性菌株出現率以Amoxicillin 98.11%(52/53),Ampicillin98.11%(52/53), Oxacillin98.11%(52/53)及Penicillin98.11(52/53)等四種為最高,只有 Ceftazidime及Netilmicin無抗藥性菌株出現;在9 株Streptococci (包括 Streptococcus uberis 2 株, Streptococcus bovis 1 株Streptococcus pyogenes 4 株,Enterococcus faecalis 2 株) 則以Streptomycin 100%(9/9)為最高,其次是Colistin和 Oxytetracycline 88.9%(8/9),Amoxicillin,Bacitracin,Ceftazidime,Netilmicin, Oxacillin 和 Vancomycin 則均無抗藥性菌株出現;在 3 株 Staphylococcus aureus 以 Colistin 100%(3/3) 最高,對Amikacin, Amoxicillin,Cephalothin,Enrofloxacin,Erythromycin,Gentamicin, Novobiotin,Oxacillin, Penicillin 和 Vacomycin 均無抗藥性菌株的出 現;在24株 CNS ( Coagulase Negative Staphylococcus) 以Penicillin 79.17%(19/24) 最高,Colistin70.83% (17/24) 次之,對 Amikacin, Amoxicillin,Cephalothin,Enrofloxacin,Erythromycin,Gentamicin, Novobiotin,Oxacillin,Penicillin 和 Vancomycin 均無抗藥性菌株的出 現. 急性乳房炎發生率在濕熱季節(5-10月)比乾冷季節(11-4月)有較 高之發生率,兩者呈極顯著差異(p<0.01) ;乳牛年齡愈大急性乳房炎發生 率愈高,急性乳房炎發生率在不同年齡間呈極顯著差異(p<0.01),經產牛比 初產牛急性乳房炎之發生率為高且兩者間呈極顯著差異(p<0.01),乳牛後 分房比前分房有較高之急性乳房炎發生率;兩者間呈極顯著差異(p<0.01). 急性乳房炎發生分房各類局部變化之出現率依次是腫88.9% (104/117),痛81.2% (95/117),熱53.9%(63/117),紅46.2%(54/117).罹患 急性乳房炎乳牛其乳房附著形態以搾乳機式乳房(milking machine udder)佔23.08%(27/117)比例最高,腿間乳房(udder in the thinghs)oyr 17.09%(20/117)為最低.在乳頭形態方面以短乳頭(short teat)發生急性 乳房炎比例最高42.74%(50/117),圓錐狀乳頭(conical teat)發生率最 少4.27%(5/117).急性乳房炎牛隻其體溫高於39.5 C 佔67.3%(68/101)大 部份急性乳房炎發生牛隻有較高之體溫反應. 急性乳房炎乳汁特性已改變 佔 89.7%(105/117),CMT 反應呈3 級佔91.5%(107/117),乳汁呈鹼性反應 佔77.8%(91/117). 為找出可鑑別Coliform bacteria 和 Non- coliform bacteria 急性乳房炎之臨床資料,將41頭Coliform 急性乳房炎 臨床資料及53頭Non-coliform 急性乳房炎臨床資料進行區分性分析( Discriminant analysis ) 結果發現有下述10個指標最為有效可用;包括 乳汁已失去原有特性,乳汁變成黃色,乳汁呈水樣變化,發病分房有熱感,發 病分房皮膚潮紅,病牛體溫大於40.5 C,發病分房曾得過乳房炎,在分娩後 四週內發生,乳汁有白色結塊,乳房附著情形是搾乳機式乳房等10項.結果 在區別Coliform bacteria 與Non-coliform bacteria所引起之急性乳房 炎,預測之正確率達88.29%(sensitivity = 0.85 ,specificity = 0.90).A survey was conducted from July,1996 to June,1996. Milk sample and clinicaldata of cows suffered from acute mastitis were collected from dairy farm in the middle and southern parts of Taiwan.108 cows and 117 quarter samples were analyzed in total. 110 strains were isolated from these samples. No pathogen wasisolated from 9 quarter samples and 2 isolates in the same sample were found in 2 cases. The distribution of important pathogens of bovine acute mastitis were as followed:Coliform bacteria 42.8%(53/110),Staphylococci 24.5%(24/110),Streptococci8.2%(9/110),Bacillus spp.5.5%(6/110), Actinomyces pyogenes 3.6%(4/110),Pseudomonas aeruginosa 2.7%(3/110)and other pathogens 7.3%(8/110). 