Physiology and Molecular Ecology of Microbial Filamentous Sulfur Formation

近年来，我们发现了一种不寻常的海洋细菌，它将硫化氢氧化成一种新型的丝状形式的元素硫，同时将二氧化碳固定到细胞材料中。 系统发生学研究将该菌归入变形菌门的弓形菌属。 微生物丝状硫产生发生在高流体流动的海洋硫化物环境中，包括沿海盐沼小溪、硫化物沉积物和世界范围内的海洋热液喷口。 在之前的研究期间，我们能够从沿海沉积物（沿海菌株pueridatus命名为“Arcobacter sulfidicus”）和深海热液环境中培养出这种产毒生物，并研究了这一有趣群体的生理学、形态学和繁殖力。本项目的总体目标是扩大对丝状硫形成的微生物生态学的研究。 到目前为止，人们对“Arcidatus Arcobacter sulfidicus”样生物的丰度、分布、多样性和功能知之甚少。 将采用荧光原位杂交（FISH）与现有和新开发的不同特异性靶向16 S rRNA的荧光标记寡核苷酸在环境样品中评估Abbands和分布。 将从硫化物海洋和热液环境中提取基因组DNA和RNA，利用变性梯度凝胶电泳、小亚基rRNA基因克隆和测序进行多样性分析。 这些数据将成为设计更精细的组、种和菌株特异性16 S rRNA探针的基础。 微电极测量将与FISH配合使用，以测量实验室和环境样品中的相关理化参数。 基于1）不存在Rubisco（许多细菌和绿色植物用于将CO2掺入细胞材料中的代谢途径的关键酶）和2）细胞的小d13 C分馏，“硫化弓形杆菌”可能通过除Calvin-Bassham-Benson循环之外的替代途径固定CO2。 因此，这种生物体似乎是第一种无色的硫氧化化能无机自养细菌，它不使用广泛存在的卡尔文-巴沙姆-本森途径进行碳固定。 这是一个具有进化意义的发现，我们建议在我们的模型沿海菌株中阐明该途径。 证据还表明，这种生物能够固氮，这将在其他研究中得到证实。这项研究的总体意义在于表征这种独特的自养硫化物氧化过程发生的参数和机制，以及在许多不同环境中负责微生物的丰度，分布和基因型多样性。 最后，这些结果将增强我们对一种新颖的、迄今为止尚未被考虑的硫循环组分的生理学和生态学的认识。 丝状硫化物的形成可能是热液喷口的一个重要过程，延伸到浅层地下生物圈，仅由无机营养物（即H2S和CO2，N2）驱动。

In recent years, we have discovered an unusual marine bacterium that oxidizes hydrogen sulfide into a novel filamentous form of elemental sulfur, while fixing CO2 into cell material. Phylogenetic studies have placed this organism into the genus Arcobacter of the epsilon subdivision of the Proteobacteria. Microbial filamentous sulfur production occurs in high fluid flow marine sulfidic environments, including coastal salt marsh creeks, sulfidic sediments, and marine hydrothermal vents worldwide. During the previous research period, we were able to cultivate this filament-producing organism from coastal sediments (coastal strain putatively named "Candidatus Arcobacter sulfidicus") and deep-sea hydrothermal environments, and have investigated the physiology, morphology and phylogeny of this interesting group. The overall goal of the present project will be to expand studies on the microbial ecology of filamentous sulfur formation. So far, little is known of the abundance, distribution, diversity, and function of "Candidatus Arcobacter sulfidicus"-like organisms in situ. Abundance and distribution will be assessed in environmental samples by employing fluorescent in situ hybridization (FISH) with existing and newly developed fluorescently labeled oligonucleotides of different specificity targeting the 16S rRNA. Genomic DNA and RNA will be extracted from sulfidic marine and hydrothermal environments for diversity analyses using denaturing gradient gel electrophoresis (DGGE), small-subunit rRNA gene cloning and sequencing. These data will form the basis for the design of more refined group-, species-, and strain-specific 16S rRNA probes. Microelectrode measurements will be used in concert with FISH to measure relevant physicochemical parameters in laboratory and environmental samples. "Candidatus Arcobacter sulfidicus" is likely to fix CO2 by an alternative pathway other than the Calvin-Bassham-Benson cycle based on 1) the absence of Rubisco, a key enzyme of a metabolic pathway used by many bacteria and green plants for incorporating CO2 into cell material, and 2) the small d13C fractionation by the cells. Thus, this organism seems to be the first colorless sulfur-oxidizing chemolithoautotrophic bacterium that does not use the widespread Calvin-Bassham-Benson pathway for carbon fixation. This is a finding of evolutionary significance and we propose to elucidate the pathway in our model coastal strain. Evidence also suggests that this organism is capable of fixing nitrogen and this will be confirmed in additional studies. The overall significance of this research lies in the characterization of the parameters and mechanisms by which this unique process of autotrophic sulfide oxidation occurs, as well as the abundance, distribution and genotypic diversity of the responsible microbes in a number of different environments. Finally, the results will enhance our knowledge of the physiology and ecology of a novel, and heretofore-unconsidered component of the sulfur cycle. Filamentous sulfur formation may be an important process at hydrothermal vents, extending into the shallow subsurface biosphere and driven by inorganic nutrients alone (i.e. H2S and CO2, N2).