Lin S, Henze S, Lundgren P, Bergman B, Carpenter EJ
Marine Sciences Research Center, State University of New York, Stony Brook, New York 11794, USA.
Appl Environ Microbiol. 1998 Aug;64(8):3052-8. doi: 10.1128/AEM.64.8.3052-3058.1998.
The mechanism by which planktonic marine cyanobacteria of the genus Trichodesmium fix N2 aerobically during photosynthesis without heterocysts is unknown. As an aid in understanding how these species protect nitrogenase, we have developed an immunofluorescence technique coupled to light microscopy (IF-LM) with which intact cyanobacteria can be immunolabeled and the distribution patterns of nitrogenase and other proteins can be described and semiquantified. Chilled ethanol was used to fix the cells, which were subsequently made permeable to antibodies by using dimethyl sulfoxide. Use of this technique demonstrated that about 3 to 20 cells (mean +/- standard deviation, 9 +/- 4) consecutively arranged in a Trichodesmium trichome were labeled with the nitrogenase antibody. The nitrogenase-containing cells were distributed more frequently around the center of the trichome and were rarely found at the ends. On average 15% of over 300 randomly encountered cells examined contained nitrogenase. The percentage of nitrogenase-containing cells (nitrogenase index [NI]) in an exponential culture was higher early in the light period than during the rest of the light-dark cycle, while that for a stationary culture was somewhat constant at a lower level throughout the light-dark cycle. The NI was not affected by treatment of the cultures with the photosynthetic inhibitor dichloro 1,3'-dimethyl urea or with low concentrations of ammonium (NH4Cl). However, incubation of cultures with 0.5 &mgr;M NH4Cl over 2 days reduced the NI. The IF technique combined with 14C autoradiography showed that the CO2 fixation rate was lower in nitrogenase-containing cells. The results of the present study suggest that (i) the IF-LM technique may be a useful tool for in situ protein localization in cyanobacteria, (ii) cell differentiation occurs in Trichodesmium and only a small fraction of cells in a colony have the potential to fix nitrogen, (iii) the photosynthetic activity (CO2 uptake) is reduced if not absent in N2-fixing cells, and (iv) variation in the NI may be a modulator of nitrogen-fixing activity.
束毛藻属的浮游海洋蓝细菌在光合作用过程中如何在没有异形胞的情况下进行需氧固氮的机制尚不清楚。为了有助于理解这些物种如何保护固氮酶，我们开发了一种与光学显微镜相结合的免疫荧光技术（IF-LM），利用该技术可以对完整的蓝细菌进行免疫标记，并描述和半定量固氮酶及其他蛋白质的分布模式。使用冷乙醇固定细胞，随后通过使用二甲基亚砜使细胞对抗体具有通透性。使用该技术表明，在束毛藻丝状体中连续排列的约3至20个细胞（平均值±标准差，9±4）被固氮酶抗体标记。含固氮酶的细胞更频繁地分布在丝状体中心周围，很少出现在末端。在随机检查的300多个细胞中，平均有15%的细胞含有固氮酶。指数生长期培养物中含固氮酶细胞的百分比（固氮酶指数[NI]）在光照期早期高于光暗循环的其余时间，而静止期培养物的NI在整个光暗循环中在较低水平上基本保持恒定。NI不受光合抑制剂二氯1,3'-二甲基脲或低浓度铵（NH4Cl）处理培养物的影响。然而，用0.5 μM NH4Cl培养培养物2天会降低NI。IF技术与14C放射自显影相结合表明，含固氮酶细胞中的CO2固定率较低。本研究结果表明：（i）IF-LM技术可能是用于蓝细菌原位蛋白质定位的有用工具；（ii）束毛藻中发生细胞分化，且群体中只有一小部分细胞具有固氮潜力；（iii）固氮细胞中的光合活性（CO2吸收）即使没有消失也会降低；（iv）NI的变化可能是固氮活性的调节剂。
