Franziska Koller

Conventional plant breeding relies on the occurrence of chromosomal crossover and spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way, as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species while at the same time allowing necessary adaptations. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs), which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break in DNA, recombinant enzymatic mutagens (REMs), such as CRISPR/Cas, additionally interfere with cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs introduce genetic changes and combinations that are unknown in the current breeding pool and that are also unlikely to occur as a result of any previously used breeding methods. The resulting genotypes may need to be considered as ‘new to the environment’. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity, and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterization can inform the following steps in risk assessment and guide requests for further data.

Conventional plant breeding relies on the occurrence of chromosomal crossover as well as spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species, while at the same time allowing necessary adaptions. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs) which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break of DNA, recombinant enzymatic mutagens (REMs) such as CRISPR/Cas additionally interfere with the cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs allow to introduce genetic changes and combinations that are unknown in the current breeding pool, and that are also unlikely to occur from any previously used breeding methods. The resulting genotypes may need to be considered as ‘new to the environment’. The reasons for the above can be identified in the mode of action of the REMs. CRISPR/Cas catalysed reactions in particular can interfere with and overcome 1) cytogenic features such as repair mechanisms; 2) factors influencing recombination and stability of the genome such as crossovers; 3) gene copies with and without proximity and 4) certain regulatory elements. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterisation can inform the following steps in risk assessment and guide requests for further data.

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.

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-Ser^Rha, anti-Thr^Rha and anti-Asn^Rha 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 (ARPs), such as fructoselysine. The gut microbiota includingEscherichia colican utilize these compounds as a nutrient source. While the degradation route for fructoselysine is well described, regulation of the corresponding pathway genesfrlABCDremained poorly understood. Here we use bioinformatics combined with molecular and biochemical analyses and show that inE. coli, fructoselysine metabolism 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 heat stress, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent tofrlABCD, 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 Summary: Thermal food processing promotes the formation of Amadori rearrangement products (ARPs), such as fructoselysine. The gut microbiota includingEscherichia colican utilize these compounds as a nutrient source. We show that inE. 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

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.