Heermann, R. | Search bioRxiv

bioRxiv2020

Transcriptional regulation of the Nε-fructoselysine metabolism in Escherichia coli by global and substrate-specific cues

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products (ARPs), such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. While the degradation route for fructoselysine is well described, regulation of the corresponding pathway genes frlABCD remained poorly understood. Here we use bioinformatics combined with molecular and biochemical analyses and show that in E. coli, fructoselysine metabolism is tightly controlled at the transcriptional level. The global regulator Crp (CAP), as well as the alternative sigma factor {sigma}32 (RpoH) contribute to promoter activation at high cAMP-levels and heat stress, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific roadblock repressor. Our study provides profound evidence that the interplay of global and substrate-specific regulation is a perfect adaptation strategy to efficiently utilize unusual substrates within the human gut environment. Abbreviated SummaryThermal food processing promotes the formation of Amadori rearrangement products (ARPs), such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. We show that in E. coli, fructoselysine metabolism is tightly controlled at the transcriptional level by global and substrate-specific regulators. Their interplay is a perfect adaptation strategy to efficiently utilize fructoselysine within the human gut environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/904318v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@15bbc3borg.highwire.dtl.DTLVardef@1a2e3eforg.highwire.dtl.DTLVardef@12261c6org.highwire.dtl.DTLVardef@412c7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Deciphering the rules underlying xenogeneic silencing and counter-silencing of Lsr2-like proteins

Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum. Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC-profile close to the transcription start site (TSS), but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters resulting in an increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability highlighting the potential for biotechnological applications. Altogether, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shapes evolutionary network expansion in this medically- and biotechnologically-relevant bacterial phylum.\n\nIMPORTANCEIn actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC-profile in combination with a short, sequence specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing thereby facilitating the integration of new genetic traits into host regulatory networks.

microbiology↗