Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.
Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.
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DOI：
10.1128/mbio.00282-15
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发表时间：
2015-05-19
期刊：
影响因子：
6.4
通讯作者：
Coates JD
中科院分区：
文献类型：
--
作者：
Clark IC;Melnyk RA;Youngblut MD;Carlson HK;Iavarone AT;Coates JD
尽管有证据表明呼吸基因水平基因转移普遍存在，但对于这些途径如何在新宿主内功能性整合却知之甚少。可移动呼吸代谢的一个例子是细菌的氯酸盐还原，它经常编码在复合转座子上。这意味着代谢的关键组分是由这些可移动元件编码的。为了验证这一点，我们在不能还原氯酸盐的希瓦氏菌属奥奈达湖菌MR - 1中异源表达了来自海藻希瓦氏菌ACDC的氯酸盐还原基因。最终能在氯酸盐上稳健生长的构建体包含cld（一个细胞色素c基因）、clrABDC以及两个功能未知的基因。尽管在将这些基因最初插入染色体后，菌株MR - 1不能在氯酸盐上生长，但通过适应性进化获得了11个能够进行氯酸盐呼吸的衍生菌株。基因组重测序表明，所有进化出的氯酸盐还原菌株都复制了一个包含氯酸盐还原基因的大片段基因组区域。还观察到拷贝数的减少以及还原氯酸盐能力的丧失，这表明这种现象是极其动态的。尽管大多数菌株包含超过六个复制区域的拷贝，但一个重复较少的单一菌株也能快速生长。这个菌株包含三个额外的突变，我们推测这些突变弥补了低拷贝数的不足。我们在未进化的菌株中组合重现了这些突变，并确定cld上游的一个单核苷酸多态性（SNP）使菌株能够在氯酸盐上生长，并且相对于在narQP和nrfA之间NarP结合序列中的第二个碱基对变化是上位性的，该变化促进了生长。
氯酸盐还原复合转座子在转移到新宿主后形成功能性代谢的能力是它们传播的一个重要部分。为了研究这一现象，我们将希瓦氏菌属奥奈达湖菌MR - 1改造成一个氯酸盐还原菌。我们确定了一组足以使菌株从质粒上获得在氯酸盐上生长能力的基因，但发现将这些基因插入染色体是无功能的。这个无功能的菌株进化为氯酸盐还原菌表明串联重复是激活的主要机制。虽然拷贝数变化是增加基因剂量的一种相对快速的方法，但复制近1兆碱基的额外DNA是有代价的。预计会出现缓解对高拷贝数需求的突变，并最终占主导地位，并且我们确定了一个缓解拷贝数要求的单核苷酸多态性（SNP）。这项研究同时使用了理性和进化的方法来深入了解一种引人入胜的呼吸代谢的进化。
Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. The ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism.