A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.     

Long-term adaptation of <Emphasis Type="Italic">Escherichia coli</Emphasis> to methanogenic co-culture enhanced succinate production from crude glycerol

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.     

Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.     

The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn’s disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn’s disease.     

Isolation of an Aptamer that Binds Specifically to E. coli

Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies.     

Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichiacoli Strains

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k_d) and transport (k_t) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k_d and k_t. The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.     

Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-l-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-l-fucose resulted in a maximum GDP-l-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer–Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.     

Atmospheric Nonthermal Plasma-Treated PBS Inactivates Escherichia coli by Oxidative DNA Damage

We recently reported that phosphate-buffered saline (PBS) treated with nonthermal dielectric-barrier discharge plasma (plasma) acquires strong antimicrobial properties, but the mechanisms underlying bacterial inactivation were not known. The goal of this study is to understand the cellular responses of Escherichia coli and to investigate the properties of plasma-activated PBS. The plasma-activated PBS induces severe oxidative stress in E. coli cells and reactive-oxygen species scavengers, α-tocopherol and catalase, protect E. coli from cell death. Here we show that the response of E. coli to plasma-activated PBS is regulated by OxyR and SoxyRS regulons, and mediated predominantly through the expression of katG that deactivates plasma-generated oxidants. During compensation of E. coli in the absence of both katG and katE, sodA and sodB are significantly overexpressed in samples exposed to plasma-treated PBS. Microarray analysis found that up-regulation of genes involved in DNA repair, and E. coli expressing recA::lux fusion was extremely sensitive to the SOS response upon exposure to plasma-treated PBS. The cellular changes include rapid loss of E. coli membrane potential and membrane integrity, lipid peroxidation, accumulation of 8-hydroxy-deoxyguinosine (8OHdG), and severe oxidative DNA damage; reveal ultimate DNA disintegration, and cell death. Together, these data suggest that plasma-treated PBS contains hydrogen peroxide and superoxide like reactive species or/and their products which lead to oxidative changes to cell components, and are eventually responsible for cell death.     

A mutant <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of <Emphasis Type="Italic">Escherichia coli</Emphasis>

l-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS–PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.     

Therapeutic stem cell‐derived alveolar‐like macrophages display bactericidal effects and resolve Pseudomonas aeruginosa‐induced lung injury

Bacterial lung infections lead to greater than 4 million deaths per year with antibiotic treatments driving an increase in antibiotic resistance and a need to establish new therapeutic approaches. Recently, we have generated mouse and rat stem cell‐derived alveolar‐like macrophages (ALMs), which like primary alveolar macrophages (1''AMs), phagocytose bacteria and promote airway repair. Our aim was to further characterize ALMs and determine their bactericidal capabilities. The characterization of ALMs showed that they share known 1''AM cell surface markers, but unlike 1''AMs are highly proliferative in vitro. ALMs effectively phagocytose and kill laboratory strains of P. aeruginosa (P.A.), E. coli (E.C.) and S. aureus, and clinical strains of P.A. In vivo, ALMs remain viable, adapt additional features of native 1''AMs, but proliferation is reduced. Mouse ALMs phagocytose P.A. and E.C. and rat ALMs phagocytose and kill P.A. within the lung 24 h post‐instillation. In a pre‐clinical model of P.A.‐induced lung injury, rat ALM administration mitigated weight loss and resolved lung injury observed seven days post‐instillation. Collectively, ALMs attenuate pulmonary bacterial infections and promote airway repair. ALMs could be utilized as an alternative or adjuvant therapy where current treatments are ineffective against antibiotic‐resistant bacteria or to enhance routine antibiotic delivery.     

Dynamical Model of Drug Accumulation in Bacteria: Sensitivity Analysis and Experimentally Testable Predictions

We present a dynamical model of drug accumulation in bacteria. The model captures key features in experimental time courses on ofloxacin accumulation: initial uptake; two-phase response; and long-term acclimation. In combination with experimental data, the model provides estimates of import and export rates in each phase, the time of entry into the second phase, and the decrease of internal drug during acclimation. Global sensitivity analysis, local sensitivity analysis, and Bayesian sensitivity analysis of the model provide information about the robustness of these estimates, and about the relative importance of different parameters in determining the features of the accumulation time courses in three different bacterial species: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The results lead to experimentally testable predictions of the effects of membrane permeability, drug efflux and trapping (e.g., by DNA binding) on drug accumulation. A key prediction is that a sudden increase in ofloxacin accumulation in both E. coli and S. aureus is accompanied by a decrease in membrane permeability.     

