A new custom microarray for sRNA profiling inEscherichia coli

Abstract

Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level.

Reprogramming of gene expression in Escherichia coli cultured on pyruvate versus glucose

“…The use of pyruvate and glucose as alternative carbon sources made it possible to compare the transcriptome profiles of E. coli cells that grow by utilizing either glycolytic (growth on M9/glucose) or gluconeogenetic (growth on M9/pyruvate) pathways. Analysis of gene expression differences was carried out by employing a custom microarray recently developed for simultaneous profiling of protein-coding genes and sRNAs in E. coli (Ruiz-Larrabeiti et al 2016). Moreover, the results of the transcriptome profiling were further compared with protein expression data obtained by two-dimensional protein gel electrophoresis.…”

“…Total RNA was isolated by using a modified hot phenol RNA extraction method (Khodursky et al 2003;Ruiz-Larrabeiti et al 2016). Briefly, aliquots of cell cultures grown in triplicate in M9/pyruvate or M9/glucose medium at 37°C with shaking (200 rpm) until OD 460 ∼ 0.4-0.6 were mixed with cold stop solution (5% phenol in ethanol) at an 8:1 ratio.…”

“…Northern blotting was performed essentially as described previously (Ruiz-Larrabeiti et al 2016). Briefly, total RNA was isolated from E. coli K-12 MG1655 strain grown in M9/glucose or M9/pyruvate.…”

“…To obtain a global overview of the gene co-expression network involved in coordination of central carbon metabolism and related pathways, we performed transcriptional profiling of E. coli cells grown in M9 minimal media supplemented with either pyruvate or glucose by using a custom microarray previously developed in our group (Ruiz-Larrabeiti et al 2016). The group of transcripts that showed at least twofold difference in the expression level in cells grown in M9/pyruvate when compared to those grown in M9/glucose were further analysed with the STRING database software (Franceschini et al 2013) to cluster the differentially expressed genes according to their biological roles.…”

Poly(A) polymerase is required for RyhB sRNA stability and function in Escherichia coli

“…PAP I adds poly(A) tails to many different classes of cellular RNAs (mRNAs, rRNAs, tRNAs, sRNAs, viral RNAs) (Régnier and Hajnsdorf 2013;Mohanty and Kushner 2016), and while the majority of E. coli ORFs undergo polyadenylation under exponential growth conditions, only a small fraction of them are polyadenylated at a specific time (Mohanty and Kushner 2006). Many sRNAs that do not require Hfq for stability and function have been shown to be polyadenylated in vivo, e.g., RNA I, Sok, Oop, SraL, SraG, and GlmY, and are subsequently degraded by exoribonucleases (Régnier and Hajnsdorf 2013;Ruiz-Larrabeiti et al 2016). Interestingly, previous data have shown that sRNAs that require Hfq for their stability, e.g., MicA and RybB, can also be targeted for degradation by PNPase and PAP I, but only when these sRNAs are not bound by Hfq (Andrade and Arraiano 2008;Andrade et al 2012;Cameron and De Lay 2016).…”