Delivery of endolysin across outer membrane of Gram-negative bacteria using translocation domain of botulinum neurotoxin.

The emergence of multidrug resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin (BoNT) to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the BoNT translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machinery to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 minutes with only 0.5 µM against an A. baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.

Conversion of Host Cell Receptor into Virus Destructor by Immunodisc to Neutralize Diverse SARS-CoV-2 Variants.

The decreasing efficacy of antiviral drugs due to viral mutations highlights the challenge of developing a single agent targeting multiple strains. Using host cell viral receptors as competitive inhibitors is promising, but their low potency and membrane-bound nature have limited this strategy. In this study, the authors show that angiotensin-converting enzyme 2 (ACE2) in a planar membrane patch can effectively neutralize all tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that emerged during the COVID-19 pandemic. The ACE2-incorporated membrane patch implemented using nanodiscs replicated the spike-mediated membrane fusion process outside the host cell, resulting in virus lysis, extracellular RNA release, and potent antiviral activity. While neutralizing antibodies became ineffective as the SARS-CoV-2 evolved to better penetrate host cells the ACE2-incorporated nanodiscs became more potent, highlighting the advantages of using receptor-incorporated nanodiscs for antiviral purposes. ACE2-incorporated immunodisc, an Fc fusion nanodisc developed in this study, completely protected humanized mice infected with SARS-CoV-2 after prolonged retention in the airways. This study demonstrates that the incorporation of viral receptors into immunodisc transforms the entry gate into a potent virucide for all current and future variants, a concept that can be extended to different viruses.

Synthesis and physicochemical characterization of acyl myricetins as potential anti-neuroexocytotic agents.

Acyl myricetins (monopropionyl-, dipropionyl-, and monooctanoyl-myricetin, termed as MP1, MP2, and MO1, respectively) were synthesized through enzymatic or non-enzymatic esterification reaction of myricetin aglycone. Structure study indicated the hydroxyl group at C4' in B-ring was highly susceptible to acylation. Over its parental myricetin, acylated compounds showed enhanced lipophilicity (from 7.4- to 26.3-fold) and oxidative stability (from 1.9- to 3.1-fold) on the basis of logP and decay rate, respectively. MO1, presenting the physicochemical superiority compared to the others, provided lowest EC50 value of 2.51 µM on inhibition of neutrotransmitter release and CC50 value of 59.0 µM, leading to widest therapeutic window. All myricetin esters did not show any irritation toxicity when assessed with a chicken embryo assay. This study describes information on acylation of myricetin that has not yet been explored, and suggests that MO1 has membrane fusion-arresting and anti-neuroexocytotic potential for industrial application due to its enhanced biological properties.

Stimulation of Surface Polysaccharide Production under Aerobic Conditions Confers Aerotolerance in Campylobacter jejuni.

The ability of a foodborne pathogen to tolerate environmental stress critically affects food safety by increasing the risk of pathogen survival and transmission in the food supply chain. Campylobacter jejuni, a leading bacterial cause of foodborne illnesses, is an obligate microaerophile and is sensitive to atmospheric levels of oxygen. Currently, the molecular mechanisms of how C. jejuni withstands oxygen toxicity under aerobic conditions have not yet been fully elucidated. Here, we show that when exposed to aerobic conditions, C. jejuni develops a thick layer of bacterial capsules, which in turn protect C. jejuni under aerobic conditions. The presence of both capsular polysaccharides and lipooligosaccharides is required to protect C. jejuni from excess oxygen in oxygen-rich environments by alleviating oxidative stress. Under aerobic conditions, C. jejuni undergoes substantial transcriptomic changes, particularly in the genes of carbon metabolisms involved in amino acid uptake, the tricarboxylic acid (TCA) cycle, and the Embden-Meyerhof-Parnas (EMP) pathway despite the inability of C. jejuni to grow aerobically. Moreover, the stimulation of carbon metabolism by aerobiosis increases the level of glucose-6-phosphate, the EMP pathway intermediate required for the synthesis of surface polysaccharides. The disruption of the TCA cycle eliminates aerobiosis-mediated stimulation of surface polysaccharide production and markedly compromises aerotolerance in C. jejuni. These results in this study provide novel insights into how an oxygen-sensitive microaerophilic pathogen survives in oxygen-rich environments by adapting its metabolism and physiology. IMPORTANCE Oxygen-sensitive foodborne pathogens must withstand oxygen toxicity in aerobic environments during transmission to humans. C. jejuni is a major cause of gastroenteritis, accounting for 400 million to 500 million infection cases worldwide per year. As an obligate microaerophile, C. jejuni is sensitive to air-level oxygen. However, it has not been fully explained how this oxygen-sensitive zoonotic pathogen survives in aerobic environments and is transmitted to humans. Here, we show that under aerobic conditions, C. jejuni boosts its carbon metabolism to produce a thick layer of bacterial capsules, which in turn act as a protective barrier conferring aerotolerance. The new findings in this study improve our understanding of how oxygen-sensitive C. jejuni can survive in aerobic environments.

