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Influence of Glucose Availability and CRP Acetylation on The ORIGINAL RESEARCH published: 23 May 2018 doi: 10.3389/fmicb.2018.00941 Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis Daniel V. Guebel 1 and Néstor V. Torres 2* 1 Biotechnology Counselling Services, Buenos Aires, Argentina, 2 Systems Biology and Mathematical Modelling Group, Department of Biochemistry, Microbiology, Cellular Biology and Genetics, Institute of Biomedical Technologies, Center for Biomedical Research of the Canary Islands, University of La Laguna, San Cristóbal de La Laguna, Spain Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple Edited by: pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, Marie-Joelle Virolle, we aimed to progress in the characterization of this regulation by dealing with three Centre National de la Recherche convergent aspects: the effector enzymes involved, the master regulator CRP, and the Scientifique (CNRS), France dependence on glucose availability. Reviewed by: Abhijit Shukla, Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 Memorial Sloan Kettering Cancer Center, United States relevant scenarios. These conditions arise when the wild type and four isogenic mutants Feng Jiang, (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and Chinese Academy of Medical R-type mutants of protein CRP) are studied under three levels of glucose availability Sciences, China (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). *Correspondence: acetylated Néstor V. Torres The Q-type emulates a permanent stage of CRP , whereas the R-type emulates [email protected] a permanent stage of CRPdeacetylated. The data were analyzed by an optimized factorial microarray method (Q-GDEMAR). Specialty section: This article was submitted to Results: (a) By analyzing one mutant against the other, we were able to unravel the true Microbial Physiology and Metabolism, genes that participate in the interaction between cobB/patZ mutations and glucose a section of the journal Frontiers in Microbiology availability; (b) Increasing stages of glucose limitation appear to be associated with the Received: 17 February 2018 up-regulation of specific sets of target genes rather than with the loss of genes present Accepted: 23 April 2018 when glucose is in excess; (c) Both CRPdeacetylated and CRPacetylated produce extensive Published: 23 May 2018 changes in specific subsets of genes, but their number and identity depend on the Citation: Guebel DV and Torres NV (2018) glucose availability; (d) In other sub-sets of genes, the transcriptional effect of CRP seems Influence of Glucose Availability and to be independent of its acetylation or deacetylation; (e) Some specific ontology functions CRP Acetylation on the Genome-Wide can be associated with each of the different sets of genes detected herein. Transcriptional Response of Escherichia coli: Assessment by an Conclusions: CRP cannot be thought of only as an effector of catabolite repression, Optimized Factorial Microarray Analysis. Front. Microbiol. 9:941. because it acts along all the glucose conditions tested (excess, limited, and exhausted), doi: 10.3389/fmicb.2018.00941 exerting both positive and negative effects through different sets of genes. Acetylation Frontiers in Microbiology | www.frontiersin.org 1 May 2018 | Volume 9 | Article 941 Guebel and Torres Integrating Acetylation Regulation of CRP does not seem to be a binary form of regulation, as there is not a univocal relationship between its activation/inhibitory effect and its acetylation/deacetylation stage. All the combinatorial possibilities are observed. During the exponential growth phase, CRP also exerts a very significant transcriptional effect, mainly on flagellar assembly and chemotaxis (FDR = 7.2 × 10−44). Keywords: Escherichia coli, post-translational modification, lysine acetylation regulation, CRP, acetyl transferase PatZ, deacetylase CobB, glucose, factorial analysis INTRODUCTION CRP is one of the three main transcriptional regulators known in E. coli as influenced by acetylation (Zhang et al., 2009). It is well known that microbial cells continuously sense their There are not many previous studies addressing catabolite external and internal environment, with this information being repression from the point of view of its regulation by filtered and integrated to trigger responses that allow them acetylation/deacetylation of CRP. Often, when studies referred to to best adapt to these stimuli (Snitkin and Segrè, 2008; Zhao regulation by acetylation, they were not actually addressing the et al., 2016). Beyond the “short-term adaptations” provided problem of glucose availability (Lima et al., 2011; Ma and Wood, by the