53 Coliform strains isolated during the whole program were selected to analyzethe antibiotic resistant patterns among 22 antibiotics, the highest resistant percentage were found on the following antibiotic:Amoxicillin 98.11%(52/53),Ampicillin 98.11%(52/53),Oxacillin 98.11%(52/53) and Penicillin 98.11%(52/53).No resistant strains were found to be against Ceftazidime and Netilmicin .The same test were performed on 9 isolated streptococci. The highest resistent percentage was found in Streptomycin 100%(9/9) and than Colistin 88.9%(8/9)and Oxytetracycline 88.9%(8/9). No resistant strains were found to be against Amoxicillin,Bacitracin,Ceftazidime,Netilmicin, Oxacillin and vancomycin. Test also conducted on the isolated Staphylococcus aureus, the highest resistent percentage was found in Colistin 100%(3/3), and no resistant strains were found to be against Amikacin, Amoxicillin, Cephalothin, Enrofloxacin, Erythromycin, Gentamicin,Novobiotin, OXacillin, Penicillin and Vacomycin.On 24 isolated coagulase negative staphylococci, highest resistent percentage was found in Penicillin 79.17%(19/24),then Colistin 70.83%(19/24). No resistant strains were found to be againstAmikacin, Cephalothin, Enrofloxacin, Erythromycin, Gentamycin, Novobiotin, Oxacillin, Penicillin and Vancomycin. On 24 isolated coagulase negative staphylococci, highest resistent percentage was found in Penicillin 79.17%(19/24). No resistant strains were found to be ahainst Amikacin, Amoxixillin,Cephalothin,Enrofloxacin,Erythromycin, Gentamycin,Novobiotin,Oxacillin,Penicillin and Vancomycin. In the aspect of clinical investigation, higher percentage of occurrence was found in hot and humid season than in cold and dry one, and the differnet was significant (p<0.01). The perscntage of occurrence raise with cow age. significant difference was detected among among cohorts(p<0.01). Posterior quarters have higher percentage of acute mastitis than anterior quarters(p<0.01). The percentage of local changes in actue mastitis were as follows:swelling 88.9%(104/117), pain 81.2%(95/117), hot 53.9%(63/117) and redness 46.2% (54/117). The forms of quarter in acute mastitis was highest in milking machine udder 23.08% (27/117), lowest in udder in the thighs 17.09%(20/117). In the forms of teat, highest was found in short teat 42.74%(50/117), lowest was in conical teat 4.27%(5/117). 67.3%(68/101) of affected cows have body temperatures higher than 39.5 C, thus most cows in acute mastitis have higher body temperature. In 89.7%(105/117) of the cases the change of milk appearance, 91.5%(107/117) with third grade of CMT reaction,and 77.8%(91/117) with basic milk. Data from 41 coliform and 53 non- coliformacute mastitis cases were analyzed using the method of discriminate analysis.Among them 10 indicators are related to coliform acute mastitism including change in milk appearance, milk color yellow, a watery consistency, hot of the affected quarter, redness of the affected quarter, elevated rectal temperature,previous mastitis in quarter, mastitis occurs within 4 weeks after calving,white flake in milk, milking-machine shape udder. Using the 10 indicators,35 out of 41 cases of acute mastitis causing from coliform pathogens can be grouped correctly, and 48 out of 53 cases of non-coliform caused ones can also be grouped corectly, the accuracy percentage is 88.29%(sensitivity=0.85,specificity==0.90