Metatranscriptomic analysis of the response of river biofilms to pharmaceutical products, using anonymous DNA microarrays

Abstract

Pharmaceutical products are released at low concentrations into aquatic environments following domestic wastewater treatment. Such low concentrations have been shown to induce transcriptional responses in microorganisms, which could have consequences on aquatic ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two rivers of differing trophic statuses and subsequently treated with environmentally relevant doses (ng/liter to microg/liter range) of four pharmaceuticals (erythromycin [ER], gemfibrozil [GM], sulfamethazine [SN], and sulfamethoxazole [SL]). To monitor functional gene expression, we constructed a 9,600-feature anonymous DNA microarray platform onto which cDNA from the biofilms was hybridized. Pharmaceutical treatments induced both positive and negative transcriptional responses from biofilm microorganisms. For instance, ER induced the transcription of several stress, transcription, and replication genes, while GM, a lipid regulator, induced transcriptional responses from several genes involved in lipid metabolism. SN caused shifts in genes involved in energy production and conversion, and SL induced responses from a range of cell membrane and outer envelope genes, which in turn could affect biofilm formation. The results presented here demonstrate for the first time that low concentrations of small molecules can induce transcriptional changes in a complex microbial community. The relevance of these results also demonstrates the usefulness of anonymous DNA microarrays for large-scale metatranscriptomic studies of communities from differing aquatic ecosystems.

FIG. 1.

Correspondence analysis biplot of anonymous microarray hybridization patterns for cDNA extracted from biofilms growing in water from Wascana Creek (a) and the South Saskatchewan River (b) and subjected to environmentally relevant doses of pharmaceutical compounds. Positions of the treatments are the mean values for the replicates (n = 2 to 3). Only the probes listed in Tables S2 to S9 in the supplemental material are depicted. ▴, probes associated exclusively with SL; ▾, probes associated exclusively with SN; ⧫, probes associated exclusively with ER; ▪, probes associated exclusively with GM; •, probes associated with multiple treatments. In a, probes in black were used in Fig. 2 or for real-time PCR quantification. In b, probes in black are the ones mentioned in Table 1. CO, control; CA, correspondence analysis.

FIG. 2.

Average log-2 expression values for selected probes following exposure of Wascana Creek biofilms to different pharmaceutical products. COG C (energy production and conversion), sum of probes 2003, 5271, 13407, 13905, and 16399; COG I (lipid transport and metabolism), sum of probes 6171, 8775, and 19275; COG M (cell envelope biogenesis, outer membrane), sum of probes 847, 14137, and 14385; COG T (signal transduction), sum of probes 1849, 5589, and 8271. DNA polymerase (COG L [replication, recombination, and repair]), probe 2027; RNA polymerase (COG K [transcription]), probe 13807; the flagellar synthesis regulator FleN (COG D [cell division and chromosome partitioning]), probe 18099; the chaperone protein DnaK (COG O [posttranslational modification, protein turnover, and chaperones]), probe 13925; ppGpp synthetase I (COG K [transcription] and COG T [signal transduction]), probe 8047; Chitinophaga pinensis, sum of probes 605, 14393, and 19439; Haliangium ochraeum, sum of probes 739 and 8467; Hyphomonas neptunium, sum of probes 8129 and 14253. See Tables S2 to S5 in the supplemental material for more details about the probes. CO, control. Error bars represent the standard deviations.