Sugar Modification of Wall Teichoic Acids Determines Serotype-Dependent Strong Biofilm Production in Listeria monocytogenes.

Biofilm production is responsible for persistent food contamination by Listeria monocytogenes, threatening food safety and public health. Human infection and food contamination with L. monocytogenes are caused primarily by serotypes 1/2a, 1/2b, and 4b. However, the association of biofilm production with phylogenic lineage and serotype has not yet been fully understood. In this study, we measured the levels of biofilm production in 98 clinical strains of L. monocytogenes at 37°C, 25°C, and 4°C. The phylogenetic clusters grouped by core genome multilocus sequence typing (cgMLST) exhibited association between biofilm production and phylogenetic lineage and serotype. Whereas clusters 1 and 3 consisting of serotype 4b strains exhibited weak biofilm production, clusters 2 (serotype 1/2b) and 4 (serotype 1/2a) were composed of strong biofilm formers. Particularly, cluster 2 (serotype 1/2b) strains exhibited the highest levels of biofilm production at 37°C, and the levels of biofilm production of cluster 4 (serotype 1/2a) strains were significantly elevated at all tested temperatures. Pan-genome analysis identified 22 genes unique to strong biofilm producers, most of which are related to the synthesis and modification of teichoic acids. Notably, a knockout mutation of the rml genes related to the modification of wall teichoic acids with l-rhamnose, which is specific to serogroup 1/2, significantly reduced the level of biofilm production by preventing biofilm maturation. Here, the results of our study show that biofilm production in L. monocytogenes is related to phylogeny and serotype and that the modification of wall teichoic acids with l-rhamnose is responsible for serotype-specific strong biofilm formation in L. monocytogenes. IMPORTANCE Biofilm formation on the surface of foods or food-processing facilities by L. monocytogenes is a serious food safety concern. Here, our data demonstrate that the level of biofilm production differs among serotypes 1/2a, 1/2b, and 4b depending on the temperature. Furthermore, sugar decoration of bacterial cell walls with l-rhamnose is responsible for strong biofilm production in serotypes 1/2a and 1/2b, commonly isolated from foods and listeriosis cases. The findings in this study improve our understanding of the association of biofilm production with phylogenetic lineage and serotype in L. monocytogenes.

Development of Fluoroquinolone Resistance through Antibiotic Tolerance in Campylobacter jejuni.

Antibiotic tolerance not only enables bacteria to survive acute antibiotic exposures but also provides bacteria with a window of time in which to develop antibiotic resistance. The increasing prevalence of Campylobacter jejuni isolates resistant to clinically important antibiotics, particularly fluoroquinolones (FQs), is a global public health concern. Currently, little is known about antibiotic tolerance and its effects on resistance development in C. jejuni. Here, we show that exposure to ciprofloxacin or tetracycline at concentrations 10 and 100 times higher than the MIC induces antibiotic tolerance in C. jejuni, whereas gentamicin or erythromycin treatment causes cell death. Interestingly, FQ resistance rapidly develops in C. jejuni after tolerance induction by ciprofloxacin and tetracycline. Furthermore, after tolerance is induced, alkyl hydroperoxide reductase (AhpC) plays a critical role in reducing FQ resistance development by alleviating oxidative stress. Together, these results demonstrate that exposure of C. jejuni to antibiotics can induce antibiotic tolerance and that FQ-resistant (FQR) C. jejuni clones rapidly emerge after tolerance induction. This study elucidates the mechanisms underlying the high prevalence of FQR C. jejuni and provides insights into the effects of antibiotic tolerance on resistance development. IMPORTANCE Antibiotic tolerance compromises the efficacy of antibiotic treatment by extending bacterial survival and facilitating the development of mutations associated with antibiotic resistance. Despite growing public health concerns about antibiotic resistance in C. jejuni, antibiotic tolerance has not yet been investigated in this important zoonotic pathogen. Here, our results show that exposure of C. jejuni to ciprofloxacin or tetracycline leads to antibiotic tolerance development, which subsequently facilitates the emergence of FQR C. jejuni. Importantly, these antibiotics are commonly used in animal agriculture. Moreover, our study suggests that the use of non-FQ drugs in animal agriculture promotes FQ resistance development, which is crucial because antibiotic-resistant C. jejuni is primarily transmitted from animals to humans. Overall, these findings increase our understanding of the mechanisms of resistance development through the induction of antibiotic tolerance.

