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This study provides a genome-wide analysis of changes in promoter-proximal polymerase II (Pol II) pausing on host genes induced by HSV-1 infection. It shows that standard measures of pausing, i.e., pausing indices, do not properly capture the complex and unsuspected alterations in Pol II pausing occurring in HSV-1 infection.

Herpes simplex virus 1 (HSV-1) infection exerts a profound shut-off of host gene expression at multiple levels. Recently, HSV-1 infection was reported to also impact promoter-proximal RNA Polymerase II (Pol II) pausing, a key step in the eukaryotic transcription cycle, with decreased and increased Pol II pausing observed for activated and repressed genes, respectively. Here, we demonstrate that HSV-1 infection induces more complex alterations in promoter-proximal pausing than previously suspected for the vast majority of cellular genes. While pausing is generally retained, it is shifted to more downstream and less well-positioned sites for most host genes. We exclude that this is an artefact of alternativede novotranscription initiation at downstream sites or read-in transcription originating from disruption of transcription termination for upstream genes. Use of downstream 2ndpause sites associated with +1 nucleosomes was previously observed upon NELF depletion. However, downstream shifts of Pol II pausing upon HSV-1 infection are much more pronounced than observed upon NELF depletion. Thus, our study reveals a novel aspect in which HSV-1 infection fundamentally reshapes host transcriptional processes with implications for our understanding of maintenance of promoter-proximal Pol II pausing in eukaryotic cells.

For canonical asparagine glycosylation, the primary amino acid sequence that directs glycosylation at specific asparagine residues is well-established. Here we reveal that a recently discovered bacterial enzyme EarP, that transfers rhamnose to a specific arginine residue in its acceptor protein EF-P, specifically recognizes a β-hairpin loop. Notably, while the in vitro rhamnosyltransferase activity of EarP is abolished when presented with linear substrate peptide sequences derived from EF-P, the enzyme readily glycosylates the same sequence in a cyclized β-hairpin mimic. Additional studies with other substrate-mimicking cyclic peptides revealed that EarP activity is sensitive to the method used to induce cyclization and in some cases is tolerant to amino acid sequence variation. Using detailed NMR approaches, we established that the active peptide substrates all share some degree of β-hairpin formation, and therefore conclude that the β-hairpin epitope is the major determinant of arginine-rhamnosylation by EarP. Our findings add a novel recognition motif to the existing knowledge on substrate specificity of protein glycosylation, and are expected to guide future identifications of rhamnosylation sites in other protein substrates., For bacterial arginine rhamnosylation, the rhamnosyltransferase EarP specifically recognizes a β-hairpin structure in the acceptor substrate.

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products, 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 used bioinformatics combined with molecular and biochemical analyses and show that fructoselysine metabolism in E. coli is tightly controlled at the transcriptional level. The global regulator CRP (CAP) as well as the alternative sigma factor σ32 (RpoH) contribute to promoter activation at high cAMP-levels and inside warm-blooded hosts, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific 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.

Despite its potential importance for bacterial virulence, protein rhamnosylation has not yet been sufficiently studied. Specific anti-SerRha, anti-ThrRha and anti-AsnRha antibodies allowed the identification of previously unknown monorhamnosylated proteins in cytosol and membrane fractions of bacterial cell lysates. Mapping of the complete rhamnoproteome in pathogens should facilitate development of targeted therapies against bacterial infections.

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products, 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 used bioinformatics combined with molecular and biochemical analyses and show that fructoselysine metabolism in E. coli is tightly controlled at the transcriptional level. The global regulator CRP (CAP) as well as the alternative sigma factor σ32 (RpoH) contribute to promoter activation at high cAMP-levels and inside warm-blooded hosts, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific 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.

Post-translational modifications (PTM) are the evolutionary solution to challenge and extend the boundaries of genetically predetermined proteomic diversity. As PTMs are highly dynamic, they also hold an enormous regulatory potential. It is therefore not surprising that out of the 20 proteinogenic amino acids, 15 can be post-translationally modified. Even the relatively inert guanidino group of arginine is subject to a multitude of mostly enzyme mediated chemical changes. The resulting alterations can have a major influence on protein function. In this review, we will discuss how bacteria control their cellular processes and develop pathogenicity based on post-translational protein-arginine modifications.

We have previously found that a Sfrp1 knock-out environment fails to support the regeneration of hematopoetic stem cells (HSC) with serial to repopulate secondary recipients Renstrom et al., [1] . In order to dissect cell specific requirements of the Sfrp1 gene we established the Sfrp1flox/flox mouse strain and deleted Sfrp1 gene in Osterix+ (Sp7) osteolineage cells. In these Osx-Cre, Sfrp1 (OS1) mice, the number of MSCs is reduced, but these show an increased proportion of colony forming units (CFU-F). Furthermore, stromal cells grown ex-vivo show increased senescence-associated beta-galactosidase staining. In addition, the CFU-F-derived stromal cells differentiated spontaneously into adipocytes. These findings indicate altered functionality of stromal cells from OS1 mice. In the hematopoietic compartment of these mice, we found a decrease in myeloid progenitors in the bone marrow (BM) with concomitant increase in CD11b+ GR1hi granulocytes in peripheral blood. Although the number of primitive CD34- CD48- CD150+ HSCs (LT-HSCs) in the BM was unchanged, LT-HSCs from OS1 mice failed to repopulate in wild type recipients. In single cell cultures, we found that LT-HSCs from OS1 mice show decreased proliferation with concomitant increased differentiation into mature myeloid cells, which was associated with increased DNA damage as shown with comet tail assays and staining for gammaH2.AX. On a molecular level, we found that LT-HSCs from OS1 mice show increased CTNNB1 protein levels. CTNNB1 regulates cell differentiation and proliferation of stem cells by binding to Ep300 or CBP respectively Miyabayashi et al., [2] . Interestingly, CBP protein level was decreased in LT-HSCs from OS1 mice while Ep300 was increased, suggesting overactivation of the CTNNB1/Ep300 axis. Indeed, blocking CTNNB1/Ep300 binding with specific PP2A inhibitor IQ-1, we not only rescued the aberrant behavior of OS1 LT-HSC in vitro, but we also restored the repopulating activity of these LT-HSCs in vivo. Our results suggest that deletion of stromal SFRP1 diminishes the repopulating activity of LT-HSCs by increasing differentiation through PP2A-mediated dephosphorylation of the phospho-Ep300-binding site with CTNNB1.