Coli Cells Research Articles

Challenging a decades-old paradigm: ProB and ProA do not channel the unstable intermediate in proline synthesis after all

The pathway for synthesis of proline in most forms of life produces a highly unstable intermediate, γ-L-glutamyl 5-phosphate (GP). For nearly 70 y, channeling of this intermediate from the active site of glutamate 5-kinase to the active site of GP reductase has been believed to protect GP from cyclization to a dead-end product. However, the evidence presented in support of this idea is not conclusive. We show that changes in the structures of the kinase or reductase that should preclude a protein-protein interaction do not compromise proline synthesis in Escherichia coli, demonstrating that channeling does not occur. We calculate that the half-life of GP is 320 ms. Although GP is indeed unstable, it should diffuse the length of an E. coli cell in less than 3 ms. Thus, most GP produced by glutamate 5-kinase should encounter the active site of GP reductase before cyclization occurs.

Antimicrobial and antibiofilm potential of α-MSH derived cationic and hydrophobic peptides against Escherichia coli: Mechanistic insight through peptide-lipopolysaccharide interactions

The prevalence of infections caused by various Gram-negative pathogens specifically Escherichia coli continuously poses a significant challenge in health care as well as community settings owing to their ability to form biofilm and escalating tolerance towards available antibiotics. While most treatment regimes are targeted at eliminating the E. coli cells, the pathogenicity factors called endotoxin (lipopolysaccharides), associated with the sepsis initiation and the leading cause of death in intensive care units globally, are often ignored. In this study, the potency of alpha-Melanocyte Stimulating Hormone based-peptides, particularly Ana-9 and Ana-10 against E. coli was investigated through microbiological, biophysical, and microscopic assays. Both Ana-9 and Ana-10 demonstrated enhanced activity against planktonic E. coli cells, and retained their activity against biofilm, which was supported by confocal microscopy. From the mechanistic perspective, spectroscopic studies indicated that the binding of peptides with LPS led to structural alteration of peptides due to their insertion into the hydrophobic environment of LPS. The electrostatic interaction of the peptide with LPS leads to outer membrane disorganization, allowing the peptide to access the inner membrane, depolarize it and ultimately inhibit the bacterial cells within the biofilm. These observations were further confirmed by atomic force and scanning electron microscopy. Thus, this study deepens our understanding of the structural characteristics of peptides attached to LPS, which could lead to the gradual improvement in developing more potent, broad-spectrum endotoxin neutralizers.

E. coli cells advance into phase-separated (biofilm-simulating) extracellular polymeric substance containing DNA, HU, and lipopolysaccharide.

Understanding the constitution and behavior of biofilms is crucial to understanding how to deal with persistent biofilms. This study, together with other recent studies from our group, elucidates a novel aspect of the extracellular polymeric substance (EPS) of Escherichia coli biofilms, by creating a simulacrum of the EPS and then demonstrating that its formation involves liquid-liquid phase separation (LLPS) of HU, DNA, and lipopolysaccharide (LPS) components, with LPS determining the liquidity of this EPS simulacrum. The findings provide insight into the nature of biofilms and into how the interplay of HU, DNA, and LPS could modulate the structural integrity and functional dynamics of biofilms.

Eliminated high lipid inhibition in the anaerobic digestion of food waste for biomethane production by engineered E. coli with cell surface display lipase

Food waste (FW) with high content of lipid typically inhibits anaerobic digestion (AD) and methane production. In this study, a novel whole-cell catalyst was created to degrade lipid by displaying lipase on the E. coli cells surface to improve FW anaerobic digestion. The methane production rose, going from 25.78 to 161.77 mL/g VS, with a greater VS removal rate of 66.3% compared to CK group (29.6%). Long-chain fatty acids (LCFAs) was similarly reduced from 1733.6 mg/L to 337 mg/L. Microbial community analysis showed the relative abundance of Acinetbacter and Hydrogenophaga were increased from 1.7% to 6.6% and 1.3%–4.9%, respectively for substrates degradation. The methanogenic Methanosarcina increased from 24.7% to 52.3% for methane production. This study provided a potential approach that might be used to lessen lipid inhibition and improve anaerobic digestion of food waste.

