Non-canonical food sources: bacterial metabolism of Maillard reaction products and its regulation.

Proteins are an important part of our regular diet. During food processing, their amino acid composition can be chemically altered by the reaction of free amino groups with sugars - a process termed glycation. The resulting Maillard reaction products (MRPs) have low bioavailability and thus predominantly end up in the colon where they encounter our gut microbiota. In the following review, we summarize bacterial strategies to efficiently metabolize these non-canonical amino acids. A particular focus will be on the complex regulatory mechanisms that allow a tightly controlled expression of metabolic genes to successfully occupy the ecological niches that result from the chemical diversity of MRPs.

Identification of Pseudomonas asiatica subsp. bavariensis str. JM1 as the first Nε -carboxy(m)ethyllysine-degrading soil bacterium.

Thermal food processing leads to the formation of advanced glycation end products (AGE) such as Nε -carboxymethyllysine (CML). Accordingly, these non-canonical amino acids are an important part of the human diet. However, CML is only partially decomposed by our gut microbiota and up to 30% are excreted via faeces and, hence, enter the environment. In frame of this study, we isolated a soil bacterium that can grow on CML as well as its higher homologue Nε -carboxyethyllysine (CEL) as sole source of carbon. Bioinformatic analyses upon whole-genome sequencing revealed a subspecies of Pseudomonas asiatica, which we named 'bavariensis'. We performed a metabolite screening of P. asiatica subsp. bavariensis str. JM1 grown either on CML or CEL and identified N-carboxymethylaminopentanoic acid and N-carboxyethylaminopentanoic acid respectively. We further detected α-aminoadipate as intermediate in the metabolism of CML. These reaction products suggest two routes of degradation: While CEL seems to be predominantly processed from the α-C-atom, decomposition of CML can also be initiated with cleavage of the carboxymethyl group and under the release of acetate. Thus, our study provides novel insights into the metabolism of two important AGEs and how these are processed by environmental bacteria.

Exceptionally versatile take II: post-translational modifications of lysine and their impact on bacterial physiology.

Among the 22 proteinogenic amino acids, lysine sticks out due to its unparalleled chemical diversity of post-translational modifications. This results in a wide range of possibilities to influence protein function and hence modulate cellular physiology. Concomitantly, lysine derivatives form a metabolic reservoir that can confer selective advantages to those organisms that can utilize it. In this review, we provide examples of selected lysine modifications and describe their role in bacterial physiology.

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-D-glucose epimerization in Pseudomonas putida KT2440.

L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-ß-L-rhamnose (dTDP-L-Rha). dTDP-L-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-L-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-L-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-keto-6-deoxy-L-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-L-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria.

The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. While previous studies focused on how the amino acid context of a Pro-Pro motif determines the stalling strength, we extend this question to the mRNA level. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. We therefore developed a luminescence reporter to detect ribosome pausing in living cells, enabling us to dissect the roles of codon choice and tRNA selection as well as to explain the genome scale observations. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers.

Synthetic post-translational modifications of elongation factor P using the ligase EpmA.

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.

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, 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.

A set of rhamnosylation-specific antibodies enables detection of novel protein glycosylations in bacteria.

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.

Molecular design of a signaling system influences noise in protein abundance under acid stress in different γ-Proteobacteria.

Bacteria have evolved different signaling systems to sense and adapt to acid stress. One of these systems, the CadABC-system, responds to a combination of low pH and lysine availability. In Escherichia coli, the two signals are sensed by the pH sensor and transcription activator CadC and the co-sensor LysP, a lysine-specific transporter. Activated CadC promotes the transcription of the cadBA operon, which codes for the lysine decarboxylase CadA and the lysine/cadaverine antiporter CadB. The copy number of CadC is controlled translationally. Using a bioinformatics approach, we identified the presence of CadC with ribosomal stalling motifs together with LysP in species of the Enterobacteriaceae family. In contrast, we identified CadC without stalling motifs in species of the Vibrionaceae family, but the LysP co-sensor was not identified. Therefore, we compared the output of the Cad system in single cells of the distantly related organisms E. coli and V. campbellii using fluorescently-tagged CadB as the reporter. We observed a heterogeneous output in E. coli, and all the V. campbellii cells produced CadB. The copy number of the pH sensor CadC in E. coli was extremely low (≤4 molecules per cell), but it was 10-fold higher in V. campbellii An increase in the CadC copy number in E. coli correlated with a decrease in heterogeneous behavior. This study demonstrated how small changes in the design of a signaling system allow a homogeneous output and, thus, adaptation of Vibrio species that rely on the CadABC-system as the only acid resistance system.Importance Acid resistance is an important property of bacteria, such as Escherichia coli, to survive acidic environments like the human gastrointestinal tract. E. coli possess both passive and inducible acid resistance systems to counteract acidic environments. Thus, E. coli evolved sophisticated signaling systems to sense and appropriately respond to environmental acidic stress by regulating the activity of its three inducible acid resistance systems. One of these systems is the Cad system that is only induced under moderate acidic stress in a lysine-rich environment by the pH-responsive transcriptional regulator CadC. The significance of our research is in identifying the molecular design of the Cad systems in different Proteobacteria and their target expression noise at single cell level during acid stress conditions.

