Growth-associated gene essentiality in Streptomyces coelicolor.

链霉菌种类有大量的特征，可能是多余的，在快速生长的水下培养。这些包括形态分化和次级代谢（如抗生素生产）。尽管如此，链霉菌发酵培养物被用于许多生物产品的生产，在这些过程中，快速、高密度的生长是至关重要的。许多实验室正在试图确定能够提高链霉菌在发酵罐中的性能的特征，从而定义能够高效合成新产品的“超级宿主”菌株的特征。coelicolor链霉菌是该属中第一个被测序的物种，对其分子生物学的了解比其他任何物种都多，因此它可能是发育成超级寄主的良好候选者。然而，许多发现很难在发酵培养中快速生长。这些想法形成了我们对了解不同基因的需求如何作为生长速率的函数而变化的兴趣的背景。我们对最近发表的S coelicolor基因组代谢网络中酶的基因编码特别感兴趣，因为我们可以进行计算机建模，预测网络中代表的每个基因的缺失对生长速度的影响。因此，基因本质上是建模研究中的一个相对术语，一些基因“比其他基因更重要”，这取决于它们的缺失对生长速度的影响。在这项研究中，我们计划将这种定量重要性概念扩展到体内实验，将两种基因重要性测量方案中的一种应用于以不同速率生长的细胞。因此，我们将确定在每种生长速率下哪些基因是必需的，并将这些发现与计算机预测进行比较。我们也有改进现有预测技术的想法，这个项目的成功结果将使我们能够第一次用实验室实验验证预测。我们将比较那些似乎与生长速度相关的基因与那些在快速生长的菌株中表达的基因，这些菌株是在趋化器中选择的，这是一种迫使细胞以我们指定的速度生长的装置。我们还将使用这个装置迫使亲本菌株以不同的速度生长，这样我们就可以确定表达对生长速度有反应的基因是否就是其重要性随生长速度而变化的基因。我们的体内基因本质实验，使用现有的技术，包括将一群突变体，其中每一个可能的基因敲除都代表了，在我们把它们放入恒温器之前，在琼脂上镀上，以确定哪些突变体可以在恒温器中以不同的生长速度存活。可以认为，这一初始电镀步骤本身是选择性的，因为只有那些能够在板上形成菌落的人才能进入恒化器。这可能是不可避免的，但不应过分削弱我们研究结果的有效性。然而，我们也计划了一种以前从未在链霉菌中尝试过的方法，这种方法将不允许基因沉默突变表现出来，直到突变体在化学调节器中，迫使它们以每个指定的速度生长。如果我们能让这种新方法起作用（我们的团队经验丰富，给了我们信心），那么它就会被使用。如果不能，我们将退回到更传统的方法。

Streptomyces species have a large number of characteristics that are, presumably, redundant during rapid growth in submerged culture. These include morphological differentiation and secondary metabolism (e.g. antibiotic production). Despite this, Streptomyces fermenter cultures are used for the production of many bioproducts, in processes in which rapid, high density growth is of paramount importance. Many laboratories are trying to identify characteristics that would enhance the performance of Streptomyces species in fermenters, thereby defining the characteristics of a 'superhost' strain, capable of synthesising new products, with high efficiency. Streptomyces coelicolor was the first species in the genus to be sequenced and more is known about its molecular biology than that of any other species, so it is potentially a good candidate for development into a superhost. However, many find it very difficult to grow at rapid rates in fermenter culture. These ideas form the background to our interest in knowing how the requirements for different genes change as a function of growth rate. We are particularly interested in genes coding for enzymes that are part of the recently-published S coelicolor genome scale metabolic network, as we can carry out computer modelling that predicts the effect of deletion of each gene, represented in the network, on growth rate. Gene essentially is therefore, a relative term in modelling studies, some genes being 'more essential than others' depending on the effect that their deletion has on the growth rate. In this study, we plan to extend this quantitative essentiality concept to in vivo experimentation, with the application of one of two gene essentiality measurement protocols to cells growing at different rates. Thus we will determine which genes are essential at each growth rate and compare these findings to the computer predictions. We also have ideas for improving on existing prediction techniques and, a successful outcome to this project will enable us to validate the predictions with laboratory experiments, for the first time. We will compare those genes that seem to have growth rate related essentiality to those that are expressed in a rapidly growing strain, selected in a chemostat, a device that forces cells to grow at the rate that we specify. We will also force the parental strain to grow at different rates, using this apparatus, so that we can determine whether the genes whose expression responds to growth rate, are the same genes whose essentiality varies with the growth rate. Our in vivo gene essentiality experiment, using existing techniques, involves plating a population of mutants, in which every possible gene knock-out is represented, onto agar before we put them in the chemostat to determine which mutants can survive in the chemostat at each of a number of growth rates. It could be argued that this initial plating step is, itself, selective as only those capable of forming colonies on plates will make it into the chemostat. It may be that this will be inevitable, and should not unduly detract from the validity of our findings. However, we also plan an approach, never previously attempted in Streptomyces, that will not allow gene silencing mutations to manifest themselves, until the mutants are in the chemostat under the conditions that force them to grow at each specified rate. If we can make this new approach work (we have a team whose breadth of experience give us confidence that we will) then it will be used. If not, we will fall back on the more conventional approach.