Antimicrobial Synergy between Aminoglycosides and Licorice Extract in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that can develop serious invasive infections. Among foodborne pathogens, L. monocytogenes exhibits the highest case fatality despite antibiotic treatment, suggesting the current therapy should be improved. Although ampicillin and gentamicin are used as a combination therapy to treat listeriosis, our results showed there is no synergy between the two antibiotics. We discovered that aqueous extract of licorice generated significant antimicrobial synergy when combined with aminoglycosides, such as gentamicin, in L. monocytogenes. In the presence of 1 mg/mL licorice extract, for instance, the minimum inhibitory concentration (MIC) of gentamicin was reduced by 32-fold. Moreover, antimicrobial synergy with licorice extract made gentamicin-resistant clinical isolates of L. monocytogenes susceptible to gentamicin. Given the common use of licorice as a food sweetener in Western countries and a herb in Oriental medicine, our findings suggest that licorice extract can be potentially used as an antibiotic adjuvant to improve the efficacy of antimicrobial treatment of listeriosis.

Neuron-recognizable characteristics of peptides recombined using a neuronal binding domain of botulinum neurotoxin.

Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2' and EGFP-RBD', exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBD' to SV2C-LD was determined to be 5.45 µM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2', respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBD' to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2', respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBD'. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.

Potentiation of ß-Lactams against Methicillin-Resistant Staphylococcus aureus (MRSA) Using Octyl Gallate, a Food-Grade Antioxidant.

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to a number of antibiotics of clinical importance and is a serious threat to public health. Since bacteria rapidly develop resistance even to newly discovered antibiotics, this study aimed to develop drug potentiators to enhance the antibacterial activity of existing antibiotics for the control of MRSA. Based on our previous studies, screening of antimicrobial synergy was conducted with gallic acid and its derivatives using checkerboard assays. Antimicrobial synergy was confirmed with MRSA isolates from clinical cases. Combinations of penicillin, ampicillin, and cephalothin with octyl gallate (OG), an antioxidant approved by the US Food and Drug Administration (FDA), consistently exhibited synergistic bacteriostatic and bactericidal activities against MRSA, rendering MRSA sensitive to ß-lactams. The fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC) indices exhibited that the antimicrobial effects of OG were synergistic. The results of a permeability assay showed that OG significantly increased the permeability of the bacterial cell wall. Despite the intrinsic resistance of MRSA to ß-lactams, the findings in this study demonstrated that OG enhanced the activity of ß-lactams in MRSA and sensitized MRSA to ß-lactams, suggesting that OG can be used as a drug potentiator to control MRSA using existing antibiotics.

CosR Regulation of perR Transcription for the Control of Oxidative Stress Defense in Campylobacter jejuni.

Oxidative stress resistance is an important mechanism to sustain the viability of oxygen-sensitive microaerophilic Campylobacter jejuni. In C. jejuni, gene expression associated with oxidative stress defense is modulated by PerR (peroxide response regulator) and CosR (Campylobacter oxidative stress regulator). Iron also plays an important role in the regulation of oxidative stress, as high iron concentrations reduce the transcription of perR. However, little is known about how iron affects the transcription of cosR. The level of cosR transcription was increased when the defined media MEMα (Minimum Essential Medium) was supplemented with ferrous (Fe2+) and ferric (Fe3+) iron and the Mueller-Hinton (MH) media was treated with an iron chelator, indicating that iron upregulates cosR transcription. However, other divalent cationic ions, such as Zn2+, Cu2+, Co2+, and Mn2+, did not affect cosR transcription, suggesting that cosR transcription is regulated specifically by iron. Interestingly, the level of perR transcription was increased when CosR was overexpressed. The positive regulation of perR transcription by CosR was observed both in the presence or in the absence of iron. The results of the electrophoretic mobility shift assay showed that CosR directly binds to the perR promoter. DNase I footprinting assays revealed that the CosR binding site in the perR promoter overlaps with the PerR box. In the study, we demonstrated that cosR transcription is increased in iron-rich conditions, and CosR positively regulates the transcription of PerR, another important regulator of oxidative stress defense in C. jejuni. These results provide new insight into how C. jejuni regulates oxidative stress defense by coordinating the transcription of perR and cosR in response to iron.