A ß-hairpin epitope as novel structural requirement for protein arginine rhamnosylation.

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 ß-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 ß-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 ß-hairpin formation, and therefore conclude that the ß-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.

Switching the Post-translational Modification of Translation Elongation Factor EF-P.

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands ß3/ß4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli. Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF-Ps from these two model organisms we demonstrate the importance of the ß3/ß4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation.

Exceptionally versatile - arginine in bacterial post-translational protein modifications.

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.

Bacterial transmembrane signalling systems and their engineering for biosensing.

Every living cell possesses numerous transmembrane signalling systems that receive chemical and physical stimuli from the environment and transduce this information into an intracellular signal that triggers some form of cellular response. As unicellular organisms, bacteria require these systems for survival in rapidly changing environments. The receptors themselves act as 'sensory organs', while subsequent signalling circuits can be regarded as forming a 'neural network' that is involved in decision making, adaptation and ultimately in ensuring survival. Bacteria serve as useful biosensors in industry and clinical diagnostics, in addition to producing drugs for therapeutic purposes. Therefore, there is a great demand for engineered bacterial strains that contain transmembrane signalling systems with high molecular specificity, sensitivity and dose dependency. In this review, we address the complexity of transmembrane signalling systems and discuss principles to rewire receptors and their signalling outputs.

Evolutionary analysis of polyproline motifs in Escherichia coli reveals their regulatory role in translation.

Translation of consecutive prolines causes ribosome stalling, which is alleviated but cannot be fully compensated by the elongation factor P. However, the presence of polyproline motifs in about one third of the E. coli proteins underlines their potential functional importance, which remains largely unexplored. We conducted an evolutionary analysis of polyproline motifs in the proteomes of 43 E. coli strains and found evidence of evolutionary selection against translational stalling, which is especially pronounced in proteins with high translational efficiency. Against the overall trend of polyproline motif loss in evolution, we observed their enrichment in the vicinity of translational start sites, in the inter-domain regions of multi-domain proteins, and downstream of transmembrane helices. Our analysis demonstrates that the time gain caused by ribosome pausing at polyproline motifs might be advantageous in protein regions bracketing domains and transmembrane helices. Polyproline motifs might therefore be crucial for co-translational folding and membrane insertion.

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P.

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-ß-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.

A Versatile Toolbox for the Control of Protein Levels Using Nε-Acetyl-l-lysine Dependent Amber Suppression.

The analysis of the function of essential genes in vivo depends on the ability to experimentally modulate levels of their protein products. Current methods to address this are based on transcriptional or post-transcriptional regulation of mRNAs, but approaches based on the exploitation of translation regulation have so far been neglected. Here we describe a toolbox, based on amber suppression in the presence of Nε-acetyl-l-lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC. The system was used to measure and compare the effects of AcK- and Nε-(tert-butoxycarbonyl)-l-lysine (BocK) dependent amber suppression in Escherichia coli. We also demonstrate here that, in combination with transcriptional regulation, the system allows protein production to be either totally repressed or gradually adjusted. To identify sequence motifs that provide improved translational regulation, we varied the sequence context of the amber codon and found that insertion of two preceding prolines drastically decreases luminescence. In addition, using LacZ as a reporter, we demonstrated that a strain encoding a variant with a Pro-Pro amber motif can only grow on lactose when AcK is supplied, thus confirming the tight translational regulation of protein output. In parallel, we constructed an E. coli strain that carries an isopropyl ß-d-1-thiogalactopyranoside (IPTG)-inducible version of the AcK-tRNA synthetase (AcKRS) gene on the chromosome, thus preventing mischarging of noncognate substrates. Subsequently, a diaminopimelic acid auxotrophic mutant (ΔdapA) was generated demonstrating the potential of this strain in regulating essential gene products. Furthermore, we assembled a set of vectors based on the broad-host-range pBBR ori that enable the AcK-dependent amber suppression system to control protein output not only in E. coli, but also in Salmonella enterica and Vibrio cholerae.

Structure-function analysis of the DNA-binding domain of a transmembrane transcriptional activator.

The transmembrane DNA-binding protein CadC of E. coli, a representative of the ToxR-like receptor family, combines input and effector domains for signal sensing and transcriptional activation, respectively, in a single protein, thus representing one of the simplest signalling systems. At acidic pH in a lysine-rich environment, CadC activates the transcription of the cadBA operon through recruitment of the RNA polymerase (RNAP) to the two cadBA promoter sites, Cad1 and Cad2, which are directly bound by CadC. However, the molecular details for its interaction with DNA have remained elusive. Here, we present the crystal structure of the CadC DNA-binding domain (DBD) and show that it adopts a winged helix-turn-helix fold. The interaction with the cadBA promoter site Cad1 is studied by using nuclear magnetic resonance (NMR) spectroscopy, biophysical methods and functional assays and reveals a preference for AT-rich regions. By mutational analysis we identify amino acids within the CadC DBD that are crucial for DNA-binding and functional activity. Experimentally derived structural models of the CadC-DNA complex indicate that the CadC DBD employs mainly non-sequence-specific over a few specific contacts. Our data provide molecular insights into the CadC-DNA interaction and suggest how CadC dimerization may provide high-affinity binding to the Cad1 promoter.