allosteric regulations working alongside the extensive 2011; Weinert et al., 2013a; Zhang et al., 2013; Baeza et al., 2014). set of protein post-translational modifications, changes in the Moreover, the mechanism of lysine-acetylation/deacetylation is transcriptional responses are the main mechanisms for achieving also present in different proteins of E. coli as a type of post- the “long-term adjustments” required (Shimizu, 2014). So, there translational modification (Ma and Wood, 2011; Liu et al., 2014; are good reasons to analyse changes in the global transcriptome Castaño-Cerezo et al., 2015; Fraiberg et al., 2015; Drazic et al., of microorganisms. To this end, one technique that is widely used 2016). is the high-density array of oligonucleotides (microarrays). Although most of the studies in Table 1 used microarrays, it In fact, microarrays enable the analysis of the complete is well known that this tool has major limitations (Guarnaccia Escherichia coli transcriptome by monitoring the labeled et al., 2014; Yang et al., 2014; Chrominski and Tkacz, 2015). hybridization of around ten thousand probes. Moreover, Many of these limitations derive from the current algorithms transcriptome analysis also provides information about the applied, such as the Empirical Bayes and Benjamini-Hochberg non-coding RNAs (anti-sense RNAs and microRNAs), which algorithms. To avoid these problems, we have developed an have increasing importance as additional layer of regulation alternative algorithm called Q-GDEMAR (Quantile-Gaussian at post-transcriptional level, not only in eukaryotes but also deconvolution of microarrays), which provides a better in bacteria (Delihas, 2015; Cech et al., 2016; Tronnet et al., sensitivity of detection together with a low false discovery rate 2016). (FDR) (Guebel et al., 2016). Here, we will focus on the analysis of changes in the Further, all the studies in Table 1 only considered pair-wise transcriptome of some E. coli strains (wild-type and isogenic comparisons. The microarray data in our analyses, however, will mutants) when these bacteria are challenged with different levels be arranged as a factorial design, thus allowing us to account for of a typical carbon source such as glucose. The analyses include the interaction between the variables. Furthermore, unlike the three well-defined scenarios: the case of unrestricted glucose studies shown in Table 1 that “summarized” their microarrays availability (exponential phase of batch culture), the case of (Irizarry et al., 2003), to optimize our study, the microarray data glucose deprivation (stationary phase of batch culture), and will be analyzed here at the probe-level. In any case, of special the stage of transition between these extremes (glucose-limited importance is the fact that all the technical variants that we chemostat culture). Of course, it must be noted that multiple will use for the microarray analyses in the present investigation experimental studies of this type have been done over the last 10 have recently been discussed and tested successfully (Guebel and years (see Table 1). Also, some reviews are available (Fic et al., Torres, 2016; Guebel et al., 2016). 2009; Hu et al., 2010; Bernal et al., 2014; Choudhary et al., 2014; Note in Table 1 the occurrence of additional sources of Drazic et al., 2016; Wolfe, 2016). What then justifies the present variability, which affect the comparability of results among study? the studies. Thus, due to the different strains used by Inspection of Table 1 reveals some important aspects that different authors, significantly different regulatory features can justify the need to perform the present study. Hence, only a few be expected, even though all the strains assayed belong to the studies simultaneously integrate the three biological scenarios E. coli taxon (Yoon et al., 2012; Castaño-Cerezo et al., 2015; Vital employed herein. Moreover, our study aims not only to analyse etal.,2015;Monketal.,2016). Moreover, the presence of different the transcriptional effect of the glucose availability, but also trademarks in the microarray studies in Table 1 might imply a to establish the extent to which these effects are specifically higher prevalence of false responses (cross-reactivity) in some regulated through acetylation/deacetylation of the CRP protein. cases due to the shorter length of probes in some kits (Chou In fact, this protein is the main protein responsible for catabolite et al., 2004). Importantly, the interpretations are conditioned repression (Deutscher, 2008). Together with RcsB and RpoB, not only by the type of computing algorithm used, but also Frontiers in Microbiology | www.frontiersin.org 2 May
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