School of Biotechnology and Biomolecular Sciences, University of New South Wales Sydney, Sydney, New South Wales, Australia.
Bioanalytical Mass Spectrometry Facility, University of New South Wales Sydney, Sydney, New South Wales, Australia.
Appl Environ Microbiol. 2019 Mar 6;85(6). doi: 10.1128/AEM.02935-18. Print 2019 Mar 15.
The canonical pathway for sucrose metabolism in haloarchaea utilizes a modified Embden-Meyerhof-Parnas pathway (EMP), in which ketohexokinase and 1-phosphofructokinase phosphorylate fructose released from sucrose hydrolysis. However, our survey of haloarchaeal genomes determined that ketohexokinase and 1-phosphofructokinase genes were not present in all species known to utilize fructose and sucrose, thereby indicating that alternative mechanisms exist for fructose metabolism. A fructokinase gene was identified in the majority of fructose- and sucrose-utilizing species, whereas only a small number possessed a ketohexokinase gene. Analysis of a range of hypersaline metagenomes revealed that haloarchaeal fructokinase genes were far more abundant (37 times) than haloarchaeal ketohexokinase genes. We used proteomic analysis of (which encodes fructokinase) and identified changes in protein abundance that relate to growth on sucrose. Proteins inferred to be involved in sucrose metabolism included fructokinase, a carbohydrate primary transporter, a putative sucrose hydrolase, and two uncharacterized carbohydrate-related proteins encoded in the same gene cluster as fructokinase and the transporter. Homologs of these proteins were present in the genomes of all haloarchaea that use sugars for growth. Enzymes involved in the semiphosphorylative Entner-Doudoroff pathway also had higher abundances in sucrose-grown cells, consistent with this pathway functioning in the catabolism of the glucose moiety of sucrose. The study revises the current understanding of fundamental pathways for sugar utilization in haloarchaea and proposes alternatives to the modified EMP pathway used by haloarchaea for sucrose and fructose utilization. Our ability to infer the function that microorganisms perform in the environment is predicated on assumptions about metabolic capacity. When genomic or metagenomic data are used, metabolic capacity is inferred from genetic potential. Here, we investigate the pathways by which haloarchaea utilize sucrose. The canonical haloarchaeal pathway for fructose metabolism involving ketohexokinase occurs only in a small proportion of haloarchaeal genomes and is underrepresented in metagenomes. Instead, fructokinase genes are present in the majority of genomes/metagenomes. In addition to genomic and metagenomic analyses, we used proteomic analysis of (which encodes fructokinase but lacks ketohexokinase) and identified changes in protein abundance that related to growth on sucrose. In this way, we identified novel proteins implicated in sucrose metabolism in haloarchaea, comprising a transporter and various catabolic enzymes (including proteins that are annotated as hypothetical).
在嗜盐古菌中，蔗糖代谢的经典途径利用了一种改良的 EMP 途径（EMP），其中酮己糖激酶和 1-磷酸果糖激酶磷酸化蔗糖水解释放的果糖。然而，我们对嗜盐古菌基因组的调查确定，并非所有已知利用果糖和蔗糖的物种都存在酮己糖激酶和 1-磷酸果糖激酶基因，这表明存在替代果糖代谢机制。在大多数利用果糖和蔗糖的物种中都鉴定到了果糖激酶基因，而只有少数物种具有酮己糖激酶基因。对一系列高盐共生体宏基因组的分析表明，嗜盐古菌果糖激酶基因的丰度（是嗜盐古菌酮己糖激酶基因的 37 倍）要高得多。我们利用 （编码果糖激酶）的蛋白质组学分析，并鉴定了与蔗糖生长相关的蛋白质丰度变化。推断与蔗糖代谢有关的蛋白质包括果糖激酶、碳水化合物初级转运蛋白、假定的蔗糖水解酶以及与果糖激酶和转运蛋白在同一基因簇中编码的两种未鉴定的碳水化合物相关蛋白。所有利用糖生长的嗜盐古菌基因组中都存在这些蛋白质的同源物。参与半磷酸化 Entner-Doudoroff 途径的酶在蔗糖生长的 细胞中也有更高的丰度，这与该途径在蔗糖葡萄糖部分的分解代谢中起作用是一致的。该研究修订了当前对嗜盐古菌糖利用基本途径的理解，并提出了替代嗜盐古菌利用蔗糖和果糖的改良 EMP 途径的方案。我们推断微生物在环境中执行的功能的能力取决于对代谢能力的假设。当使用基因组或宏基因组数据时，代谢能力是根据遗传潜力推断出来的。在这里，我们研究了嗜盐古菌利用蔗糖的途径。涉及酮己糖激酶的经典嗜盐古菌果糖代谢途径仅存在于一小部分嗜盐古菌基因组中，在宏基因组中代表性不足。相反，果糖激酶基因存在于大多数基因组/宏基因组中。除了基因组和宏基因组分析外，我们还利用 （编码果糖激酶但缺乏酮己糖激酶）的蛋白质组学分析，并鉴定了与蔗糖生长相关的蛋白质丰度变化。通过这种方式，我们在嗜盐古菌中鉴定到了参与蔗糖代谢的新蛋白质，包括转运蛋白和各种分解代谢酶（包括被注释为假设的蛋白质）。