Synthesis of colicin Ia neoglycoproteins: tools towards glyco-engineering of bacterial cell surfaces.

Colicins are antimicrobial proteins produced by certain strains of Escherichia coli that function as offensive weapons against closely-related competitor strains. Their bactericidal properties and narrow bacterial targeting range has made them of therapeutic interest. Furthermore, the applications of engineered non-bactericidal colicins are of interest as a cell surface-directed protein anchor for decorating E. coli with biomolecules. We previously demonstrated that an engineered non-bacteriocidal colicin E9 could be used to label bacterial cells with multiple biomolecules including glycans. Herein we extend our approach to colicin Ia, constructing mannose-presenting colicin la neoglycoproteins, through N-terminal organocatalyst-mediated protein aldol ligation (OPAL), or maleimide ligation targeting an internal cysteine. This work further highlights the potential utility of engineered colicins for non-genetic glyco-engineering of the E. coli cell surface.

Heterotrophic nitrification by Alcaligenes faecalis links organic and inorganic nitrogen metabolism.

Heterotrophic nitrification remains a mystery for decades. It has been commonly hypothesized that heterotrophic nitrifiers oxidize ammonia to hydroxylamine and then to nitrite in a way similar to autotrophic AOA and AOB. Recently, heterotrophic nitrifiers from Alcaligenes were found to oxidize ammonia to hydroxylamine and then to N2 ("dirammox", direct ammonia oxidation) by the gene cluster dnfABC with a yet-to-be-reported mechanism. The role of a potential glutamine amidotransferase DnfC clues the heterotrophic ammonia oxidation might involving in glutamine. Here, we found Alcaligenes faecalis JQ135 could oxidize amino acids besides ammonia. We discovered that glutamine is an intermediate of the dirammox pathway and the glutamine synthetase gene glnA is essential for both A. faecalis JQ135 and the Escherichia coli cells harboring dnfABC gene cluster to oxidize amino acids and ammonia. Our study expands understanding of heterotrophic nitrifiers and challenges the classical paradigm of heterotrophic nitrification.

E. coli FtsN coordinates synthesis and degradation of septal peptidoglycan by partitioning between a synthesis track and a denuded glycan track.

The E. coli cell division protein FtsN was proposed to coordinate septal peptidoglycan (sPG) synthesis and degradation to ensure robust cell wall constriction without lethal lesions. Although the precise mechanism remains unclear, previous work highlights the importance of two FtsN domains: the E domain, which interacts with and activates the sPG synthesis complex FtsWIQLB, and the SPOR domain, which binds to denuded glycan (dnG) strands, key intermediates in sPG degradation. Here, we used single-molecule tracking of FtsN and FtsW (a proxy for the sPG synthesis complex FtsWIQLB) to investigate how FtsN coordinates the two opposing processes. We observed dynamic behaviors indicating that FtsN's SPOR domain binds to dnGs cooperatively, which both sequesters the sPG synthesis complex on dnG (termed as the dnG-track) and protects dnGs from degradation by lytic transglycosylases (LTs). The release of the SPOR domain from dnGs leads to activating the sPG synthesis complex on the sPG-track and simultaneously exposing those same dnGs to degradation. Furthermore, FtsN's SPOR domain self-interacts and facilitates the formation of a multimeric sPG synthesis complex on both tracks. The cooperative self-interaction of the SPOR domain creates a sensitive switch to regulate the partitioning of FtsN between the dnG- and sPG-tracks, thereby controlling the balance between sequestered and active populations of the sPG synthesis complex. As such, FtsN coordinates sPG synthesis and degradation in space and time.

Recombinant C-Terminal Catalytic Domain of Rat L-Gulono Lactone Oxidase Produced in Bacterial Cells Is Enzymatically Active.

The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the 'in-gel' staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, Km values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively.

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Synonymous and non-synonymous codon substitutions can alleviate dependence on GroEL for folding.

