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

由于过去在环中性pH下分离铁氧化微生物的困难，很难证明原核生物具有在中性pH下在铁上自养生长的能力。最近，在pH为6的铁氧化条件下，纯化的细菌培养物已经被PI分离出来。本研究将重点利用其中的菌株ES-1作为模式生物，研究中性ph下铁氧化的生理生化。目标是建立这种微生物是一种化学岩石自养菌，并开始剖析其从铁氧化中保存能量和固定二氧化碳的代谢途径。研究将旨在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耐受性的限制，并为体外生化和分子研究培养足够的细胞生物量。进一步的研究将旨在纯化一种可能负责铁氧化的铁氧化还原酶，并确定电子传递系统的其他组分。嗜酸趋化自养铁氧化亚铁硫杆菌的电子传递链的两个组分将被用来在菌株ES-1中寻找类似的蛋白质。这是一种假定的质周电子传递蛋白和作为初始铁氧化酶的酶。我们将利用分子技术在菌株ES-1中寻找这些蛋白的基因，如果存在的话，我们将尝试用常规的生化技术纯化这些蛋白。我们还将采用分子方法来确定菌株ES-1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）；RuBPC/O基因亚基的杂交研究和PCR扩增都将进行。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。铁是地壳中含量第四丰富的元素；它也可能是所有生物体中最重要的微量元素。我们对微生物在中性ph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认识。由于铁的丰富，它的循环影响其他重要的元素循环，包括碳、氧和硫。铁氧化细菌经常出现在工业和市政供水系统中，它们会导致管道堵塞和生活供水变色，对经济产生重大影响。因此，了解这些生物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.