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

9723459爱默生由于过去在中性pH下分离铁氧化微生物的困难，很难证明原核生物有能力在中性pH下在铁上生长。最近，PI分离了在pH 6下通过铁氧化生长的纯细菌培养物。本研究将侧重于使用其中之一，菌株ES-1，作为一个模式生物，研究在中性pH值的铁氧化的生理和生物化学。目标是建立这种微生物是一个chemolithoautotroph，并开始解剖的代谢途径，它保存能量从铁氧化，并固定CO2。 研究的目的是开发一种连续培养系统，用于确定在Fe 2+上生长的细胞的摩尔生长产量，确定Fe 2+和O2耐受性的限度，以及为体外生物化学和分子研究生长足够的细胞生物量。 进一步的研究将旨在纯化可能负责铁氧化的铁氧化还原酶，并鉴定电子传递系统的其他组分。 嗜酸性chemolithoautorophic铁氧化剂氧化亚铁硫杆菌的电子传递链的两个组件将被用来启动在菌株ES-1中的类似蛋白质的搜索。 这些是rusticyanin，一种假定的周质电子传递蛋白和作为初始Fe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，并尝试使用常规生物化学技术纯化蛋白质（如果存在）。 还将采取分子方法来确定ES-1菌株中是否存在关键的CO2固定酶核酮糖1-5、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PCR扩增。 总之，这些研究将为未来研究一种独特的原核代谢类型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体最重要的微量元素。 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本知识。 由于铁的大量存在，它的循环影响其他重要的元素循环，包括碳、氧和硫。 铁氧化细菌通常存在于工业和市政配水系统中，它们可导致管道结垢和家庭供水变色，并产生重大的经济影响。 因此，了解这些生物体的基本代谢过程将有助于我们对它们进行积极或消极的控制。

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.