Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L(-1) was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as 'persisters', are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Here we present a highly effective persister enrichment method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We used our enrichment method to monitor outgrowth of Escherichia coli persisters at the single-cell level, thereby conclusively demonstrating that persister awakening is a stochastic phenomenon. We anticipate that our approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported.

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100-400 s-1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.

Clostridium botulinum is an anaerobic sporeforming bacterium that is notorious for producing a potent neurotoxin. Spores of C. botulinum can survive mild food processing treatments and subsequently germinate, multiply, produce toxin and cause botulism. Control of spore germination and outgrowth is therefore essential for the safety of mildly processed foods. However, little is known about the process of spore germination in group II C. botulinum (gIICb), which are a major concern in chilled foods because they are psychrotrophic. The classical model of spore germination states that germination is triggered by the binding of a germinant molecule to a cognate germinant receptor. Remarkably, unlike many other sporeformers, gIICb has only one predicted canonical germinant receptor although it responds to multiple germinants. Therefore, we deleted the gerBAC locus that encodes this germinant receptor to determine its role in germination. Surprisingly, the deletion did not affect germination by any of the nutrient germinants, nor by the non-nutrient dodecylamine. We conclude that one or more other, so far unidentified, germinant receptors must be responsible for nutrient induced germination in gIICb. Furthermore, the gerBAC locus was strongly conserved with intact open reading frames in 159 gIICb genomes, suggesting that it has nevertheless an important function.

Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information.Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics' efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

Clinicians are increasingly confronted with the limitations of antibiotics to clear bacterial infections in patients. It has long been assumed that only antibiotic resistance plays a pivotal role in this phenomenon. Indeed, the worldwide emergence of antibiotic resistance is considered one of the major health threats of the 21st century. However, the presence of persister cells also has a significant influence on treatment outcomes. These antibiotic-tolerant cells are present in every bacterial population and are the result of the phenotypic switching of normal, antibiotic-sensitive cells. Persister cells complicate current antibiotic therapies and contribute to the development of resistance. In the past, extensive research has been performed to investigate persistence in laboratory settings; however, antibiotic tolerance under conditions that mimic the clinical setting remain poorly understood. In this study, we optimized a mouse model for lung infections with the opportunistic pathogen Pseudomonas aeruginosa. In this model, mice are intratracheally infected with P. aeruginosa embedded in seaweed alginate beads and subsequently treated with tobramycin via nasal droplets. A diverse panel of 18 P. aeruginosa strains originating from environmental, human, and animal clinical sources was selected to assess survival in the animal model. Survival levels were positively correlated with the survival levels determined via time-kill assays, a common method to study persistence in the laboratory. We showed that survival levels are comparable and thus that the classical persister assays are indicative of antibiotic tolerance in a clinical setting. The optimized animal model also enables us to test potential antipersister therapies and study persistence in relevant settings. IMPORTANCE The importance of targeting persister cells in antibiotic therapies is becoming more evident, as these antibiotic-tolerant cells underlie relapsing infections and resistance development. Here, we studied persistence in a clinically relevant pathogen, Pseudomonas aeruginosa. It is one of the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.), which are considered major health threats. P. aeruginosa is mostly known to cause chronic lung infections in cystic fibrosis patients. We mimicked these lung infections in a mouse model to study persistence under more clinical conditions. It was shown that the survival levels of natural P. aeruginosa isolates in this model are positively correlated with the survival levels measured in classical persistence assays in vitro. These results not only validate the use of our current techniques to study persistence but also open opportunities to study new persistence mechanisms or evaluate new antipersister strategies in vivo.

CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering.

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy.

Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem, as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. In this study, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which assumes that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof of concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections, which will set the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure, and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

Bacterial persistence is a potential cause of antibiotic therapy failure. Antibiotic-tolerant persisters originate from phenotypic differentiation within a susceptible population, occurring with a frequency that can be altered by mutations. Recent studies have proven that persistence is a highly evolvable trait and, consequently, an important evolutionary strategy of bacterial populations to adapt to high-dose antibiotic therapy. Yet, the factors that govern the evolutionary dynamics of persistence are currently poorly understood. Theoretical studies predict far-reaching effects of bottlenecking on the evolutionary adaption of bacterial populations, but these effects have never been investigated in the context of persistence. Bottlenecking events are frequently encountered by infecting pathogens during host-to-host transmission and antibiotic treatment. In this study, we used a combination of experimental evolution and barcoded knockout libraries to examine how population bottlenecking affects the evolutionary dynamics of persistence. In accordance with existing hypotheses, small bottlenecks were found to restrict the adaptive potential of populations and result in more heterogeneous evolutionary outcomes. Evolutionary trajectories followed in small-bottlenecking regimes additionally suggest that the fitness landscape associated with persistence has a rugged topography, with distinct trajectories toward increased persistence that are accessible to evolving populations. Furthermore, sequencing data of evolved populations and knockout libraries after selection reveal various genes that are potentially involved in persistence, including previously known as well as novel targets. Together, our results do not only provide experimental evidence for evolutionary theories, but also contribute to a better understanding of the environmental and genetic factors that guide bacterial adaptation to antibiotic treatment.

A set of bifunctional oxidase-peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low-micromolar range. The peroxidase fusions of eugenol oxidase and 5-hydroxymethylfurfural oxidase could be used for dioxygen-driven, one-pot, two-step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase-peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.