tRNA‐dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum

Aminoacyl‐phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala‐PG and a novel alanylated lipid, Alanyl‐diacylglycerol (Ala‐DAG). Ala‐DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala‐PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.     

GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway

The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine‐5′‐monophosphate (GMP) formation: conversion from xanthosine‐5′‐monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche.     

Hydrogen exchange of disordered proteins in Escherichia coli

A truly disordered protein lacks a stable fold and its backbone amide protons exchange with solvent at rates predicted from studies of unstructured peptides. We have measured the exchange rates of two model disordered proteins, FlgM and α-synuclein, in buffer and in Escherichia coli using the NMR experiment, SOLEXSY. The rates are similar in buffer and cells and are close to the rates predicted from data on small, unstructured peptides. This result indicates that true disorder can persist inside the crowded cellular interior and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic rates of exchange.     

Divergent evolution of a bifunctional de novo protein

Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn‐IF, was chosen from a library of binary patterned 4‐helix bundles. Syn‐IF was shown previously to rescue two different auxotrophic strains of E. coli: ΔilvA and Δfes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn‐IF, was evolved for faster rescue of either ΔilvA or Δfes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity.     

Evidence of land‐sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole‐genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype‐host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.