Biphasic polysaccharide metabolism of Alteromonas macleodii 83-1 analyzed by RNA-Seq

Polysaccharides from macroalgae are important bacterial nutrient source and central biogeochemical component in the oceans. To illuminate the cellular mechanisms of polysaccharide degradation by marine bacteria, growth of Alteromonas macleodii 83-1 on a mix of laminarin, alginate and pectin was characterized using transcriptomics, proteomics and exometabolomics. A. macleodii 83-1 showed two distinct growth stages, with exponential growth during laminarin utilization followed by maintenance during simultaneous alginate/pectin utilization. The biphasic growth coincided with major temporal shifts in gene expression and metabolite secretion, enabling to define major/accessory polysaccharide utilization loci, reconstruct the complete degradation pathways for each polysaccharide, as well as identify temporal phenotypes in other relevant traits. FT-ICR-MS revealed a distinct suite of secreted metabolites for each growth phase, with pyrroloquinoline quinone exclusively produced with alginate/pectin. The finding of substrate-unique phenotypes indicates an exquisite adaptation to polysaccharide utilization with probable relevance for the degradation of macroalgal biomass, which comprises a complex mix of polysaccharides. Moreover, substrate-unique exometabolomes possibly influence metabolic interactions with other community members. Overall, the presence of fine-tuned genetic machineries for polysaccharide degradation and the widespread detection of related CAZymes in global locations indicate an ecological relevance of A. macleodii in marine polysaccharide cycling and bacteria-algae interactions. Overall design: Alteromonas macleodii 83-1 cells were grown on a mix of three polysaccharides (laminarin, pectin, alginate) in triplicates (Mix1-3). As control, cells were grown on glucose also in triplicates (Glucose1-3). For both incubations, biomass samples were taken at two time points; T1 (13h) and T2 (21h of incubation) and transcriptomics analyses were performed by doing RNA-Seq. Please note that the FASTA file contains the nucleotide sequences of all genes of the genome, which were used as a reference to obtain transcript abundance counts by aligning them onto the gene sequences. The genome sequence is deposited in the DOE JGI-IMG/MER database (accession number 2716884210).大型藻类（macroalgae）来源的多糖是海洋中关键的细菌营养底物与核心生物地球化学组分。 为阐明海洋细菌降解多糖的细胞机制，本研究采用转录组学（transcriptomics）、蛋白质组学（proteomics）与外代谢组学（exometabolomics）技术，对麦克劳德交替单胞菌（Alteromonas macleodii）83-1在昆布多糖（laminarin）、褐藻胶（alginate）与果胶（pectin）混合底物上的生长特征进行了系统表征。 该菌株展现出两个截然不同的生长阶段：在利用昆布多糖的过程中处于指数生长期，随后在同时利用褐藻胶与果胶的阶段进入生长维持期。 双相生长与基因表达及代谢物分泌的显著时间动态变化高度吻合，借此得以界定主要/辅助多糖利用基因座（polysaccharide utilization loci），重构各多糖的完整降解通路，并识别其他相关性状的时间依赖性表型。 傅里叶变换离子回旋共振质谱（FT-ICR-MS）分析显示，每个生长阶段均存在独特的分泌代谢物组，其中吡咯喹啉醌（pyrroloquinoline quinone）仅在利用褐藻胶与果胶时合成。 底物特异性表型的发现表明，该菌株对多糖利用具备精细的适应性机制，这可能与由多种多糖复合构成的大型藻类生物质降解过程密切相关。 此外，底物特异性外代谢组可能会调控与其他群落成员之间的代谢互作。 综上，用于多糖降解的精细调控遗传装置的存在，以及相关碳水化合物活性酶（CAZymes）在全球范围内的广泛检出，表明麦克劳德交替单胞菌在海洋多糖循环及菌藻互作中具有重要的生态学意义。 实验总体设计：将麦克劳德交替单胞菌83-1接种于由昆布多糖、果胶、褐藻胶组成的复合底物中，设置3次生物学重复（编号Mix1-3）；以葡萄糖为单一底物培养菌株作为对照，同样设置3次生物学重复（编号Glucose1-3）。两类培养体系均在两个时间点采集菌体样本：T1（培养13小时）与T2（培养21小时），并通过RNA测序（RNA-Seq）开展转录组学分析。 请注意：本研究提供的FASTA格式文件包含该菌株全基因组的所有基因核苷酸序列，可作为参考序列，通过将测序reads比对至基因序列上以获取基因转录丰度计数。该基因组序列已提交至美国能源部联合基因组研究所（DOE JGI）IMG/MER数据库，登录号为2716884210。