Inducible plasmid display system for high-throughput selection of proteins with improved solubility.

Soluble expression of enzymes inside the cell is a prerequisite for the successful biotransformation of valuable products. Some key enzymes involved in biotransformation processes, however, are hardly expressed in their soluble forms. Here, we propose an inducible plasmid display, which is a molecular evolution strategy coupled with a high-throughput screening and/or selection method, as a simple and powerful tool for improving the solubility of target enzymes. Specifically, the Oct-1 DNA-binding domain and intein (i.e., auto-processing domain) were employed as anchoring and protein trans-splicing motifs to develop the system, in which the probability of protein trans-splicing is dependent on the soluble property of target proteins. The applicability of inducible plasmid display was investigated using an α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori, a highly insoluble and unstable enzyme in the cytoplasmic space of Escherichia coli, as a model protein. One round of the overall inducible plasmid display process, which consists of in vivo production of FucT2 mutants and in vitro screening, enabled soluble expression of FucT2 and selection of plasmids containing the corresponding genetic information. The inducible plasmid display developed in this study will contribute to the rapid and efficient screening and/or selection of soluble proteins.

Development of fluorescent Escherichia coli for a whole-cell sensor of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL), a major component of fucosylated human milk oligosaccharides, is beneficial to human health in various ways like prebiotic effect, protection from pathogens, anti-inflammatory activity and reduction of the risk of neurodegeneration. Here, a whole-cell fluorescence biosensor for 2'-FL was developed. Escherichia coli (E. coli) was engineered to catalyse the cleavage of 2'-FL into L-fucose and lactose by constitutively expressing α-L-fucosidase. Escherichia coli ∆L YA, in which lacZ is deleted and lacY is retained, was employed to disable lactose consumption. E. coli ∆L YA constitutively co-expressing α-L-fucosidase and a red fluorescence protein (RFP) exhibited increased fluorescence intensity in media containing 2'-FL. However, the presence of 50 g/L lactose reduced the RFP intensity due to lactose-induced cytotoxicity. Preadaptation of bacterial strains to fucose alleviated growth hindrance by lactose and partially recovered the fluorescence intensity. The fluorescence intensity of the cell was linearly proportional to 1-5 g/L 2'-FL. The whole-cell sensor will be versatile in developing a 2'-FL detection system.

Comparison of Fitness Cost and Virulence in Chromosome- and Plasmid-Mediated Colistin-Resistant Escherichia coli.

Five types of Escherichia coli strains were obtained and sequenced: colistin-susceptible (CL-S) strains, in vitro induced colistin-resistant (CL-IR) strains, mcr-1-negative colistin-resistant strains from livestock (CL-chrR), mcr-1-positive colistin-resistant strains (CL-mcrR), and mcr-1-transferred transconjugants (TC-mcr). Amino acid alterations of PmrAB, PhoPQ, and EptA were identified, and their mRNA expression was measured. Their growth rate was evaluated, and an in vitro competition assay was performed. Virulence was compared through serum resistance and survival in macrophages and Drosophila melanogaster. CL-IR and CL-chrR strains were colistin-resistant due to amino acid alterations in PmrAB, PhoPQ, or EptA, and their overexpression. All colistin-resistant strains did not show reduced growth rates compared with CL-S strains. CL-IR and CL-chrR strains were less competitive than the susceptible strain, but CL-mcrR strains were not. In addition, TC-mcr strains were also significantly more competitive than their respective parental susceptible strain. CL-IR strains had similar or decreased survival rates in human serum, macrophages, and fruit flies, compared with their parental, susceptible strains. CL-chrR strains were also less virulent than CL-S strains. Although CL-mcrR strains showed similar survival rates in human serum and fruit fly to CL-S strains, the survival rates of TC-mcr strains decreased significantly in human serum, macrophages, and fruit flies, compared with their susceptible recipient strain (J53). Chromosome-mediated, colistin-resistant E. coli strains have a fitness cost, but plasmids bearing mcr-1 do not increase the fitness burden of E. coli. Along with high usage of polymyxins, the no fitness cost of mcr-1-positive strains may facilitate rapid spread of colistin resistance.