Insight into mechanisms of pig lncRNA FUT3-AS1 regulating E. coli F18-bacterial diarrhea

Escherichia coli F18 is a common conditional pathogen that is associated with a variety of infections in humans and animals. LncRNAs have emerged as critical players in pathogen infection, but their role in the resistance of the host to bacterial diarrhea remains unknown. Here, we used piglets as animal model and identified an antisense lncRNA termed FUT3-AS1 as a host regulator related to E. coli F18 infection by RNA sequencing. Downregulation of FUT3-AS1 expression contributed to the enhancement of E. coli F18 resistance in IPEC-J2 cells. FUT3-AS1 knockdown reduced FUT3 expression via decreasing the H4K16ac level of FUT3 promoter. Besides, the FUT3-AS1/miR-212 axis could act as a competing endogenous RNA to regulate FUT3 expression. Functional analysis demonstrated that target FUT3 plays a vital role in the resistance of IPEC-J2 cells to E. coli F18 invasion. A Fut3-knockout mice model was established and Fut3-knockout mice obviously improved the ability of resistance to bacterial diarrhea. Interestingly, FUT3 could enhance E. coli F18 susceptibility by activating glycosphingolipid biosynthesis and toll-like receptor signaling which are related to receptor formation and immune response, respectively. In summary, we have identified a novel biomarker FUT3-AS1 that modulates E. coli F18 susceptibility via histone H4 modifications or miR-212/FUT3 axis, which will provide theoretical guidance to develop novel strategies for combating bacterial diarrhea in piglets.     

FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium –copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium -copy number plasmid vectors in E. coli.     

Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli

Lipoxygenase (LOX; EC 1.13.11.12,) is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this study, we described the expression and characterization of Pseudomonas aeruginosa LOX in Escherichia coli. The recombinant LOX was successfully expressed and secreted by E. coli using its endogenous signal peptide. When induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (final concentration) at 20 °C for 47 h, the titer of the recombinant enzyme reached 3.89 U/mL. In order to characterize the catalytic properties, the recombinant LOX was purified to homogeneity on Q High Performance and Mono Q5/50GL sequentially. The molecular weight of the LOX was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The K _m and V _max of the recombinant enzyme were 48.9 μM and 0.226 μmol/min, respectively. The purified enzyme exhibited a maximum activity at 25 °C and pH 7.5. High-performance liquid chromatography analysis of the linoleic acid hydroperoxides produced by recombinant LOX revealed that the LOX from P. aeruginosa falls into linoleic acid 13(S)-LOX. To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms, and the achieved LOX yield is the highest ever reported.     

Heterologous Expression of Mannanase and Developing a New Reporter Gene System in Lactobacillus casei and Escherichia coli

Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli.     

Genome wide association study of Escherichia coli bloodstream infection isolates identifies genetic determinants for the portal of entry but not fatal outcome

Escherichia coli is an important cause of bloodstream infections (BSI), which is of concern given its high mortality and increasing worldwide prevalence. Finding bacterial genetic variants that might contribute to patient death is of interest to better understand infection progression and implement diagnostic methods that specifically look for those factors. E. coli samples isolated from patients with BSI are an ideal dataset to systematically search for those variants, as long as the influence of host factors such as comorbidities are taken into account. Here we performed a genome-wide association study (GWAS) using data from 912 patients with E. coli BSI from hospitals in Paris, France. We looked for associations between bacterial genetic variants and three patient outcomes (death at 28 days, septic shock and admission to intensive care unit), as well as two portals of entry (urinary and digestive tract), using various clinical variables from each patient to account for host factors. We did not find any association between genetic variants and patient outcomes, potentially confirming the strong influence of host factors in influencing the course of BSI; we however found a strong association between the papGII operon and entrance of E. coli through the urinary tract, which demonstrates the power of bacterial GWAS when applied to actual clinical data. Despite the lack of associations between E. coli genetic variants and patient outcomes, we estimate that increasing the sample size by one order of magnitude could lead to the discovery of some putative causal variants. Given the wide adoption of bacterial genome sequencing of clinical isolates, such sample sizes may be soon available.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Enhancing extracellular protein production in Escherichia coli by deleting the d‐alanyl‐d‐alanine carboxypeptidase gene dacC

d ‐Alanyl‐d ‐alanine carboxypeptidase DacC is important for synthesis and stabilization of the peptidoglycan layer of Escherichia coli. In this work, dacC of E. coli BL21 (DE3) was successfully deleted, and the effects of this deletion on extracellular protein production in E. coli were investigated. The extracellular activities and fluorescence value of recombinant amylase, green fluorescent protein, and α‐galactosidase of the deletion mutants were increased by 82.3, 29.1, and 37.7%, respectively, compared with that of control cells. The outer membrane permeability and intracellular soluble peptidoglycan accumulation of deletion mutant were also enhanced compared with those of control cells, respectively. Based on fluorescence‐assisted cell sorting analyses, we found that the morphology of the E. coli deletion mutant cells was altered compared with that of control cells. Local transparent bulges in the poles of the E. coli mutant with deletion of the dacC gene were found by transmission electron microscopy analysis. These bulges in the poles could explain the improvement in the production of extracellular protein by the E. coli mutant with deletion of the dacC gene. These findings provide important insights into the extracellular production of proteins using E. coli as microbial cell factories.