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP-driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non-synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non-synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E . coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Comparative Study of the Antibacterial Effects of S-Nitroso-N-acetylcysteine and Sodium Nitrite against Escherichia coli and Their Application in Beef Sausages.

This study investigated the antibacterial effects of S-nitroso-N-acetylcysteine (SNAC) and sodium nitrite (NaNO2) against Escherichia coli and their application in beef sausages. Both SNAC and NaNO2 demonstrated pH-responsive antibacterial activity, with SNAC showing greater efficacy than NaNO2 (p < 0.05) at the same pH (3, 5, and 7). The reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced in E. coli by SNAC were significantly higher than those induced by NaNO2 (p < 0.05), and both ROS and RNS values increased as the pH decreased. In addition, a lower pH led to more pores on the E. coli cell surface and increased membrane permeability, resulting in a more pronounced inhibitory effect. When applied to a beef sausage, SNAC-treated sausages had significantly lower total colony counts and carbonyl content compared to NaNO2-treated ones (p < 0.05). Consequently, SNAC shows great potential as a replacement for NaNO2 in meat products.

Construction of a high-efficiency GjCCD4a mutant and its application for de novo biosynthesis of five crocins in Escherichia coli

Crocins are bioactive natural products that rarely exist in plants. High costs and resource shortage severely limit its development and application. Synthetic biology studies on crocins are of considerable global interest. However, the lack of high-efficiency genetic tools and complex cascade biocatalytic systems have substantially hindered progress in crocin biosynthesis-related research. Based on mutagenesis, a high-efficiency GjCCD4a mutant (N212m) was constructed with a catalytic efficiency that was 25.08-fold higher than that of the wild-type. Solubilized GjCCD4a was expressed via fusion with an MBP tag. Moreover, N212m and ten other genes were introduced into Escherichia coli for the de novo biosynthesis of five crocins. The engineered E57 strain produced crocins III and V with a total yield of 11.50 mg/L, and the E579 strain produced crocins I-V with a total output of 8.43 mg/L at shake-flask level. This study identified a marvelous genetic element (N212m) for crocin biosynthesis and achieved its de novo biosynthesis in E. coli using glucose. This study provides a reference for the large-scale production of five crocins using E. coli cell factories.

Effects of producing high levels of hyperthermophile-specific C25,C25-archaeal membrane lipids in Escherichia coli

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

The cytochrome P450 gene CYP4g1 driven by high temperature confers abamectin tolerance on Liriomyza trifolii through promoting cuticular hydrocarbons biosynthesis

Liriomyza trifolii, an invasive pest, poses a substantial threat to horticultural and vegetable plants. It spreads rapidly, especially in hot weather, leading to large-scale outbreaks with strong thermotolerance and insecticide resistance. In this study, mortality and LtCYP4g1 expression in L. trifolii were evaluated after thermal and insecticides exposure. Furthermore, functional verification of LtCYP4g1 was conducted through RNA interference and bacterial survival assays in Escherichia coli containing recombinant LtCYP4g1 protein. Results indicated that a short time exposure to high temperature incresed insecticide tolerance of L. trifolii, attributed to decreased mortality and induced LtCYP4g1 expression; LtCYP4g1 was involved in stimulating synthesis of cuticular hydrocarbons (CHCs) and elevating epicuticle lipid content and thickness, and E. coli cells overexpressing LtCYP4g1 exhibited significant tolerance to thermal and insecticide stress. In general, P450-mediated tolerance of L. trifolii was enhanced by high temperature, with LtCYP4g1 playing a role in promoting biosynthesis of CHCs for thickening epidermal lipid barrier and reducing cuticular penetration. This study provides a framework for delving into the function of CYP450s in insecticide detoxification and illustrates the role of global warming in driving the evolution of L. trifolii.

Flagellar Motility is Mutagenic.

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but to also promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an E. coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation frequencies. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.

Single-Use, Metabolite Absorbing, Resonant Transducer (SMART) Culture Vessels for Label-Free, Continuous Cell Culture Progression Monitoring.

