Metatranscriptomic array analysis of “Candidatus Accumulibacter phosphatis”-enriched EBPR Sludge

Here we report a metatranscriptomic analysis of gene expression and regulation of “Candidatus Accumulibacter”-enriched lab-scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. The objectives were to investigate which genes were expressed during EBPR and which genes were differentially expressed between the early stage of anaerobic and aerobic phases (defined as 15 min after acetate addition and 15 min after switching to aeration respectively). A medium density oligonucleotide Combimatrix microarray, CustomArrayTM 12K, (Combimatrix Corporation, Mukilteo, WA) was designed by Combimatrix to target many predicted genes identified during the metabolism reconstruction of "Candidatus Accumulibacter phosphatis" and the flanking community members, bacterial community composition markers (such as 16S rRNA) and viral genes from viral metagenomic analysis of the same bioreactor. The 12,544 spots on the array consisted of 10,644 spots for user-designed probes, 326 spots for manufacture-designed quality controls, 70 spots for negative controls and 1,504 empty spots. A total of 3,548 unique probes, each with triplicate spots were randomly distributed on the array. Among them, 2,108 probes were designed to target 1,300 predicted genes from the bacterial metagenome. The remaining 1,440 probes targeted contigs from the viral metagenome. Among the 1,300 bacterial genes, 404 genes were targeted by 3 different probes and the remaining 896 genes were targeted by only one probe. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). Alexa Fluor 647-labeled aRNA from the anaerobic and aerobic samples were sequentially hybridized on the microarray to perform comparison of gene expression. Briefly, after the first hybridization and imaging, the hybridized aRNA on the array was stripped off under stringent conditions. The effectiveness of stripping was verified by imaging the array. After this, a second hybridization with a different sample was hybridized to this stripped microarray. The array manufacturer indicated that an array slide could be used with good reproducibility up to four times. The replicability of sequential uses was confirmed in our study. In this way, Alexa Fluor 647-labeled aRNA from the anaerobic and aerobic samples were sequentially hybridized on the same slide. Therefore, the comparison of gene expression profiles between anaerobic and aerobic samples was performed on two consecutive hybridizations on the same slide to minimize slide- and hybridization-dependent variation. A total of four hybridizations with alternating anaerobic and aerobic samples were applied on a single slide. Two microarray slides were used. Therefore, six pairs of such comparison on two separate slides were conducted (Hybridization 1 vs 2, 2 vs 3, 3 vs 4 on Slide 1, and Hybridization 5 vs 6, 6 vs 7 and 7 vs 8 on Slide 2).本研究报道了针对强化生物除磷（enhanced biological phosphorus removal, EBPR）过程中，富集候选聚磷菌（Candidatus Accumulibacter）的实验室规模污泥的基因表达与调控的宏转录组学分析（metatranscriptomic analysis）。研究设计了中密度寡核苷酸微阵列（medium density oligonucleotide microarrays），其探针靶向绝大多数被推测与EBPR表型密切相关的预测基因。 本研究的核心目标为：解析EBPR过程中表达的基因，以及厌氧阶段早期与有氧阶段早期（分别定义为乙酸盐投加后15 min与曝气启动后15 min）间的差异表达基因。 本研究使用的微阵列为Combimatrix公司设计制备的CustomArray™ 12K（Combimatrix Corporation, Mukilteo, WA）中密度寡核苷酸微阵列，其探针靶向三类序列：一是在候选聚磷菌（Candidatus Accumulibacter phosphatis）代谢重构及侧翼群落成员分析中鉴定得到的大量预测基因；二是用于表征细菌群落组成的标记基因（如16S rRNA）；三是来自同一生物反应器病毒宏基因组分析（viral metagenomic analysis）的病毒基因。 该微阵列共计12544个点样，其中10644个点样为用户设计的探针，326个点样为厂商设计的质量对照，70个点样为阴性对照，剩余1504个为空白点样。总计3548种独特探针，每种均设置三次重复点样并随机分布于阵列中。其中2108个探针靶向细菌宏基因组中的1300个预测基因：404个基因分别由3种不同探针靶向，剩余896个基因仅由1种探针靶向；余下1440个探针则靶向病毒宏基因组中的重叠群（contigs）。 研究分别在厌氧阶段早期与有氧阶段早期采集RNA样本，即乙酸盐投加后15 min与曝气启动后15 min。将厌氧与有氧样本的Alexa Fluor 647标记的扩增RNA（aRNA）依次进行微阵列杂交，以实现基因表达水平的比较。 具体流程如下：首次杂交并成像后，在严格条件下洗脱阵列上已杂交的aRNA，随后通过成像验证洗脱效果；之后将另一样本的aRNA杂交至该已洗脱的微阵列。该阵列厂商声称，一张阵列玻片可重复使用至多4次且重现性良好，本研究也验证了多次使用的可重复性。 通过该方式，厌氧与有氧样本的Alexa Fluor 647标记的aRNA可依次杂交至同一张玻片上，因此厌氧与有氧样本的基因表达谱比较可通过同一张玻片上的两次连续杂交完成，以最小化玻片与杂交过程引入的系统误差。 单张玻片上可进行总计4次交替使用厌氧与有氧样本的杂交实验。本研究共使用2张微阵列玻片，因此在两张独立玻片上共计完成6组此类比较：玻片1上为杂交1 vs 2、2 vs 3、3 vs 4；玻片2上为杂交5 vs 6、6 vs 7、7 vs 8。