Co-introduction of plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, increases fitness and virulence of bacterial host.

BACKGROUND: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. METHODS: Different plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. RESULTS: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring blaNDM-1 and blaOXA-232. CONCLUSIONS: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.

The mgtCBR mRNA Leader Secures Growth of Salmonella in Both Host and Non-host Environments.

Upon intracellular cues, bacterial mRNA leaders often form secondary structures that determine expression of a downstream protein-coding region(s), thereby providing bacteria with a mechanism to control the amounts of necessary proteins in the right locales. Here we describe a polycistronic mRNA leader that secures bacterial growth by preventing dysregulated expression of the protein-coding regions. In Salmonella, the mgtCBR mRNA encodes the virulence protein MgtC and the Mg2+ transporter MgtB. A mutant designed to produce leaderless mgtCBR mRNA induced MgtC and MgtB in conditions that promote mgtC transcription. The dysregulated expression of MgtC and MgtB impaired bacterial growth under all such non-host environments. While MgtC, but not MgtB, normally reduces ATP levels in a process requiring the F1F0 ATP synthase, dysregulated MgtC and MgtB reduced ATP levels independently of the F1F0 ATP synthase, which correlated with the mutant's growth defect. The mutant showed dysregulated MgtC expression and attenuated survival inside macrophages. While MgtB normally does not affect the phenotype, MgtB impaired intramacrophage survival of the mutant in the presence of MgtC. We provide an example showing that a polycistronic mRNA leader prevents the dysregulated function of protein-coding regions to allow bacteria to proliferate across complex niches.

Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids.

Whole cell biocatalysts can be used to convert fatty acids into various value-added products. However, fatty acid transport across cellular membranes into the cytosol of microbial cells limits substrate availability and impairs membrane integrity, which in turn decreases cell viability and bioconversion activity. Because these problems are associated with the mechanism of fatty acid transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was generated to promote substrate endocytosis. Caveolin-1 ( CAV1) expression in Escherichia coli increased both the fatty acid transport rate and intracellular fatty acid concentrations via endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-hydroxyoctadec-9-enoic acid) to ( Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of hydrophobic substrates.

ATP reduction by MgtC and Mg2+ homeostasis by MgtA and MgtB enables Salmonella to accumulate RpoS upon low cytoplasmic Mg2+ stress.

RpoS is one of several alternative sigma factors known to alter gene expression profiles by RpoS-associated RNA polymerase in response to a variety of stresses. The enteric bacteria Salmonella enterica and Escherichia coli accumulate RpoS under low Mg2+ concentrations via a common mechanism in which the PhoP regulator activates expression of antiadaptor proteins that, by sequestering the adaptor RssB, prevent RpoS degradation by the protease ClpXP. Here, we demonstrate that this genetic program alone does not fully support RpoS accumulation when cytoplasmic Mg2+ concentration drops to levels that impair protein synthesis. Under these circumstances, only S. enterica continues RpoS accumulation in a manner dependent on other PhoP-activated programs (i.e. ATP reduction by the MgtC protein and Mg2+ import by the MgtA and MgtB transporters) that maintain translation homeostasis. Moreover, we provide evidence that the mgtC gene, which is present in S. enterica but not in E. coli, is responsible for the differences in RpoS accumulation between these two bacterial species. Our results suggest that bacteria possess a mechanism to control RpoS accumulation responding to cytoplasmic Mg2+ levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

Engineering of α-1,3-fucosyltransferases for production of 3-fucosyllactose in Escherichia coli.

Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose. The C-terminus of 1,3-FTs is composed of heptad repeats, responsible for dimerization of the enzymes, and a peripheral membrane anchoring region. It has long been thought that truncation of most heptad repeats, retaining just 1 or 2, helps the soluble expression of 1,3-FTs. However, whether the introduction of truncated version of 1,3-FTs enhances the production of 3-FL in a metabolically engineered strain, is yet to be tested. In this study, the effect of these structural components on the production of 3-FL in Escherichia coli was evaluated through systematic truncation and elongation of the C-terminal regions of three 1,3-FTs from Helicobacter pylori. Although these three 1,3-FTs contained heptad repeats and membrane-anchoring regions of varying lengths, they commonly exhibited an optimal performance when the number of heptad repeats was increased, and membrane-binding region was removed. The production of 3-FL could be increased 10-20-fold through this simple strategy.

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regarding the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening.