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Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH 4 + through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K + . Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.

Microbial natural products, particularly those produced by filamentous Actinobacteria , underpin the majority of clinically used antibiotics. Unfortunately, only a few new antibiotic classes have been discovered since the 1970s, which has exacerbated fears of a postapocalyptic world in which antibiotics have lost their utility. Excitingly, the genome sequencing revolution painted an entirely new picture, one in which an average strain of filamentous Actinobacteria harbors 20 to 50 natural product biosynthetic pathways but expresses very few of these under laboratory conditions. Development of methodology to access this "hidden" biochemical diversity has the potential to usher in a second Golden Era of antibiotic discovery. The proliferation of genomic data has led to inconsistent use of "cryptic" and "silent" when referring to biosynthetic gene clusters identified by bioinformatic analysis. In this Perspective, we discuss this issue and propose to formalize the use of this terminology., (Copyright © 2020 Hoskisson and Seipke.)

To avoid an antibiotic resistance crisis, we need to develop antibiotics at a pace that matches the rate of evolution of resistance. However, the complex functions performed by antibiotics-combining, e.g., penetration of membranes, counteraction of resistance mechanisms, and interaction with molecular targets-have proven hard to achieve with current methods for drug development, including target-based screening and rational design. Here, we argue that we can meet the evolution of resistance in the clinic with evolution of antibiotics in the laboratory. On the basis of the results of experimental evolution studies of microbes in general and antibiotic production in Actinobacteria in particular, we propose methodology for evolving antibiotics to circumvent mechanisms of resistance. This exploits the ability of evolution to find solutions to complex problems without a need for design. We review evolutionary theory critical to this approach and argue that it is feasible and has important advantages over current methods for antibiotic discovery., (Copyright © 2019 Wollein Waldetoft et al.)

The expansion of the genetic repertoire of an organism by gene duplication or horizontal gene transfer (HGT) can aid adaptation. Streptomyces bacteria are prolific producers of bioactive specialized metabolites that have adaptive functions in nature and have found extensive utility in human medicine. While the biosynthesis of these specialized metabolites is directed by dedicated biosynthetic gene clusters, little attention has been focused on how these organisms have evolved robustness in their genomes to facilitate the metabolic plasticity required to provide chemical precursors for biosynthesis during the complex metabolic transitions from vegetative growth to specialized metabolite production and sporulation. Here, we examine genetic redundancy in actinobacteria and show that specialized metabolite-producing bacterial families exhibit gene family expansion in primary metabolism. Focusing on a gene duplication event, we show that the two pyruvate kinases in the genome of Streptomyces coelicolor arose by an ancient duplication event and that each has evolved altered enzymatic kinetics, with Pyk1 having a 20-fold-higher k cat than Pyk2 (4,703 s -1 compared to 215 s -1 , respectively), and yet both are constitutively expressed. The pyruvate kinase mutants were also found to be compromised in terms of fitness compared to wild-type Streptomyces These data suggest that expanding gene families can help maintain cell functionality during metabolic perturbation such as nutrient limitation and/or specialized metabolite production. IMPORTANCE The rise of antimicrobial-resistant infections has prompted a resurgence in interest in understanding the production of specialized metabolites, such as antibiotics, by Streptomyces The presence of multiple genes encoding the same enzymatic function is an aspect of Streptomyces biology that has received little attention; however, understanding how the metabolic expansion influences these organisms can help enhance production of clinically useful molecules. Here, we show that expanding the number of pyruvate kinases enables metabolic adaptation, increases strain fitness, and represents an excellent target for metabolic engineering of industrial specialized metabolite-producing bacteria and the activation of cryptic specialized metabolites., (Copyright © 2018 Schniete et al.)