Physiology and Biochemistry of Microaerophilic Iron- oxidizing Bacteria Growing at Neutral pH

9723459艾默生由于过去在中性pH下分离氧化铁微生物有困难，因此很难证明原核生物有能力在中性pH下在铁上进行岩石自养生长。最近，在pH值为6的情况下，似乎通过铁氧化而生长的细菌的纯培养物已经被PI分离出来。本研究将以其中的一株菌株ES-1为模式生物，研究中性pH条件下铁氧化的生理生化过程。我们的目标是确定这种微生物是一种化学自养微生物，并开始剖析代谢途径，通过这些途径，它可以从铁氧化中保存能量，并固定二氧化碳。研究的目标将是开发一种连续培养系统，以确定在Fe2+上生长的细胞的摩尔生长量，确定Fe2+和O2的耐受性极限，并为体外生化和分子研究培养足够的细胞生物量。进一步的研究将致力于提纯一种可能导致铁氧化的铁氧化还原酶，并确定电子传递系统的其他成分。嗜酸性化学自体铁氧化剂氧化亚铁硫杆菌的电子传输链的两个组成部分将被用于在菌株ES-1中开始寻找类似的蛋白质。它们是黄花青素，一种假定的周质电子传递蛋白，以及作为初始铁氧化酶的酶。我们将使用分子技术在ES-1菌株中寻找这些蛋白质的基因，如果存在的话，将尝试使用传统的生化技术来纯化这些蛋白质。还将采用分子方法来确定在ES-1菌株中是否存在关键的二氧化碳固定酶--核酮糖1-5，即二磷酸羧化酶/加氧酶(RuBPC/O)；将进行RuBPC/O基因亚基的杂交研究和基因的PCR扩增。总之，这些研究将为未来研究一种独特类型的原核代谢的代谢生物化学奠定基础。铁是地壳中第四丰富的元素；它也可能是所有生物最重要的微量元素。我们对微生物在中性pH值下氧化铁所起的作用知之甚少。这项研究的意义在于，它将增加我们对生命在控制铁循环中所起作用的基础知识。由于铁的丰富，它的循环会影响其他重要的元素循环，包括碳、氧和硫。工业和市政供水系统中经常发现氧化铁细菌，它们会造成管道结垢和家庭供水变色，造成重大的经济影响。因此，了解这些生物体的基本代谢过程将有助于我们努力对它们施加积极或消极的控制。

9723459 Emerson Because of past difficulties with isolating iron-oxidizing microbes at circumneutral pH, it has been difficult to prove that prokaryotes have the capacity to grow lithoautotrophically on iron at neutral pH. Recently, pure cultures of bacteria that appear to grow lithotrophically by Fe-oxidation at pH 6 have been isolated by the PI. This research will focus on using one of these, strain ES-1, as a model organism to study the physiology and biochemistry of iron oxidation at neutral pH. The goals are to establish that this microbe is a chemolithoautotroph, and to begin to dissect the metabolic pathways by which it conserves energy from Fe-oxidation, and fixes CO2 . Studies will be aimed at developing a continuous culture system for determining molar growth yields of cells growing on Fe2+, determining limits for Fe2+ and O2 tolerance, and for growing enough cell biomass for in vitro biochemical and molecular studies. Further studies will be aimed at purifying an Fe-oxidoreductase enzyme that could be responsible for Fe-oxidation, and identifying other components of the electron transport system. Two components of the electron transport chain of the acidophilic chemolithoautorophic Fe-oxidizer Thiobacillus ferrooxidans will be used to initiate a search for analogous proteins in strain ES-1. These are rusticyanin, a putative periplasmic electron transport protein and the enzyme that serves as the initial Fe-oxidase. We will search for the genes for these proteins in strain ES-1 by using molecular techniques, and will attempt to purify the proteins, if present, using conventional biochemical techniques. A molecular approach will also be taken to establish whether or not the key CO2 fixation enzyme, ribulose 1-5, bisphosphate carboxylase/oxygenase (RuBPC/O), is present in strain ES-1; both hybridization studies and PCR amplification of genes for RuBPC/O gene subunits will be performed. Together these studies will lay the groundwork for future investigation of the metabolic bi ochemistry of a unique type of prokaryotic metabolism. Iron is the fourth most abundant element in the Earth's crust; it is also perhaps the most important trace element for all living organisms. We understand very little about the role that microorganisms play in oxidizing iron at neutral pH. The signficance of this research is that it will increase our fundamental knowledge about the role life plays in controlling the iron cycle. Due to the great abundance of iron its cycling influences other important elemental cycles including carbon oxygen and sulfur. Iron-oxidizing bacteria are often found in industrial and municipal water distribution systems where they can cause fouling of pipelines and discoloration of domestic water supplies with significant economic impact. Thus understanding the fundamental metabolic processes of these organisms will help in our efforts to exert positive or negative control over them.