Secreted metabolites are an important class of bio-process analytical technology (PAT) targets that can correlate to cell conditions. However, current strategies for measuring metabolites are limited to discrete measurements, resulting in limited understanding and ability for feedback control strategies. Herein, a continuous metabolite monitoring strategy is demonstrated using a single-use metabolite absorbing resonant transducer (SMART) to correlate with cell growth. Polyacrylate is shown to absorb secreted metabolites from living cells containing hydroxyl and alkenyl groups such as terpenoids, that act as a plasticizer. Upon softening, the polyacrylate irreversibly conformed into engineered voids above a resonant sensor, changing the local permittivity which is interrogated, contact-free, with a vector network analyzer. Compared to sensing using the intrinsic permittivity of cells, the SMART approach yields a 20-fold improvement in sensitivity. Tracking growth of many cell types such as Chinese hamster ovary, HEK293, K562, HeLa, and E. coli cells as well as perturbations in cell proliferation during drug screening assays are demonstrated. The sensor is benchmarked to show continuous measurement over six days, ability to track different growth conditions, selectivity to transducing active cell growth metabolites against other components found in the media, and feasibility to scale out for high throughput campaigns.

Exploring operational boundaries for acoustic concentration of cell suspensions

The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion.Key points• Acoustic focusing in a microchannel was used to concentrate cell suspensions• The parameters influencing focusing at high concentrations were studied• Three different cell strains were successfully concentratedGraphical abstract

Trans-cinnamaldehyde inhibits Escherichia coli growth by regulating lipopolysaccharide accumulation

Colibacillosis is among the most commonly occurring and economically devastating bacterial diseases of poultry worldwide owing to the misuse and overuse of antibiotics. Trans-cinnamaldehyde exhibits effective inhibition on multidrug-resistant Escherichia coli by disrupting the integrity of its cell wall. However, the mechanism underlying this inhibition is unclear. We found that trans-cinnamaldehyde inhibited E. coli efficiently with a minimum inhibitory concentration (MIC) of 140 μg/mL. Trans-cinnamaldehyde disrupted the integrity of the E. coli cell wall due to an imbalance between lipopolysaccharide (LPS) and phospholipids. The structural change in the cell wall disrupted its stability, further causing the leakage of alkaline phosphatase (AKP) and β-galactosidase, proteins, and DNA/RNA, and altering biofilms in the E. coli CVCC 1567 and the E. coli (047). This finally resulted in the death of E. coli. Inhibition by trans-cinnamaldehyde was more significant on the wall of E. coli CVCC 1567 because of a more severe loss of high molecular weight proteins and an easier dissolution of its cells. Trans-cinnamaldehyde affected gene expression of enzymes regulating LPS synthesis (lpxC, lpxA, lpxK, lpxM, yejM, ftsH, lapB), ultimately altering (LPS) content. Indeed, this effect is particularly pronounced in the genes expression of E. coli (047). In conclusion, trans-cinnamaldehyde is a potential novel drug candidate for preventing and treating infectious diseases caused by resistant E. coli clinical strains.

Visible light photocatalytic response of Fe doped TiO2: Inactivation of Escherichia coli

Heterogeneous photocatalysis has been widely investigated for the removal of pollutants and pathogenic microorganisms. Various modified photocatalysts were developed for the beneficial use of solar light. E. coli inactivation by Fe doping of TiO2 (P-25) and sol–gel prepared (SynTiO2) was achieved upon solar irradiation in both isotonic (IsoT) and water matrix (WM) conditions. First order kinetic rate constants were comparatively higher for bare SynTiO2 both in IsoT (k = 0.254 min−1) and WM (k = 0.180 min−1) solutions. WM exerted an overall retardation effect for all photocatalysts except for 0.50 % Fe-TiO2 and 0.25 % Fe-SynTiO2. Release of intracellular components was followed by spectroscopic parameters as well as dissolved organic carbon (DOC) contents. E. coli cell destruction was related to the released protein, carbohydrate, and K+ ion contents. Solar photocatalytic inactivation of E. coli using novel sol–gel prepared TiO2 and Fe doped sol–gel TiO2 was successfully demonstrated as an alternative and promising method for disinfection purposes.